CN102516392B

CN102516392B - Cancer-targeted super antigen fusion protein, and preparation method and application thereof

Info

Publication number: CN102516392B
Application number: CN201110378799.4A
Authority: CN
Inventors: 孙嘉琳
Original assignee: Individual
Current assignee: Nanjing Double Bond Targeted Drug Technology Co ltd; Sun Jialin
Priority date: 2011-11-25
Filing date: 2011-11-25
Publication date: 2014-05-28
Anticipated expiration: 2031-11-25
Also published as: CN102516392A; WO2013075553A1

Abstract

The invention discloses a cancer-targeted super antigen fusion protein, and a preparation method and application thereof. The fusion protein contains: a) a ligand which promotes the growth of cancer cells and interacts with a cancer cell overexpression receptor; and b) super antigen Staphylococcus aureus enterotoxin E and mutants thereof which can cause anti-cancer immunologic reaction, wherein the Staphylococcus aureus enterotoxin E is abbreviated to SEE, and the mutants of the Staphylococcus aureus enterotoxin E are abbreviated to SEE-X, and X=1-3. Compared with SEA, the super antigen SEE disclosed by the invention is more stable; and SEE-1, SEE-2 and SEE-3, the mutants of SEE subjected to large-scale multi-point mutation reconstruction, reduce the interaction with MHC. Thus, the fusion protein composed of the SEE mutants and TGF-alpha, EGF, VEGF, GnRH or GRP indicates very high anti-cancer and anti-tumor biological activity.

Description

A kind of cancer-targeted super antigen fusion rotein and Preparation method and use

Technical field

The present invention relates to a kind of super antigen fusion protein and Preparation method and use, particularly relate to a kind of cancer-targeted super antigen and mutant fusion protein thereof and Preparation method and use.

Background technology

Super antigen fusion protein is applied to the drug research of cancer, for example cytokine and superantigen constructed fusion rotein (Chinese Patent Application No. 200310109829.7, " a kind of can in order to the super antigen fusion protein of anticancer therapy and production method thereof "; Chinese Patent Application No. 201010118438.1, " cell factor-super-antigen fusion protein is being prepared the application of solid tumor resisting medicine "; Chinese Patent Application No. 201010585818.6, " cell factor-super-antigen fusion protein is being prepared the application of cancer therapy drug "; PLoSOne 6, e16642,2011), hormone and superantigen constructed fusion rotein (Chinese Patent Application No. 200510078775.1 " cancer-targeted super antigen fusion protein and production method thereof ") and antibody and superantigen constructed fusion rotein (Proc Natl Acad Sci USA, 91,8945-8949,1994; Proc Natl Acad Sci USA, 92,9791-9795,1995), hormone in cytokine and hormone-super antigen fusion protein in cell factor-super-antigen fusion protein be by with cancer cells on the cytokine receptor of great expression and the interaction of hormone receptor and fusion rotein is navigated on cancer cells, and the cytotoxicity of another part of fusion rotein to be superantigen can induce t cell responses of attacking cancer cells.

Above-mentioned superantigen is staphylococcus aureus toxin A (Staphylococcal-enterotoxin A, SEA), it can produce the side effect of the lymphocytic reaction of general, there is research (Proc Natl Acad Sci USA, 94,2489-2494,1997) it has been carried out to the transformation of a point mutation.In addition, it is reported also unstable (J Biol Chem, 275,1665-1672,2000) of SEA itself.Improve the anticancer effect of super antigen fusion protein, can select the new superantigen of thinking except SEA.

In addition, activated material such as compound, nucleic acid, the polypeptide etc. that have utility value can improve its activity or utility value by the structure of transforming them, take enzyme as example, can remove or the means such as increase improve the catalytic activity of enzyme, heat-resisting, low temperature resistant, acidproof, alkaline-resisting, organic solvent-resistant etc. by the displacement of extensive multipoint mutation, fragment, amino acid.So by the large-scale point mutation transformation of superantigen, expect to create the superantigen that biological activity is higher.

Summary of the invention

The object of the invention is to overcome deficiency of the prior art, a kind of cancer-targeted super antigen fusion rotein that biological activity is higher is provided.

Second object of the present invention is to provide a kind of preparation method of super antigen fusion protein.

The 3rd object of the present invention is to provide a kind of purposes of super antigen fusion protein.

Technical scheme of the present invention is summarized as follows:

A kind of cancer-targeted super antigen fusion rotein, this fusion rotein contains:

A) promote growth of cancer cells the part with cancer cells overexpression acceptor interaction, described part is the polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor;

B) can cause superantigen staphylococcus aureus enterotoxin E (Staphylococcal-enterotoxin E, SEE) and the mutant thereof that antitumor immune reacts, described staphylococcus aureus enterotoxin E represents with abbreviation SEE; The mutant of described staphylococcus aureus enterotoxin E represents with abbreviation SEE-X, described X=1-3.

Described cytokine is selected from transforminggrowthfactor-α (the Transforming growth factor-α that is abbreviated as TGF-α, TGF-α), be abbreviated as Urogastron (the Epidermal growth factor of EGF, EGF), be abbreviated as the vascular endothelial growth factor (Vascular endothelial cell growth factor, VEGF) of VEGF; Described hormone is selected from the gonadotropin releasing hormone (Gonadotropin-releaslng hormone, GnRH) of being abbreviated as GnRH; The polypeptide of the non-hormone of the described non-antibody acellular factor is selected from the gastrin releasing peptide (Gastrin-releasing peptide, GRP) of being abbreviated as GRP.

Described SEE is the aminoacid sequence shown in SEQ ID No.2; Described SEE-1 is the aminoacid sequence shown in SEQ ID No.4; Described SEE-2 is the aminoacid sequence shown in SEQ ID No.6; Described SEE-3 is the aminoacid sequence shown in SEQ ID No.8.

Described SEE-1 is that the one or more amino acid on 20,21,24,27,34,39,40,1,42,44,49,74,75,78,79,81,83,84,217,220,222,223,225,227 on SEE are replaced.

Described SEE-2 is that the one or more amino acid on 20,21,24,27,34,35,39,40,41,42,44,45,46,49,74,75,78,79,81,83,84,188,190,217,219,220,222,223,224,225,227 on SEE are replaced.

Described SEE-3 is that the one or more amino acid on 20,21,24,27,34,35,36,39,40,41,42,44,45,46,49,62,74,75,77,78,79,81,83,84,188,190,217,218,219,220,221,222,223,224,225,227 on SEE are replaced.

