CN112592395A

CN112592395A - Construction, preparation and application of plasmodium falciparum RIFIN recombinant protein PfRIFIN-54

Info

Publication number: CN112592395A
Application number: CN202011460834.2A
Authority: CN
Inventors: 陈军虎; 卡森; 崔延冰; 陈绅波; 沈海默; 徐斌; 周晓农
Original assignee: National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention
Current assignee: National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-04-02

Abstract

The invention relates to a plasmodium falciparum RIFIN recombinant protein coded by a plasmodium falciparum RIFIN gene PF3D7_0833100, which is named as PfRIFIN-54, the gene sequence of the recombinant protein is shown as SEQ ID No.1, and the amino acid sequence of the recombinant protein is shown as SEQ ID No. 4. A recombinant plasmid containing the gene PF3D7_ 0833100. A protein chip containing protein PfriFIN-54. The invention also discloses a preparation method of the plasmodium falciparum RIFIN recombinant protein. Compared with a control PfMSP1-42, the protein of the invention not only has better ability of specifically recognizing the serum of patients infected with plasmodium falciparum, but also shows obvious difference in the serum immunoreaction of severe patients and non-severe patients infected with plasmodium falciparum, and the positive rate of the immunoreaction of the serum of severe patients is obviously lower than that of the serum immunoreaction of non-severe patients.

Description

Construction, preparation and application of plasmodium falciparum RIFIN recombinant protein PfRIFIN-54

Technical Field

The invention belongs to the field of biological gene engineering, relates to a plasmodium falciparum RIFIN protein, and particularly relates to construction, preparation and application of a plasmodium falciparum RIFIN recombinant protein coded by a plasmodium falciparum RIFIN gene PF3D7_ 0833100.

Background

Genetic engineering (also known as gene splicing technology and DNA recombination technology) is based on molecular genetics as a theoretical basis, and by means of modern methods in molecular biology and microbiology, genes from different sources are recombined into a new gene expression host, so that the protein product with biological activity can be produced in large quantities. The gene engineering technology provides a powerful means for the research of the structure and the function of the gene, and is one of the most widely applied technical means in the field of molecular biology at present.

Plasmodium falciparum (Plasmodium falciparum) has become the most dangerous Plasmodium because of its high mortality rate and resistance to widespread antimalarial drugs, and is predominant in the world in continental africa where malaria is the most severe. The main causes of severe malaria morbidity and mortality from plasmodium falciparum infection are severe anemia due to plasmodium exponential growth causing infected erythrocytosis, blockage of the microvascular vasculature due to adhesion of mature plasmodium falciparum infected erythrocytes to microvascular endothelial cells, and obstruction of blood flow due to rosettes formed by infected and uninfected erythrocytes. Among its pathogenic mechanisms, the RIFIN proteins (p. falciparum-encoded recombinant antibodies of polypeptides) of the family of variant surface antigens play an important role in mediating adhesion of infected erythrocytes and rosette formation in the microvascular vasculature, thereby blocking blood flow and blocking blood vessels leading to severe malaria. The expression of RIFIN protein on the surface of infected erythrocytes is one of important immune targets, but the RIFIN protein can escape from the attack of the immune system due to the polygenic and genetic polymorphism of the repeatedly scattered gene family rif (recurrent intermittent family). Meanwhile, the combination of RIFIN protein and immune cell surface inhibitory receptor leukocyte immunoglobulin-like receptor B1(leukocyte immunoglobulin receptor B1, LILRB1), leukocyte-associated immunoglobulin-like receptor 1(leukocyte-associated immunoglobulin-like receptor 1, LAIR1) and the like can down-regulate immune response to achieve the purpose of immune evasion.

In recent years, with the progress of the research on the molecular mechanism of plasmodium falciparum, the development of vaccines capable of inducing effective protective immunity has become a key to the prevention and implementation of malaria elimination programs. The RIFIN proteins of the variant surface antigen family may also be important biological detection and malaria vaccine candidates.

Disclosure of Invention

The invention aims to provide a construction, preparation and function verification method of plasmodium falciparum RIFIN recombinant protein coded by a plasmodium falciparum RIFIN gene PF3D7_0833100, and the construction, preparation and function verification method of the plasmodium falciparum RIFIN recombinant protein coded by the plasmodium falciparum RIFIN gene PF3D7_0833100 aim to solve the technical problem of poor effect on treating plasmodium falciparum infection in the prior art.

The invention provides a plasmodium falciparum RIFIN recombinant protein coded by a plasmodium falciparum RIFIN gene PF3D7_0833100, which is named as PfRIFIN-54, the gene sequence of the recombinant protein is shown as SEQ ID No.1, and the amino acid sequence of the recombinant protein is shown as SEQ ID No. 4.

The invention also provides a recombinant plasmid containing the plasmodium falciparum RIFIN gene PF3D7_ 0833100.

The invention also provides a protein chip containing the plasmodium falciparum RIFIN protein, and the amino acid sequence of the protein chip is shown as SEQ ID No. 4.

The invention also provides a specific primer for PCR amplification of the plasmodium falciparum RIFIN protein gene, wherein the upstream sequence of the specific primer is shown as SEQ ID NO: 2 is shown in the specification; the downstream sequence is shown as SEQ ID NO: 3, respectively.

The invention also provides a preparation method of the plasmodium falciparum RIFIN recombinant protein coded by the plasmodium falciparum RIFIN gene PF3D7_0833100, which comprises the following steps:

1) the specific primer used for the plasmodium falciparum RIFIN protein gene is used for PCR amplification of the plasmodium falciparum RIFIN gene segment, and the sequence of the obtained plasmodium falciparum RIFIN gene segment is shown as SEQ ID No. 1;

2) connecting the gene fragment obtained in the step (1) with a vector pEU-E01-His-N2 to construct and obtain a recombinant plasmid pEU-E01-His-N2-PfRIFIN-54, respectively transferring the recombinant plasmid into competent escherichia coli E.coli DH5 alpha, and verifying and confirming the obtained recombinant strain through PCR and agarose gel electrophoresis;

3) inoculating the recombinant strain obtained in the step (2) and taking pEU-E01-His-N2 as a vector into an LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12 h;

4) extracting plasmids from the bacterial liquid obtained in the step (3) by using a kit to obtain a large amount of recombinant plasmids pEU-E01-His-N2-PfRIFIN-54;

5) and (4) expressing the protein obtained in the step (4) in a wheat germ cell-free protein expression system to obtain plasmodium falciparum RIFIN protein PfRIFIN-54, and detecting the expressed protein by Western Blot (WB).

Further, in step 1), designing SEQ ID NO: 2 and SEQ ID NO: 3 or the complementary strand thereof is used as a primer, total DNA of plasmodium falciparum is extracted, and PCR amplification is carried out by taking the total DNA as a template; the PCR amplification reaction condition is pre-denaturation at 98 ℃ for 2.0 min; denaturation at 98 ℃ for 20sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1.0min, 35 cycles; extension at 72 ℃ for 10min and final storage at 4 ℃.

Further, in the step 2), the plasmid vector pEU-E01-His-N2 is subjected to double enzyme digestion by Xho I and BamH I, and is recovered and purified; performing In-Fusion connection according to the concentration of the PCR product obtained In the step 1) and the enzyme digestion fragment of the plasmid vector to construct a recombinant plasmid pEU-E01-His-N2-PfRIFIN-54 for prokaryotic expression of the RIFIN encoding gene of the plasmodium falciparum; transforming the recombinant plasmid into escherichia coli DH5 alpha competent cells by a heat shock method, coating the cells on an LB agar plate containing ampicillin after culturing, randomly selecting bacterial colonies on the plate, and carrying out PCR identification and sequencing identification on a bacterial liquid after shaking culture;