Described this fusion rotein is: by the TGF-α-SEE shown in SEQ ID No.10; By the EGF-SEE shown in SEQ ID No.18; By the VEGF-SEE shown in SEQ ID No.26; By the GnRH-SEE shown in SEQ ID No.34; By the GRP-SEE shown in SEQ ID No.42; By the TGF-α-SEE-1 shown in SEQ ID No.12; By the EGF-SEE-1 shown in SEQ ID No.20; By the VEGF-SEE-1 shown in SEQ ID No.28; By the GnRH-SEE-1 shown in SEQ ID No.36; By the GRP-SEE-1 shown in SEQ ID No.44; By the TGF-α-SEE-2 shown in SEQ ID No.14; By the EGF-SEE-2 shown in SEQ ID No.22; By the VEGF-SEE-2 shown in SEQ ID No.30; By the GnRH-SEE-2 shown in SEQ ID No.38; By the GRP-SEE-2 shown in SEQ ID No.46; By the TGF-α-SEE-3 shown in SEQ ID No.16; By the EGF-SEE-3 shown in SEQ ID No.24; By the VEGF-SEE-3 shown in SEQ ID No.32; By the GnRH-SEE-3 shown in SEQ ID No.40; Or by the GRP-SEE-3 shown in SEQ ID No.48.

Recombinant vectors, contains the nucleotide sequence of fusion rotein described in coding claim 7, and described nucleotides sequence is classified the nucleotide sequence SEQ ID No.9 of coding SEQ ID No.10 as; The nucleotide sequence SEQ ID No.11 of coding SEQ ID No.12; The nucleotide sequence SEQ ID No.13 of coding SEQ ID No.14; The nucleotide sequence SEQ ID No.15 of coding SEQ ID No.16; The nucleotide sequence SEQ ID No.17 of coding SEQ ID No.18; The nucleotide sequence SEQ ID No.19 of coding SEQ ID No.20; The nucleotide sequence SEQ ID No.21 of coding SEQ ID No.22; The nucleotide sequence SEQ ID No.23 of coding SEQ ID No.24; The nucleotide sequence SEQ ID No.25 of coding SEQ ID No.26; The nucleotide sequence SEQ ID No.27 of coding SEQ ID No.28; The nucleotide sequence SEQ ID No.29 of coding SEQ ID No.30; The nucleotide sequence SEQ ID No.31 of coding SEQ ID No.32; The nucleotide sequence SEQ ID No.33 of coding SEQ ID No.34; The nucleotide sequence SEQ ID No.35 of coding SEQ ID No.36; The nucleotide sequence SEQ ID No.37 of coding SEQ ID No.38; The nucleotide sequence SEQ ID No.39 of coding SEQ ID No.40; The nucleotide sequence SEQ ID No.41 of coding SEQ ID No.42; The nucleotide sequence SEQ ID No.43 of coding SEQ ID No.44; The nucleotide sequence SEQ ID No.45 of coding SEQ ID No.46; Or shown in the nucleotide sequence SEQ IDNo.47 of coding SEQ ID No.48.

A preparation method for cancer-targeted super antigen fusion rotein, cultivates the host cell that contains above-mentioned recombinant vectors, collects and expresses a kind of cancer-targeted super antigen fusion rotein.

A kind of cancer-targeted super antigen fusion rotein is in the application of the medicine of preparation treatment cancer and anti entity tumour.

Advantage of the present invention:

Superantigen SEE is than more stable (the J Biol Chem of SEA, 275,1665-1672,2000), and reduced and the interaction of MHC through improved SEE mutant SEE-1, the SEE-2 of extensive multipoint mutation and SEE-3, so the fusion rotein being made up of SEE mutant and TGF-α, EGF, VEGF, GnRH or GRP shows very high anticancer and antineoplastic biological activity.

Accompanying drawing explanation

To be TGF-α suppress S180 tumor growth with SEE or SEE mutant composition fusion rotein to Fig. 1, expression be the tumor weight after tumor growth curve and mouse are dissected.

To be EGF suppress S180 tumor growth with SEE or SEE mutant composition fusion rotein to Fig. 2, expression be the tumor weight after tumor growth curve and mouse are dissected.

To be VEGF suppress S180 tumor growth with SEE or SEE mutant composition fusion rotein to Fig. 3, expression be the tumor weight after tumor growth curve and mouse are dissected.

The T cell that Fig. 4 utilizes immunohistochemical methods to detect, brown point is T cell.(in figure: 4-1 is TGF-α-SEE-3 administration group; 4-2 is EGF-SEE-3 administration group; 4-3 is VEGF-SEE-3 administration group)

The interferon-γ by T emiocytosis (IFN-γ) that Fig. 5 utilizes immunohistochemical methods to detect, brown part is IFN-γ.(in figure: 5-1 is TGF-α-SEE-3 administration group; 5-2 is EGF-SEE-3 administration group; 5-3 is VEGF-SEE-3 administration group)

Fig. 6 is that 6nRH and SEE or SEE mutant composition fusion rotein kill and wound Hep G2 liver cancer cell.

Fig. 7 is that GRP and SEE or SEE mutant composition fusion rotein kill and wound A549 lung carcinoma cell.

The cancer cells of being attacked by T cell that Fig. 8 shows, maxicell is cancer cells, minicell is T cell.(in figure: 8-1 is GnRH-SEE-3 group; 8-2 is GRP-SEE-3 group)

Embodiment

Strategic thought of the present invention is as follows:

Although can improve the performance of SEA by a point mutation, find a biological activity that can increase substantially superantigen than the better superantigen of SEA the structure construction that carries out the multipoint mutation of albumen, obtain better cancer therapy effect.

The superantigen of streptococcus aureus origin has SEA, SEB, SEC, SED, SEF, SEG, SEH, SEI, SEJ etc., they structurally have certain similarity (Infect Immun, 66,3337-3348,1998), so the also reference mutually of their space structure, for example SEA (EMBO J, 14,3292-3301,1995; J Biol Chem, 271,32212-32216,1996; J Mol Biol, 269,270-280,1997; Structure, 10,1619-1626,2002), SEB and SEC (Nature, 368,711-718,1994; Nature, 384,188-192,1996; J Mol Biol, 277,61-79,1998; Structure, 11,1151-1161,2003), SHE (EMBO J, 20,3306-3312,2001; J Mol Biol, 302,527-537,2000), and other related documents (Nature, 346,471-473,1990; Infect Immun, 59,2126-2134,1991; J Exp Med, 175,415-424,1992; Proc Natl Acad Sci USA, 89,7727-7731,1992; J Exp Med, 177,175-184,1993; Infect Immun, 61,2059-2068,1993; J Biol Chem, 275,1665-1672,2000; Trends Microbiol, 8,369-375,2000; Immunity, 14,331-344,2001; Clin Exp Immunol, 133,299-306,2003; J Mol Biol, 333,893-905,2003; J Biol Chem, 278,50412-50421,2003; Scand J Immunol, 59,345-355,2004; Semin Immunol, 19,262-271,2007), superantigen comprise the 20-27 amino acid fragment in SEE and SEA be considered to T cell on TCR interact, thereby activated T cell; The amino acid fragments such as 34-49,74-84,187-190 and 217-227 are considered to interact with histocompatibility antigen MHC.Can find one than the more effective superantigen of SEA and improve its biological activity by large-scale multipoint mutation by these information.