further, in step 3): inoculating the Escherichia coli DH5a recombinant strain containing pEU-E01-His-N2-PfRIFIN-54 sequenced in the step 2) into an LB liquid culture medium, culturing at 37 ℃ and 200rpm for 12h, collecting the strain, extracting the recombinant plasmid pEU-E01-His-N2-PfRIFIN-54 by using a kit, and placing the obtained DNA at-30 ℃ or-80 ℃ for later use;

further, in step 4): expressing the recombinant plasmid pEU-E01-His-N2-PfRIFIN-54 obtained in the step 3) and a malt germ cell-free expression system to obtain a plasmodium falciparum RIFIN protein PfRIFIN-54, and identifying a protein expression result by using Western-blot.

The invention also provides application of the plasmodium falciparum RIFIN recombinant protein coded by the plasmodium falciparum RIFIN gene PF3D7_0833100 in preparing a kit for detecting severe or non-severe plasmodium falciparum infected patients.

The plasmodium falciparum RIFIN protein is named as PfRIFIN-54, is derived from plasmodium falciparum (P.falciparum 3D7), has the nucleotide sequence length of 1044bp, and is shown as SEQ ID No. 1; the amino acid sequence has a length of 347aa and is shown as SEQ ID No. 4.

The invention obtains a gene sequence of PfRIFIN-54 through a plasmodium falciparum genome sequence, obtains a PfRIFIN-54 gene fragment by utilizing specific primer PCR, thereby constructing a recombinant expression plasmid pEU-E01-His-N2-PfRIFIN-54, and performs serum immune reaction through a protein chip to perform functional verification on the PfRIFIN-54.

Compared with the prior art, the invention has the advantages of positive and obvious technical effect. Compared with the control PfMSP1-42, the protein of the invention not only has better ability of specifically recognizing the serum of the patient infected with plasmodium falciparum, but also shows obvious difference on the serum immunoreaction of severe and non-severe patients infected with plasmodium falciparum, and the positive rate of the immunoreaction of the serum of the severe patients is obviously lower than that of the serum immunoreaction of the non-severe patients.

Drawings

FIG. 1: schematic diagram of PCR amplification result of PfriFIN-54 gene fragment, wherein: m is DL,2000DNA Leader (Takara); lane 1 shows the PfRIFIN-54 gene fragment.

FIG. 2: schematic representation of colony PCR results for recombinant strain e.coli DH5 α/pEU-E01-His-N2-PfRIFIN-54, wherein: m is DNA marker (DL,2000DNA Leader, Takara); lanes 1-4 are E.coli DH5 α/pEU-E01-His-N2-PfRIFIN-54 recombinant strains.

FIG. 3: PF3D 7-0833100 cell-free expression of western-blot results are shown schematically, in which: m is Protein marker (Page Ruler Protein Ladder 26616, Thermo).

FIG. 4: schematic diagram of antibody serum immunoreaction analysis results, wherein: SMG represents the serum sample antibody response of severe patients with falciparum malaria, and NMG represents the serum sample antibody response of non-severe patients with falciparum malaria.

Detailed Description

The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

EXAMPLE 1 preparation of Plasmodium falciparum RIFIN protein PfRIFIN-54

1 Material

1.1 Plasmodium falciparum genomic DNA

Is derived from Plasmodium falciparum PF3D 7.

1.2 host strains Escherichia coli DH5 alpha, plasmid pEU-E01-His-N2 from Tiangen Biochemical technology limited and Nippon, CellFree Sciences company, respectively.

1.3 Primary reagent and tool enzymes

2 method

2.1 amplification and cloning of Plasmodium falciparum RIFIN protein PfRIFIN-54 Gene

2.1.1 PCR amplification of Plasmodium falciparum RIFIN protein PfRIFIN-54 Gene

According to the Sequence (shown in SEQ ID NO. 1) of plasmodium falciparum RIFIN protein PfRIFIN-54 gene (PF3D7_0833100GeneID:9221922, NCBI Reference Sequence: XM _002808685.1), a pair of specific primers are designed by using primer design software of Shanghai Yingjun Biotechnology limited company, wherein the lower case letters in the specific primers are respectively 15 bases which are homologous with the tail end of a digestion vector, and the single underline indicates that the digestion sites are respectively Xho I and BamH I digestion sites. The specific primer is synthesized by Shenzhen Hua Dagen Co. The primer sequences are as follows:

upstream: 5' -gggcggatatctcgagCAAATTCCATCCATCACACC-3' (shown in SEQ ID NO: 2);

downstream: 5' -gcggtacccgggatccttaTTCTTCTAATAGTTTTATATATTG-3' (shown in SEQ ID NO: 3)

PCR amplification was performed using Plasmodium falciparum genomic DNA as a template. The reaction condition is pre-denaturation at 98 ℃ for 2.0 min; denaturation at 98 ℃ for 20 sec; annealing at 55 deg.C for 30sec, extending at 72 deg.C for 1.0min, and performing 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃. Phusion high fidelity DNA polymerase was purchased from Finnzymes under Thermo Scientific flag. The reaction system is 20.0 mul, in particular to 4.0 mul of 5x Phusion HF Buffer (Phusion high fidelity Buffer); 0.4. mu.l of 10mM dNTPs; p F (upstream primer) (10. mu.M each) 1. mu.l; p R (downstream primer) (10. mu.M each) 1. mu.l; template DNA (DNA Template) 1. mu.l; phusion DNA Polymerase (Phusion DNA Polymerase) 0.2. mu.l; nuclean-free water (Nuclease-free water) 12.4. mu.l. (the PCR system reagents are all from Phusion Hi-Fi DNA polymerase kit from Finnzymes under Thermo Scientific flag)

2.1.2 recombinant plasmid expressed by Plasmodium falciparum RIFIN protein PfRIFIN-54

Construction and identification of pEU-E01-His-N2-PfRIFIN-54

The plasmid pEU-E01-His-N2 is cut by endonuclease Xho I and BamH I respectively, and the PCR product and the concentration of the cut plasmid pEU-E01-His-N2 are connected by an In-Fusion cloning method to construct a recombinant plasmid pEU-E01-His-N2-PfriFIN-54 of the plasmodium falciparum RIFIN protein PfriFIN-54 coding gene without cell expression. The method comprises the following specific steps:

1) mu.l of PCR reaction product was taken and 2. mu.l of cloning Enhancer (cloning promoter) purchased from Clontech was added;

2) placing the mixture in the previous step into a PCR instrument, incubating for 15min at 37 ℃ and incubating for 15min at 80 ℃;

3) the PCR product was ligated with the digested pEU-E01-His-N2 empty plasmid using the In-Fusion cloning method. The reaction condition is incubation for 15min at 37 ℃; incubating at 50 deg.C for 15 min; taking out and putting on ice for conversion and use. The In-Fusion enzyme was purchased from Clontech. The Reaction system (reagents used were all from an In-Fusion enzyme kit, available from Clontech) was 10. mu.l, specifically 5 XIn-Fusion Reaction Buffer (In-Fusion Reaction Buffer) 2. mu.l; 1. mu.l of In-Fusion Enzyme; vector 2. mu.l (. about.100 ng); insert 2. mu.l; dH₂O (distilled water) (if necessary) 3. mu.l; the joined recombinant plasmid

pEU-E01-His-N2-PfRIFIN-54 was transformed into E.coli DH5 alpha competent cells by heat shock method, cultured and plated on ampicillin-containing (Amp)⁺100 ug/ml) on an LB agar plate, randomly picking colonies on the plate, culturing the colonies by shaking, and performing colony PCR identification and sequencing identification. The bacterial sample with the correct colony PCR identification is sent to Huada Gene company for sequencing analysis.