First the present invention selects SEE, and it has very large similarity but than SEA more stable (J Biol Chem, 275,1665-1672,2000), in the aminoacid sequence of SEE, can introduce extensive multipoint mutation compared with SEA, point mutation according to being:

1. strengthen the binding ability of TCR, the effect of strengthening superantigen and TCR just can improve the ability of superantigen inducing T cell, can consider to adopt the amino acid in SEA same position here.

2. reduce and the binding ability of histocompatibility antigen MHC, the interaction of superantigen SEE and histocompatibility antigen MHC as synantibody and polypeptide antigen interaction similar, be complementary interaction.The mode of its effect is:

1) interaction of polare Aminosaeren, i.e. the interaction of the basic aminoacids in acidic amino acid and the histocompatibility antigen in superantigen, or the interaction of acidic amino acid in basic aminoacids and histocompatibility antigen in superantigen;

2) interaction of nonpolar hydrophilic amino acid, i.e. interaction between nonpolar hydrophilic amino acid in superantigen and histocompatibility antigen;

3) interaction of nonpolar hydrophobic amino acid, i.e. interaction between nonpolar hydrophobic amino acid in superantigen and histocompatibility antigen;

4) the amino acid whose interaction of 1-3 needs suitable space above, and amino acid whose side chain lengths is suitable, people for a change above interactional amino acid whose length also can reduce these amino acid whose interactions.

So, by changing the amino acid whose character of superantigen and histocompatibility antigen interaction zone, for example change in superantigen and the interactional amino acid whose polarity of histocompatibility antigen and length etc. can reduce or weaken the interaction between superantigen and histocompatibility antigen.

Although disclosed the position of point mutation in the present invention embodiment 1 below, in fact can also on the position on Huo Qi side, these positions, introduce the amino acid of other similar physico-chemical property, also can obtain similar result.

As another part of modified version super antigen fusion protein be selected from cancer cells surface on the interactional part of acceptor (Receptor) (Ligand) of great expression, the for example polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor, in the present invention, cytokine is TGF-α, EGF or VEGF, hormone GnRH, the polypeptide of the non-hormone of the non-antibody acellular factor is GRP.

Superantigen is the superantigen SEE of staphylococcus aureus enterotoxin E, shown in SEQ ID No.2;

Superantigen is that wherein mutant SEE-1 is shown in SEQ ID No.4 through the SEE mutant of multipoint mutation transformation, and mutant SEE-2 is shown in SEQ ID No.6, and mutant SEE-3 is shown in SEQ ID No.8;

Described multipoint mutation occurs in 20,21,24,27,34,35,36,39,40,41,42,44,45,46,49,62,74,75,77,78,79,81,83,84,188,190,217,218,219,220,221,222,223,224,225,227;

The amino acid of described multipoint mutation is, on 20, and Arg → Gly; On 21, Asn → Thr; On 24, Ser → Gly; On 27, Arg → Lys; On 34, Glu → Ser or Glu → Ala; On 35, Lys → Ser; On 36, Ala → Gly; On 39, Glu → Ser; On 40, Asn → Ser or Asn → Ala; On 41, Lys → Glu; On 42, Glu → Lys; On 44, Asp → Ala or Asp → Ser; On 45, Asp → Ala; On 46, Gln → Ser; On 49, Glu → Thr or Glu → Ser; On 62, Pro → Ser; On 74, Lys → Thr or Lys → Ser; On 75, Asp → Ala or Asp → Thr; On 77, Thr → Ser; On 78, Asn → Ser; On 79, Lys → Glu; On 81, Lys → Glu; On 83, Lys → Ser; On 84, Lys → Ser or Lys → Thr; On 188, Ser → Thr; On 190, Glu → Thr; On 217, Lys → Thr or Lys → Ser; On 218, Thr → Ser; On 219, Ile → Leu; On 220, Asn → Ser; On 221, Ser → Thr; On 222, Glu → Thr or Glu → Ser; On 223, Asn → Ser; On 224, Leu → Ile; On 225, His → Ser; On 227, Asp → Ser.

Part-modified version super antigen fusion protein of utilization of the present invention and cancer cells acceptor interaction, wherein fused protein can be navigated on tumour cell with for example polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor of part of cancer cells acceptor interaction, modified version superantigen causes more powerful antitumor immune reaction around tumour cell, it is the cell-mediated cytotoxicity (Superantigen-dependent-cellular-cytotoxicity, SDCC) that superantigen relies on.Utilize this method just such fused protein can be navigated to specifically and on tumour cell and around tumour cell, cause anticancer cell toxicant immune response.

Below by specific embodiment, the present invention is further illustrated.

Experiment JEG-3: be murine sarcoma tumour Sarcoma (S180) tumour cell, human liver cancer cell Hep G2 and the human lung cancer cell A549 for setting up mouse-borne tumor model as the JEG-3 using in the embodiment of the present invention, these JEG-3 are purchased from ATCC.

Have been reported EGF Receptor EGFR on the S180 cancer cells of mouse and vegf receptor VEGFR can with the EGF in people source and VEGF interact (PLoS One 6, e16642,2011; Chinese Patent Application No. 201010118438.1, " cell factor-super-antigen fusion protein is being prepared the application of solid tumor resisting medicine "), and the acceptor of TGF-α is identical with EGF, all EGFR, so S180 cancer cells can be used for the experiment of TGF, EGF or VEGF and SEE or SEE mutant composition fusion rotein.

Liver cancer cell is expressed acceptor GnRH-R (Endocrinology, 136,75-84,1995 of GnRH; Cancer Res, 60,3701-3705,2000), so Hep G2 cancer cells can be used for the experiment of GnRH and SEE or SEE mutant composition fusion rotein.

Lung carcinoma cell is expressed acceptor GRP-R (Oncogene, 20,1563-1569,2001 of GRP; Ann Oncol, 18,1457-1466,2007), so A549 cancer cells can be used for the experiment of GRP and SEE or SEE mutant composition fusion rotein.

Embodiment 1 designs the mutant of SEE

Because SEA and SEE have very large similarity (Infect Immun, 66,3337-3348 in sequence, 1998), so the research of the space structure of SEA and other superantigen etc. can be used as reference (EMBO J, 14 of SEE, 3292-3301,1995; J Biol Chem, 271,32212-32216,1996; J Mol Biol, 269,270-280,1997; Structure, 10,1619-1626,2002; Nature, 368,711-718,1994; Nature, 384,188-192,1996; J Mol Biol, 277,61-79,1998; Structure, 11,1151-1161,2003; EMBO J, 20,3306-3312,2001; J Mol Biol, 302,527-537,2000), in addition also with reference to related documents (Nature, 346,471-473,1990; Infect Immun, 59,2126-2134,1991; J Exp Med, 175,415-424,1992; Proc Natl Acad Sci USA, 89,7727-7731,1992; J Exp Med, 177,175-184,1993; Infect Immun, 61,2059-2068,1993; J Biol Chem, 275,1665-1672,2000; Trends Microbiol, 8,369-375,2000; Immunity, 14,331-344,2001; Clin Exp Immunol, 133,299-306,2003; J Mol Biol, 333,893-905,2003; J Biol Chem, 278,50412-50421,2003; Scand J Immunol, 59,345-355,2004; Semin Immunol, 19,262-271,2007), by these information and SEE gene information (J Bacteriol, 170,2954-2960,1988; GenBank, M21319) determine the position of SEE point mutation, in the amino acid fragment of 20-27,34-49,74-84,187-190 and the 217-227 of the position of the point mutation that can consider in SEE, design as follows:

1. adopt the amino acid segment of being combined with TCR in SEA, because SEA has ability (Proc Natl Acad Sci USA, 91,8945-8949,1994 of very powerful inducing T cell; Proc Natl Acad Sci USA, 92,9791-9795,1995), design 4 amino acid whose point mutation of the 20-27 position in SEE sequence here: on 20, Arg → Gly; On 21, Asn → Thr; On 24, Ser → Gly; On 27, Arg → Lys.The point mutation of the mutant (SEE-1, SEE-2 and SEE-3) of all SEE on this region is all identical.