The recombinant strain E.coli DH5 alpha/pEU-E01-His-N2-PfRIFIN-54 which is correctly sequenced is preserved for later use.

2.2 expression and validation of recombinant plasmid of Plasmodium falciparum RIFIN protein PfRIFIN-54 in wheat germ cell-free (WGCF) protein expression System

2.2.1 preparation of recombinant plasmid pEU-E01-His-N2-PfRIFIN-54

The recombinant Plasmid pEU-E01-His-N2-PfRIFIN-54 is extracted by using a QIAGEN Plasmid middle extraction Kit (25), and the specific steps are as follows:

1) the recombinant bacterium containing the plasmid pEU-E01-His-N2-PfRIFIN-54 was inoculated into 50ml LB liquid medium (Amp)⁺100. mu.g/ml) at 37 ℃ and 200rpm for 12 hours;

2) and (3) collecting thalli: centrifuging at 4 deg.C and 6000Xg for 15min, and removing supernatant;

3) resuspend the cells with 4ml Buffer P1;

4) adding 4ml Buffer P2, inverting for 4-6 times to mix the solution completely, standing at room temperature for 5min (changing Lyseblue reagent solution into blue);

5) adding 4ml of precooled Buffer P3, reversing the upper part and the lower part for 4 to 6 times to completely mix, and standing on ice for 15min (the blue color disappears);

6) centrifuging at 4 deg.C for 30min to obtain supernatant with impurities, and centrifuging for 15 min;

7) the QIAGEN-tip column was equilibrated with 4ml Buffer QBT to allow the liquid to drain naturally by gravity;

8) pouring the supernatant obtained in the step 5 into a balanced column, and draining the supernatant by natural flow (impurities are not poured);

9) the column was washed with 2 × 10ml Buffer QC and drained (2 washes, 10ml each);

10) eluting the DNA on the column into a clean 15ml centrifuge tube by using 5ml of Buffer QF, and if the DNA is more than 45kb, heating the elution Buffer to 65 ℃ in advance to improve the elution efficiency;

11) DNA precipitation: adding 3.5ml isopropanol into the eluted DNA solution, mixing well, centrifuging at no less than 15000Xg at 4 deg.C for 30min, and carefully removing the supernatant;

12) washing the DNA precipitate with 2ml 70% ethanol, centrifuging at no less than 15000Xg at 4 deg.C for 15min, and carefully removing the supernatant;

13) air-dried (left for 5-10min), and then resuspended with 200. mu.l of nucleic-Free Water, the obtained DNA was assayed for concentration using NanODROP 2000(Thermo), and the remaining DNA was left at-30 ℃ or-80 ℃ for further use.

2.2.2 expression of recombinant plasmid of Plasmodium falciparum RIFIN protein PfRIFIN-54 in Wheat Germ Cell Free (WGCF) expression System

The method comprises the following specific steps:

1) 10 mul of transcription system, adding pEU-E01-His-N2-PfRIFIN-54 plasmid with the final concentration of 100 ng/mul, 0.125 mul of RNase inhibitor, 0.125 mul of SP6 RNase, 25mmol NTP mix 1 mul, 5x transcription Buffer 2 mul, adding Nuclear-Free Water to 10 mul, and standing at 37 ℃ for 6 h;

2) expression (translation) system: adding 10 μ l of 1) transcription system into 10 μ l WEPRO and 1.6 μ l CK enzyme (diluted), mixing, adding into 206 μ l SubMix, and standing at 15 deg.C for 20 h;

3) after the expression is finished, mixing evenly, identifying by Western-blot, and keeping the residual protein at-80 ℃ for later use.

2.2.3 validation of Plasmodium falciparum RIFIN protein PfRIFIN-54

The method comprises the following specific steps:

western-blot identification was performed on the Plasmodium falciparum RIFIN protein PF3D 7-0833100 expressed in a malt germ cell-free (WGCF) expression system. The method comprises the following specific steps:

1) after SDS-PAGE electrophoresis, the plasmodium falciparum RIFIN protein PfRIFIN-54 is electrically transferred to a PVDF membrane (Millipore);

2) cutting the membrane into strips, placing in 5% skimmed milk powder solution (3% BSA) for blocking for 1h (dissolving with TBST), rinsing PVDF membrane with TBST for 5min each time for 3 times;

3) diluting primary antibody (Penta, His, Ab) and TBST according to a certain ratio (1:2000), placing on a shaking table for reacting for 1h, and rinsing the PVDF membrane with TBST for 5min each time for 3 times;

4) diluting the secondary antibody (Goat-a-Mouse IgG-HRP) and TBST according to a certain ratio (1:2000), placing on a shaking table for reacting for 1h, and rinsing the PVDF membrane with TBST for 5min each time for 3 times;

5) the color developing solution (DAB: PBS: 30% H) is prepared according to the proportion₂O₂2mg:3ml:0.9 mul), placing the rinsed PVDF membrane into a tray containing a color development solution for color development, immediately rinsing with PBS after a target strip appearsThe reaction was stopped and blotted dry with filter paper.

3 results

3.1 amplification and cloning of Plasmodium falciparum RIFIN protein PfRIFIN-54 Gene

3.1.1 PCR amplification of Plasmodium falciparum RIFIN protein PfRIFIN-54 Gene

The plasmodium falciparum genomic DNA is used as a template to carry out PCR amplification, and a target fragment (shown in figure 1) with 1044bp consistent with the expected length is amplified, which indicates that the PfRIFIN-54 gene is successfully amplified.

3.1.2 recombinant plasmid for Plasmodium falciparum RIFIN protein PfRIFIN-54 expression

Construction and identification of pEU-E01-His-N2-PfRIFIN-54

The target gene fragment is connected with the restriction enzyme pEU-E01-His-N2 no-load plasmid through an In-Fusion cloning method to construct a recombinant plasmid pEU-E01-His-N2-PfRIFIN-54, and the colony PCR result (shown In figure 2) and the sequencing result are consistent with the size of the target gene fragment through colony PCR and sequencing identification, which indicates that the recombinant plasmid is successfully constructed.

3.2 expression and validation of recombinant plasmid of Plasmodium falciparum RIFIN protein PfRIFIN-54 in wheat germ cell-free (WGCF) protein expression System

Western blot analysis results of the Plasmodium falciparum RIFIN protein PfRIFIN-54 expressed in a wheat germ cell-free (WGCF) protein expression system show that the Plasmodium falciparum RIFIN protein PfRIFIN-54 can be specifically recognized by an anti-His antibody, and a single and obvious band appears on a PVDF membrane (see figure 3).

Example 2 preparation of Plasmodium falciparum RIFIN protein PfRIFIN-54 protein chip and serum immunoreaction

1 Material

1.1 preparation of protein chip plectrum and rail, from CapitalBio of Beijing.

1.2 human serum samples of falciparum malaria patients and normal humans, collected from the malaria endemic area.

1.3 Alexa Fluor 546goat anti-human IgG from Invitrogen.

2 method

2.1 preparation of chip of protein PfRIFIN-54 of Plasmodium falciparum RIFIN

1) The crude protein PfRIFIN-54 was mixed with protein spot buffer (CapitalBio, beijing) at a ratio of 1:1 and 50 μ Ι _ of the protein mixture was transferred to a 384 well plate;

2) pf MSP1-42 as positive control, and wheat germ expression system without plasmid vector as negative control;

3) spotting was performed using a 48Smart array (CapitalBio, Beijing) protein chip spotter.