2. the combination of minimizing and histocompatibility antigen MHC, becomes acidity or basic aminoacids polar neutral amino acid (Ser or Thr) or nonpolar hydrophobic amino acid (Ala) or changes the character (basic aminoacids becomes acidic amino acid or acidic amino acid becomes basic aminoacids) of its polarity or change amino acid whose side chain lengths.

2.1.SEE-1 the design of mutant

On 34, Glu → Ser; On 39, Glu → Ser; On 40, Asn → Ser; On 41, Lys → Glu; On 42, Glu → Lys; On 44, Asp → Ala; On 49, Glu → Thr; On 74, Lys → Thr; On 75, Asp → Ala; On 78, Asn → Ser; On 79, Lys → Glu; On 81, Lys → Glu; On 83, Lys → Ser; On 84, Lys → Ser; On 217, Lys → Thr; On 220, Asn → Ser; On 222, Glu → Thr; On 223, Asn → Ser; On 225, His → Ser; On 227, Asp → Ser.Finally obtain the aminoacid sequence (SEQ ID NO.4) of SEE-1.

2.2.SEE-2 the design of mutant

On 34, Glu → Ser; On 35, Lys → Ser; On 39, Glu → Ser; On 40, Asn → Ser; On 41, Lys → Glu; On 42, Glu → Lys; On 44, Asp → Ser; On 45, Asp → Ala; On 46, Gln → Ser; On 49, Glu → Thr; On 74, Lys → Thr; On 75, Asp → Ala; On 78, Asn → Ser; On 79, Lys → Glu; On 81, Lys → Glu; On 83, Lys → Ser; On 84, Lys → Ser; On 188, Ser → Thr; On 190, Glu → Thr; On 217, Lys → Ser; On 219, Ile → Leu; On 220, Asn → Ser; On 222, Glu → Thr; On 223, Asn → Ser; On 224, Leu → Ile; On 225, His → Ser; On 227, Asp → Ser.Finally obtain the aminoacid sequence (SEQ ID NO.6) of SEE-2.

2.3.SEE-3 the design of mutant

On 34, Glu → Ala; On 35, Lys → Ser; On 36, Ala → Gly; On 39, Glu → Ser; On 40, Asn → Ala; On 41, Lys → Glu; On 42, Glu → Lys; On 44, Asp → Ser; On 45, Asp → Ala; On 46, Gln → Ser; On 49, Glu → Ser; On 62, Pro → Ser; On 74, Lys → Ser; On 75, Asp → Thr; On 77, Thr → Ser; On 78, Asn → Ser; On 79, Lys → Glu; On 81, Lys → Glu; On 83, Lys → Ser; On 84, Lys → Thr; On 188, Ser → Thr; On 190, Glu → Thr; On 217, Lys → Ser; On 218, Thr → Ser; On 219, Ile → Leu; On 220, Asn → Ser; On 221, Ser → Thr; On 222, Glu → Ser; On 223, Asn → Ser; On 224, Leu → Ile; On 225, His → Ser; On 227, Asp → Ser.Finally obtain the aminoacid sequence (SEQ ID NO.8) of SEE-3.

The structure of embodiment 2 superantigens and mutant expression vector thereof

The DNA nucleotide sequence of coding SEE derives from its gene information (J Bacteriol, 170,2954-2960,1988, GenBank database, M21319), and mutant is according to the design of embodiment 1, entrust TAKARA company synthetic DNA fragment, it comprises the sequence fragment of SEE gene or its mutant and a connection peptides (shown in SEQ ID NO.50) and HindIII and below several bases of XhoI limiting enzyme point above in whole segment, synthetic nucleic acid fragment is inserted respectively to T carrier and carries out DNA sequencing evaluation, and then after processing with HindIII and XhoI by double digestion method, fragment is inserted respectively on pET22b plasmid, obtain the carrier pET22b-SEE-1 that expresses the carrier pET22b-SEE of SEE gene and the mutant of SEE, pET22b-SEE-2 and pET22b-SEE-3.By the corresponding protein sequence of these vector expressions, SEE is shown in SEQ ID NO.2, and SEE-1 is shown in SEQ ID NO.4, and SEE-2 is shown in SEQ ID NO.6, and SEE-3 is shown in SEQ ID NO.8.

The nucleotide sequence of coding SEE is shown in SEQ ID NO.1, and the nucleotide sequence of coding SEE-1 is shown in SEQ ID NO.3, and the nucleotide sequence of coding SEE-2 is shown in SEQ ID NO.5, and the nucleotide sequence of coding SEE-3 is shown in SEQ ID NO.7.

The nucleotide sequence of coding connection peptides is shown in SEQ ID NO.49.

The structure of the expression vector of embodiment 3TGF-α and SEE or SEE mutant composition fusion rotein

According to the information (NM_003236) of the TGF-α gene in the people source of GenBank database, entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise TGF-α gene and before TGF-α, increase again the several bases that limit restriction enzyme site BamHI and EcoRI, at the restriction restriction enzyme site that has comprised SalI and HindIII below of segment.This synthetic nucleic acid fragment is inserted to T carrier and carries out DNA sequencing evaluation, and then after processing with BamHI and HindIII by double digestion method, this fragment is inserted respectively to the pET22b-SEE in embodiment 2, pET22b-SEE-1, the carrier of pET22b-SEE-2 and pET22b-SEE-3, so just produce expression vector pET22b-TGF-α-SEE, pET22b-TGF-α-SEE-1, pET22b-TGF-α-SEE-2 and pET22b-TGF-α-SEE-3, can express TGF-α-SEE (SEQ ID NO.10), TGF-α-SEE-1 (SEQ ID NO.12), TGF-α-SEE-2 (SEQ ID NO.14) and TGF-α-SEE-3 (SEQ ID NO.16) fusion rotein.

The structure of the expression vector of embodiment 4EGF and SEE or SEE mutant composition fusion rotein

According to the information (NM_001963 and NM_001178130) of the EGF gene in the people source of GenBank database, entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise EGF gene and before EGF, increase again the several bases that limit restriction enzyme site BamHI and EcoRI, at the restriction restriction enzyme site that has comprised SalI and HindIII below of segment.This synthetic nucleic acid fragment is inserted to T carrier and carries out DNA sequencing evaluation, and then after processing with BamHI and HindIII by double digestion method, this fragment is inserted respectively to the pET22b-SEE in embodiment 2, pET22b-SEE-1, the carrier of pET22b-SEE-2 and pET22b-SEE-3, so just produce expression vector pET22b-EGF-SEE, pET22b-EGF-SEE-1, pET22b-EGF-SEE-2 and pET22b-EGF-SEE-3, can express EGF-SEE (SEQ ID NO.18), EGF-SEE-1 (SEQ ID NO.20), EGF-SEE-2 (SEQ ID NO.22) and EGF-SEE-3 (SEQ ID NO.24) fusion rotein.

The structure of the expression vector of embodiment 5VEGF and SEE or SEE mutant composition fusion rotein

According to information (NM_003376) and the reference papers (J.Biol.Chem. of the VEGF gene in the people source of GenBank database, 266,11947-11954,1991), entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise VEGF gene (121 amino acid) and before VEGF, increase again the several bases that limit restriction enzyme site BamHI and EcoRI, at the restriction restriction enzyme site that has comprised SalI and HindIII below of segment.This synthetic nucleic acid fragment is inserted to T carrier and carries out DNA sequencing evaluation, and then after processing with BamHI and HindIII by double digestion method, this fragment is inserted respectively to the pET22b-SEE in embodiment 2, pET22b-SEE-1, the carrier of pET22b-SEE-2 and pET22b-SEE-3, so just produce expression vector pET22b-VEGF-SEE, pET22b-VEGF-SEE-1, pET22b-VEGF-SEE-2 and pET22b-VEGF-SEE-3, can VEGF expression-SEE (SEQ ID NO.26), VEGF-SEE-1 (SEQ ID NO.28), VEGF-SEE-2 (SEQ ID NO.30) and VEGF-SEE-3 (SEQ ID NO.32) fusion rotein.

The structure of the expression vector of embodiment 6GnRH and SEE or SEE mutant composition fusion rotein

According to the information (NM_021081 and NM_000825 etc.) of the GnRH gene in the people source of GenBank database, entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise GnRH gene and before GnRH, increase again the several bases that limit restriction enzyme site BamHI and EcoRI, at the restriction restriction enzyme site that has comprised SalI and HindIII below of segment.This synthetic nucleic acid fragment is inserted to T carrier and carries out DNA sequencing evaluation, and then after processing with BamHI and HindIII by double digestion method, this fragment is inserted respectively to the pET22b-SEE in embodiment 2, pET22b-SEE-1, the carrier of pET22b-SEE-2 and pET22b-SEE-3, so just produce expression vector pET22b-GnRH-SEE, pET22b-GnRH-SEE-1, pET22b-GnRH-SEE-2 and pET22b-GnRH-SEE-3, can express GnRH-SEE (SEQ ID NO.34), GnRH-SEE-1 (SEQ ID NO.36), GnRH-SEE-2 (SEQ ID NO.38) and GnRH-SEE-3 (SEQ ID NO.40) fusion rotein.

The structure of the expression vector of embodiment 7GRP and SEE or SEE mutant composition fusion rotein

According to the information (NM_002091, NM_001012512 and NM_001012513 etc.) of the GRP gene in the people source of GenBank database, entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise GnRH gene and before GnRH, increase again the several bases that limit restriction enzyme site BamHI and EcoRI, at the restriction restriction enzyme site that has comprised SalI and HindIII below of segment.This synthetic nucleic acid fragment is inserted to T carrier and carries out DNA sequencing evaluation, and then after processing with BamHI and HindIII by double digestion method, this fragment is inserted respectively to the pET22b-SEE in embodiment 2, pET22b-SEE-1, the carrier of pET22b-SEE-2 and pET22b-SEE-3, so just produce expression vector pET22b-GRP-SEE, pET22b-GRP-SEE-1, pET22b-GRP-SEE-2 and pET22b-GRP-SEE-3, can express GRP-SEE (SEQ ID NO.42), GRP-SEE-1 (SEQ ID NO.44), GRP-SEE-2 (SEQ ID NO.46) and GRP-SEE-3 (SEQ ID NO.48) fusion rotein.

Expression, denature and renature and the purifying of embodiment 8 various fusion roteins

By various expression plasmid pET22b-TGF-α-SEE, pET22b-TGF-α-SEE-1, pET22b-TGF-α-SEE-2, pET22b-TGF-α-SEE-3, pET22b-EGF-SEE, pET22b-EGF-SEE-1, pET22b-EGF-SEE-2, pET22b-EGF-SEE-3, pET22b-VEGF-SEE, pET22b-VEGF-SEE-1, pET22b-VEGF-SEE-2, pET22b-VEGF-SEE-3, pET22b-GnRH-SEE, pET22b-GnRH-SEE-1, pET22b-GnRH-SEE-2, pET22b-GnRH-SEE-3, pET22b-GRP-SEE, pET22b-GRP-SEE-1, pET22b-GRP-SEE-2 and pET22b-GRP-SEE-3 proceed to e. coli bl21 (DE3) with electroporation respectively, utilize microbiotic Amp (Ampicillin) screening positive bacteria.Next the process of the expression of various albumen, denature and renature and purifying is roughly the same, operates as follows:

The e. coli bl21 (DE3) that contains expression plasmid first carries out extensive 37 ℃ of cultivations, then add IPTG (Isopropylthio-β-D-galactoside) to make it concentration and reach 1mM and spend the night 30 ℃ and cultivate, thus induction expression protein.The 2nd day centrifugation medium and collect thalline, use supersonic method breaking cell wall, and centrifugal collection occlusion body precipitates, and the albumen is here to exist with occlusion body form.The urea-denatured dissolving of 6M for occlusion body albumen, then carry out multistage dialysis, dialysis solution is urea for example 3M, 2M and the 1M progressively diluting, next be 0.5M urea, 0.4M L-arginine, 375 μ M Sleep-promoting factor B GSSG, 1.875mM reduced glutathion GSH, after dialysis, carry out centrifugally, gained supernatant liquor is exactly the renaturation solution of albumen.Carry out purifying for albumen with HisBind Purification Kit test kit (Novagen company), first rinse gel column with Binding Buffer, also in protein solution, add Binding Buffer simultaneously, by protein sample upper prop, with Wash Buffer rinsing, then using Elute Buffer wash-out, identify with protein electrophorese, is that the albumen of a band is for later experiment by purity.Obtain so various high purity fusion rotein TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2, TGF-α-SEE-3, EGF-SEE, EGF-SEE-1, EGF-SEE-2, EGF-SEE-3, VEGF-SEE, VEGF-SEE-1, VEGF-SEE-2, VEGF-SEE-3, GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3.