2.2 Plasmodium falciparum RIFIN protein PF3D7_0833100 protein chip serum immune response

Multiple sera from severe patients (SMG), non-severe patients (NMG), and normal human sera (NCG) infected with plasmodium falciparum were prepared as positive and negative serum controls, respectively. The method comprises the following specific steps:

1) blocking the protein chip obtained in 2.1 with 3% BSA (PBS) at 4 ℃ overnight, incubating at 37 ℃ for 1h, centrifuging at 1000-1500rpm at 4 ℃ for 3min, and spin-drying the blocking solution;

2) washing with PBS-T for 3 times, each time for 3min, centrifuging at 1000-1500rpm at 4 deg.C for 3min, and spin-drying;

3) incubating with primary antigen (falciparum malaria human serum and normal human serum (1:100 (adjustable) and PBS dilution)) at 37 deg.C for 1h, centrifuging at 1000-;

4) washing with PBS-T for 3 times, each time for 3min, centrifuging at 1000-1500rpm at 4 deg.C for 3min, and spin-drying;

5) diluting the secondary antibody (Alexa Fluor 546 gat anti-human IgG) with PBS (1:200), incubating at 37 ℃ for 1h, centrifuging at 1000 ℃ and 1500rpm at 4 ℃ for 3min, and spin-drying;

6) washing with PBS-T for 3 times, each time for 3min, centrifuging at 1000-1500rpm at 4 deg.C for 3min, and spin-drying;

7) the protein chips were scanned at 532nm using a LuxScan HT24 microarray scanner (CapitalBio, Beijing).

Statistical analysis of data

Protein chip fluorescence data were extracted using Microarray Image Analysis SpotData-Ver2.0(CapitalBio, Beijing) software, and experimental data were collated and statistically analyzed using Excel spreadsheet processing software and GraphPad Prism-Ver7.0.

4 results

The detection of IgG of a protein chip is respectively carried out on the serum of severe patients and non-severe patients infected by plasmodium falciparum and the normal human serum by using the plasmodium falciparum RIFIN protein PfRIFIN-54 (as shown in figure 4), the result shows that the protein shows obvious difference on the serum immunoreaction of severe patients and non-severe patients infected by plasmodium falciparum, as shown in figure 4, compared with the control PfMSP1-42, the protein not only has higher immunological recognition on the serum of patients infected by plasmodium falciparum, but also shows obvious difference on the serum immunoreaction of severe patients and non-severe patients infected by plasmodium falciparum, and the positive rate of the immunoreaction on the serum of severe patients is obviously lower than that of the serum immunoreaction of non-severe.

Discussion of 5

The invention takes plasmodium falciparum genome DNA as a template, adopts an In-Fusion cloning method to successfully clone and obtain recombinant plasmids constructed by plasmodium falciparum RIFIN protein PfRIFIN-54 and a cell-free expression vector pEU-E01-His-N2, obtains PfRIFIN-54 recombinant protein through a wheat germ cell-free (WGCF) protein expression system, and is proved by Western-blot identification to be specifically identified by an anti-His antibody, a clear band appears at 38.7KDa, and the molecular weight of the band is consistent with that of a target protein.

The gene engineering technology provides convenience for developing a preparation and detection method of recombinant antigen for detecting human serum antibody, and the protein chip technology provides a simple, convenient and effective method for researching the interaction between protein and protein, even the interaction between DNA-protein and RNA-protein, and screening protein targets of drug action. The invention discloses the obvious immunological difference of the protein in severe patients and non-severe patients, and the positive rate of the immune response of the serum of the severe patients is obviously lower than the positive rate of the immune response of the serum of the non-severe patients. In conclusion, the protein of the invention participates in the immune evasion process in severe plasmodium falciparum infection, so that the immune system of a human body cannot recognize the antigen and cannot generate antibodies, thereby leading the red blood cells infected with the plasmodium falciparum to generate immune evasion. Therefore, the invention can be used as the basis for researching the immune evasion mechanism of the plasmodium falciparum, provides the basis for further researching the pathogenic mechanism of the plasmodium falciparum, and has important application value in the aspects of elimination and control of the malaria.