Embodiment 9 mouse tumors form experiment

First select 450 of male ICR mouses, 4-5 week, 18-22g, mice sarcoma cell S180 buys from ATCC, first carries out vitro culture, then is expelled to ICR mouse peritoneal, carries out large scale culturing in body, last, takes out Intraabdominal S180 cell, by 2x10 ⁶mice sarcoma cell S180 is inoculated in the oxter, right side of ICR mouse.

Embodiment 10TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2 and TGF-α-SEE-3 fusion rotein suppress mouse tumor experiment

Get 150 tumor-bearing mices in embodiment 9, be divided into 5 groups, every group 30, after inoculated tumour cell, within the 2nd, 4,6,8 days, inject respectively TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2 and TGF-α-SEE-3 fusion rotein, dosage is 100pmol, a control group injecting normal saline, kills mouse on the 9th day.Result shows that TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2 and TGF-α-SEE-3 have restraining effect to the growth of tumour, wherein, TGF-α-SEE-2 and TGF-α-SEE-3 have shown better effect, tumour occurs organizing late 1-2 days than other, and very little tumour (Fig. 1) appears in TGF-α-SEE-3 medication group the 7th talent.

Embodiment 11EGF-SEE, EGF-SEE-1, EGF-SEE-2 and EGF-SEE-3 fusion rotein suppress mouse tumor experiment

Get 150 tumor-bearing mices in embodiment 9, be divided into 5 groups, every group 30, after inoculated tumour cell, within the 2nd, 4,6,8 days, inject respectively EGF-SEE, EGF-SEE-1, EGF-SEE-2 and EGF-SEE-3 fusion rotein, dosage is 100pmol, a control group injecting normal saline, kills mouse on the 9th day.Result shows that EGF-SEE, EGF-SEE-1, EGF-SEE-2 and EGF-SEE-3 have restraining effect to the growth of tumour, wherein, EGF-SEE-2 and EGF-SEE-3 have shown better effect, tumour occurs organizing late 1-2 days than other, and very little tumour (Fig. 2) appears in EGF-SEE-3 medication group the 7th talent.

Embodiment 12VEGF-SEE, VEGF-SEE-1, VEGF-SEE-2 and VEGF-SEE-3 fusion rotein suppress mouse tumor experiment

Get 150 tumor-bearing mices in embodiment 9, be divided into 5 groups, every group 30, the 2nd, 4,6,8 days difference injection of VEGF-SEE, VEGF-SEE-1, VEGF-SEE-2 and VEGF-SEE-3 fusion roteins after inoculated tumour cell, dosage is 100pmol, a control group injecting normal saline, kills mouse on the 9th day.Result shows that VEGF-SEE, VEGF-SEE-1, VEGF-SEE-2 and VEGF-SEE-3 have restraining effect to the growth of tumour, wherein, VEGF-SEE-2 and VEGF-SEE-3 have shown better effect, tumour occurs organizing late 1-2 days than other, and very little tumour (Fig. 3) appears in VEGF-SEE-3 medication group the 7th talent.

The lymphocytic detection of T in embodiment 13 tumor tissues

The mouse S180 tumor tissues of the S180 tumor tissues of various process in embodiment 10-12 fusion rotein administration mouse and control group physiological saline is cut into small pieces, makes paraffin section with paraffin embedding, then carry out immunohistochemical assay.In order to detect the T cell in tumor tissues, use the anti-cd 3 antibodies of Santa Cruz Biotechnolog company, then and avidin-biotin-perxidase complex (Zymed company) anti-with two, finally uses Diaminobenzidine (DAB) colour developing.Fig. 4 is (in figure: 4-1 is TGF-α-SEE-3 administration group; 4-2 is EGF-SEE-3 administration group; 4-3 is VEGF-SEE-3 administration group) experimental result of the fusion rotein that just formed by SEE-3, the T cell that utilizes ImmunohistochemistryMethods Methods to detect, brown point is T cell.

Through testing: also all found the existence of T cell in the mouse tumor tissue of the administration group of TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2, EGF-SEE, EGF-SEE-1, EGF-SEE-2, VEGF-SEE, VEGF-SEE-1 and VEGF-SEE-2 fusion rotein.

The detection of embodiment 14 tumor tissues internal interference element-γ (IFN-γ)

Adopt immunohistochemical method similar to Example 13 to detect the interior cytokine IFN-γ by T emiocytosis of tumor tissues in embodiment 10-12, antibody is the anti-IFN-gamma antibodies of Santa Cruz Biotechnolog company.Fig. 5 is (in figure: 5-1 is TGF-α-SEE-3 administration group; 5-2 is EGF-SEE-3 administration group; 5-3 is VEGF-SEE-3 administration group) interferon-γ by T emiocytosis (IFN-γ) that utilizes immunohistochemical methods to detect, brown part is IFN-γ.

Through testing: also all found the existence by the IFN-γ of induced t cell in the mouse tumor tissue of the administration group of TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2, EGF-SEE, EGF-SEE-1, EGF-SEE-2, VEGF-SEE, VEGF-SEE-1 and VEGF-SEE-2 fusion rotein.

Embodiment 15 calf serums and fusion rotein hatch experiment

The calf serum dilution that GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and the GRP-SEE-3 fusion rotein of the high density using in cancer cells culture experiment all cultivated with cancer cells, then at 37 ℃, hatch 5 hours, these fusion roteins that use in the experiment of later all cancer cells all pass through the processing of hatching of calf serum.Hatching the existence that experiment is the basic antibody (IgM and IgG etc.) in order to check the anti-superantigen in opposing proteasome degradation in calf serum and thermally-stabilised etc. and serum here, whether the biological activity of fusion rotein changes.

The inducer T lymphocyte activity of embodiment 16GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein

Human peripheral blood cell is purchased from Tianjin Blood Center, obtains T lymphocyte (J Clin Invest, 91 with Ficoll density centrifugation and nylon hair column method, 1490-1498,1993), these T cells are used DMEM culture medium culturing on 6 orifice plates, and add 10% calf serum.There is 5x10 in each hole ⁵individual T cell, then add GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and the GRP-SEE-3 fusion rotein of 5pmol amount, these albumen all pass through hatching of calf serum, then use the IFN-γ test kit (R & D Systems company) of enzyme-linked immunosorbent assay (ELISA) to detect the content of the IFN-γ in substratum.

Table 1 stimulates the ability of T cell for analyzing GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2 and GnRH-SEE-3 fusion rotein, detection be cytokine IFN-γ.

That table 1 detects is cytokine IFN-γ (pg/ml)

Result shows that GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2 and GnRH-SEE-3 fusion rotein have the vigor that stimulates T cell, and wherein, GnRH-SEE-2 and GnRH-SEE-3 fusion rotein have shown the vigor of stronger stimulation T cell.

Table 2 stimulates the ability of T cell for analyzing GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein, detection be cytokine IFN-γ.

That table 2 detects is cytokine IFN-γ (pg/ml)

Result shows that GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein have the vigor that stimulates T cell, and wherein, GRP-SEE-2 and GRP-SEE-3 fusion rotein have shown the vigor of stronger stimulation T cell.

Embodiment 17GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein kill and wound cancer cells

On 6 orifice plates, with liver cancer cell Hep G2 and the lung cell A549 in DMEM culture medium culturing human cancer cell source, cancer cells number is 5x10 ⁵individual cancer cells/hole, then adds T cell, is that 10 times of cancer cells are 5x10 ⁶individual T cells/well, add respectively again GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and the GRP-SEE-3 fusion rotein of hatching through calf serum of various dosage, wherein, GnRH super antigen fusion protein joins liver cancer cell Hep G2 culture hole, and GRP super antigen fusion protein joins lung cell A549 culture hole.

Cytotoxicity or cancer cells fragmentation effect adopt MTT (Methabenzthiazuron) method to measure (Immunology, 82,117-125,1994), cell growth inhibition 100-[(A _test-A _b)/(A _c-A _b)] calculating of x100 formula, A _testrefer to the growth of cancer cells of T cell under adding, A _brefer to and in hole, only have substratum, A _crefer to growth of cancer cells.The number of times of every kind of cancer cells killing experiments is all more than 20 later.

Fig. 6 is experimental result, shows that GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3 have the ability of killing hepatoma cell Hep G2, and wherein GnRH-SEE-2 and GnRH-SEE-3 just have higher cancer cells lethal effect under very low dosage.

Fig. 7 is experimental result, shows that GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 have the ability of killing and wounding lung cell A549, and wherein GRP-SEE-2 and GRP-SEE-3 just have higher cancer cells lethal effect under very low dosage.

Fig. 8 is (in figure: 8-1 is GnRH-SEE-3 group; 8-2 is GRP-SEE-3 group) show the cancer cells of being attacked by T cell, maxicell is cancer cells, minicell is T cell.

Through testing: the phenomenon of also having observed the cancer cells of T cell attack in GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GRP-SEE, GRP-SEE-1 and GRP-SEE-2 group.

The present invention has selected a kind of superantigen SEE and mutant thereof, makes SEE mutant have more powerful induced t cell ability than SEE, more can resist the antibody neutralization in blood.TGF-α-the SEE being formed by SEE, EGF-SEE, VEGF-SEE, GnRH-SEE and GRP-SEE, TGF-α-the SEE-2 being formed by SEE-2, EGF-SEE-2, VEGF-SEE-2, GnRH-SEE-2 and GRP-SEE-2 and the TGF-α-SEE-3 being formed by SEE-3, EGF-SEE-3, VEGF-SEE-3, GnRH-SEE-3 and GRP-SEE-3 fusion rotein have antitumous effect, wherein, TGF-α-SEE-2, EGF-SEE-2, VEGF-SEE-2, GnRH-SEE-2, GRP-SEE-2TGF-α-SEE-3, EGF-SEE-3, VEGF-SEE-3, GnRH-SEE-3 and GRP-SEE-3 fusion rotein have shown stronger cancer cells and the antineoplastic biological activity of killing and wounding.

Mutant SEE-1, the SEE-2 of the superantigen SEE here and SEE-3 are selected from SEE and have introduced extensive multiple point mutation, because SEA and SEE have more than 80% homogeny on aminoacid sequence, so can also can obtain from SEA the mutant of SEE through point mutation.By the molecular biology working method of range gene point mutation, on existing SEE mutant, introduce point mutation and obtain the superantigen that biological activity is higher in addition.

Determined point mutation position from embodiment 1, the amino acid that can introduce other similar physico-chemical property on these positions also can obtain the biological activity identical or higher with SEE mutant.

From larger scope, SEB, SEC, SED, SEF, SEG, SEH, SEI, the SEJ etc. of such as Staphylococcus aureus enterotoxin family of other superantigen, SPE-A, the SPE-B of streptococcus toxin, SPE-C, shock syndrome toxin (Shock syndrome toxin), the superantigen in viral protein source can be strengthened by multipoint mutation means the neutralizing effect of T cytositimulation ability, the stability of superantigen and the superantigen of opposing antibodies in blood of superantigen.

Can select and the interactional peptide molecule of cancer cells surface receptor as another part of SEE and mutant fusion protein thereof, such polypeptide has Prostatropin (Basic fibroblast growth factor, bFGF) and FGF family, for example interleukin II of interleukin-family (Interleukin), interleukin 3, interleukin 4, interleukin-6, interleukin 8, interleukin 11, Interleukin-13, rHuGM-CSF (Granulocyte-macrophage colony-stimulating factor, GM-CSF), Heparin-binding EGF like growth factor (Heparin-binding EGF-like growth factor, HB-EGF), rhIGF-1 (Insulin-like growth factor, IGF), pHGF (Hepatocyte growth factor, HGF), Thr6 PDGF BB (Platelet-derived growth factor, PDGF), nerve growth factor (Nerve growth factor, NGF), placenta growth factor (Placental growth factor, PGF), STEM CELL FACTOR (Stem cell factor, SCF), Heregulin, erbB part, various chemokines (Chemokine) and family thereof, Ephrin family, angiogenin (Angiopoietin), thrombopoietin (Thrombopoietin) and the blood plasma VII factor (Factor VII), plasma urokinase-type plasminogen activator (Urokinase-type plasminogen activator), growth hormone releasing hormone (Growth hormone releasing hormone), melanotropin α-MSH, prolactin (Prolactin), prolactin releasing hormone (PRH) (Prolactin releasing hormone), tethelin (Growth hormone), follicle stimulating hormone (Follicle stimulating hormone), placental lactogen hormone (Placental lactogen), chorionic-gonadotropin hormone (Chorionic gonadotropin) and corticotropin releasing hormone (Corticotropin releasing hormone), Somatostatin (Somatostatin), Asialoglycoprotein (Asialoglycoprotein), low-density lipoprotein (Low density lipoprotein) and Transferrins,iron complexes (Transferrin) etc., and the aminoacid sequence of these albumen has nature and the artificial varient etc. of more than 70% homogeny.Many tumor tissues are these material acceptors of overexpression all, thereby the peptide molecule parts such as chemokine, enzyme, hormone and other albumen just can be connected to form fused protein with costimulatory molecules as cytokine, and costimulatory molecules is navigated in tumor tissues.

Fused protein can also be connected the polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor etc. respectively by means such as chemical crosslink reactions with the protein fragments of superantigen above, and for example covalent linkage connects to build.Can carry out a part of polypeptide fragment of chemically modified, damaged fused protein and other polypeptide is connected to the first-class a series of transformation of these protein for fused protein.Fused protein after purifying can improve its space structure including disulfide linkage by the denature and renature process of a series of protein, improves its biological activity.

From larger scope, on the polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor adopting in experiment here cancer cells surface corresponding thereto, the acceptor of overexpression is actually interactional relation between a kind of part and acceptor, the avidity of utilizing this part and acceptor, navigates to tumor tissues by superantigen.Except cytokine, hormone or polypeptide, other every protein or peptide molecule that can be corresponding with the acceptor of cancer cells overexpression be the specific localization that part also can be used for cancer cells.Except the part that acceptor said and on cancer cells is corresponding above, screen from methods such as phage displays (Phage display) with cancer cells acceptor have avidity and have the protein of artificial screening of antagonistic action or polypeptide and screen by other method can be directly and the protein of cancer cells surface interaction or peptide molecule can form fused protein with costimulatory molecules.

As its formulation of medicine can be emulsifying agent, liposome, dispersion agent, stablizer etc. make together various injections, oral, apply ointment or plaster and the form of medication of the medicine such as surgical procedure.Except fused protein itself can be used as medicine, the nucleotide fragments of encoding fusion protein matter or carrier can also be applied as gene therapy form.

In specific embodiments of the invention, TGF-α-SEE, EGF-SEE and VEGF-SEE that described cell factor-super-antigen fusion protein is made up of SEE, TGF-α-SEE-1, the EGF-SEE-1 and the VEGF-SEE-1 that are formed by the mutant SEE-1 of SEE, TGF-α-SEE-2, the EGF-SEE-2 and the VEGF-SEE-2 that are formed by the mutant SEE-2 of SEE, and TGF-α-SEE-3, the EGF-SEE-3 and the VEGF-SEE-3 that are formed by the mutant SEE-3 of SEE; The GnRH-SEE that described hormone-super antigen fusion protein is made up of SEE, the GnRH-SEE-1 being formed by the mutant SEE-1 of SEE, the GnRH-SEE-2 being formed by the mutant SEE-2 of SEE, and the GnRH-SEE-3 being formed by the mutant SEE-3 of SEE; The GRP-SEE that the polypeptide-super antigen fusion protein of the non-hormone of the described non-antibody acellular factor is made up of SEE, the GRP-SEE-1 being formed by the mutant SEE-1 of SEE, the GRP-SEE-2 being formed by the mutant SEE-2 of SEE, and the GRP-SEE-3 being formed by the mutant SEE-3 of SEE.These fusion roteins can induce and stimulate T lymphocyte to come anti-sarcoma S180, Hep G2 liver cancer and A549 lung cancer respectively, but, as long as the upper acceptor of expressing TGF-α, EGF, VEGF, GnRH and GRP of the cancer cells (cell, bone, brain, intestines and skin etc. in various internal organs, blood) of other kind, can adopt the fusion rotein here to carry out killing tumor cells, thereby be applied to the treatment of various cancers or malignant tumour.

The description of above embodiment and preferred implementation belongs to of the present invention the schematically illustrating that claim is limited, rather than restriction the present invention.It needs to be noted, only otherwise depart from aim of the present invention, all apparent changes and have the similar invention of equivalent substitution, within being all included in protection scope of the present invention.

Claims

1. a cancer-targeted super antigen fusion rotein, is characterized in that this fusion rotein contains:

Described cytokine be selected from be abbreviated as TGF-α transforminggrowthfactor-α, be abbreviated as EGF Urogastron, be abbreviated as the vascular endothelial growth factor of VEGF; Described hormone is selected from the gonadotropin releasing hormone of being abbreviated as GnRH; The polypeptide of the non-hormone of the described non-antibody acellular factor is selected from the gastrin releasing peptide of being abbreviated as GRP;

B) can cause superantigen staphylococcus aureus enterotoxin E and the mutant thereof that antitumor immune reacts, described staphylococcus aureus enterotoxin E represents with abbreviation SEE; The mutant of described staphylococcus aureus enterotoxin E represents with abbreviation SEE-X, described X=1-3;

Described SEE-1 is the aminoacid sequence shown in SEQ ID No.4;

Described SEE-2 is the aminoacid sequence shown in SEQ ID No.6;

Described SEE-3 is the aminoacid sequence shown in SEQ ID No.8.

2. a kind of cancer-targeted super antigen fusion rotein according to claim 1, is characterized in that described this fusion rotein is:

By the TGF-α-SEE-1 shown in SEQ ID No.12;

By the EGF-SEE-1 shown in SEQ ID No.20;

By the VEGF-SEE-1 shown in SEQ ID No.28;

By the GnRH-SEE-1 shown in SEQ ID No.36;

By the GRP-SEE-1 shown in SEQ ID No.44;

By the TGF-α-SEE-2 shown in SEQ ID No.14;

By the EGF-SEE-2 shown in SEQ ID No.22;

By the VEGF-SEE-2 shown in SEQ ID No.30;

By the GnRH-SEE-2 shown in SEQ ID No.38;

By the GRP-SEE-2 shown in SEQ ID No.46;

By the TGF-α-SEE-3 shown in SEQ ID No.16;

By the EGF-SEE-3 shown in SEQ ID No.24;

By the VEGF-SEE-3 shown in SEQ ID No.32;

By the GnRH-SEE-3 shown in SEQ ID No.40;

Or by the GRP-SEE-3 shown in SEQ ID No.48.

3. recombinant vectors, is characterized in that containing the nucleotide sequence of fusion rotein described in coding claim 2, and described nucleotides sequence is classified the nucleotide sequence SEQ ID No.9 of coding SEQ ID No.10 as;

The nucleotide sequence SEQ ID No.11 of coding SEQ ID No.12;

The nucleotide sequence SEQ ID No.13 of coding SEQ ID No.14;

The nucleotide sequence SEQ ID No.15 of coding SEQ ID No.16;

The nucleotide sequence SEQ ID No.17 of coding SEQ ID No.18;

The nucleotide sequence SEQ ID No.19 of coding SEQ ID No.20;

The nucleotide sequence SEQ ID No.21 of coding SEQ ID No.22;

The nucleotide sequence SEQ ID No.23 of coding SEQ ID No.24;

The nucleotide sequence SEQ ID No.25 of coding SEQ ID No.26;

The nucleotide sequence SEQ ID No.27 of coding SEQ ID No.28;

The nucleotide sequence SEQ ID No.29 of coding SEQ ID No.30;

The nucleotide sequence SEQ ID No.31 of coding SEQ ID No.32;

The nucleotide sequence SEQ ID No.33 of coding SEQ ID No.34;

The nucleotide sequence SEQ ID No.35 of coding SEQ ID No.36;

The nucleotide sequence SEQ ID No.37 of coding SEQ ID No.38;

The nucleotide sequence SEQ ID No.39 of coding SEQ ID No.40;

The nucleotide sequence SEQ ID No.41 of coding SEQ ID No.42;

The nucleotide sequence SEQ ID No.43 of coding SEQ ID No.44;

The nucleotide sequence SEQ ID No.45 of coding SEQ ID No.46;

Or shown in the nucleotide sequence SEQ ID No.47 of coding SEQ ID No.48.

4. a kind of preparation method of cancer-targeted super antigen fusion rotein described in claim 1, is characterized in that cultivating the host cell that contains recombinant vectors described in claim 3, collects and expresses fusion rotein claimed in claim 1.

5. fusion rotein claimed in claim 1 is in the application of the medicine of preparation treatment cancer and anti entity tumour.