Although the embodiments of the present invention have been described and illustrated, it should be understood that they have been presented by way of example only, and not limitation, and that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Chinese disease prevention and control center for prevention and control of parasitic diseases institute (national center for research on tropical diseases)

<120> construction, preparation and application of plasmodium falciparum RIFIN recombinant protein PfRIFIN-54

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1044

<212> DNA

<213> Plasmodium falciparum

<400> 1

atgcaaattc catccatcac accacaccat acaccaacta tcaggttatt atgcgaatgc 60

gaattatatg tgcctaattg tgacaacgat cctgaaatga aaagggtgat gcaacaattt 120

catgatcgta caacacaaag gtttcaagaa tacgatgaaa gattgcaaga aagacgacaa 180

atatgtaaag ataaatgtga taaggaaatc caaaaaatta ttttaaaaga taaattagaa 240

aaagaattaa tggacaaatt tgctacgtta gacacggata tacaaagtga cgccattcca 300

acatgtattt gcgaaaaatc gttagcagat aaagtggaaa gaggatgctt gagatgtggt 360

tatggtctag gaagtgttgc accaatgatt ggattaactg gttcagttgc tgtaaatgtg 420

tggaaaactg cagaacttgc agcggctatg gaattagcta aacaagcagg tgctgcggcc 480

ggtattaaag caggacattt ggcgggtact aaagttgtta ttgatcaatt acacacattg 540

ggtatatact ttgtaggtaa taaacaattg gaaacaatta ttgatgtaac aaattatatg 600

aatgtgtctg tcattaatga taatgtttat tctcattata tcacgtcatg tacacctagt 660

gttgttaatg gtcgccctgt cggtactttt aaatttagtg gtcctgtttg caatttggtt 720

cagccaaatc atcaaggtat atgggataga agtttagcac aagctattat aaaaaaaaag 780

gtagaagaag ctgttgcaga aggtacacaa gctgctgaaa ccgaagctgc tagggtgact 840

gccactaaaa cagcagcatt tgaagcaaaa aacatagccg aagtagaagc tgcaactact 900

tcctactata ctcctataat agcatccatc gttgcaatag taatcatagt tttaattatg 960

gtgataatat ataagatttt acgttatcga cgaaaaaaaa aaatgaagaa aaaactccaa 1020

tatataaaac tattagaaga atag 1044

<210> 2

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gggcggatat ctcgagcaaa ttccatccat cacacc 36

<210> 3

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcggtacccg ggatccttat tcttctaata gttttatata ttg 43

<210> 4

<211> 347

<212> PRT

<213> Plasmodium falciparum

<400> 4

Met Gln Ile Pro Ser Ile Thr Pro His His Thr Pro Thr Ile Arg Leu

1 5 10 15

Leu Cys Glu Cys Glu Leu Tyr Val Pro Asn Cys Asp Asn Asp Pro Glu

20 25 30

Met Lys Arg Val Met Gln Gln Phe His Asp Arg Thr Thr Gln Arg Phe

35 40 45

Gln Glu Tyr Asp Glu Arg Leu Gln Glu Arg Arg Gln Ile Cys Lys Asp

50 55 60

Lys Cys Asp Lys Glu Ile Gln Lys Ile Ile Leu Lys Asp Lys Leu Glu

65 70 75 80

Lys Glu Leu Met Asp Lys Phe Ala Thr Leu Asp Thr Asp Ile Gln Ser

85 90 95

Asp Ala Ile Pro Thr Cys Ile Cys Glu Lys Ser Leu Ala Asp Lys Val

100 105 110

Glu Arg Gly Cys Leu Arg Cys Gly Tyr Gly Leu Gly Ser Val Ala Pro

115 120 125

Met Ile Gly Leu Thr Gly Ser Val Ala Val Asn Val Trp Lys Thr Ala

130 135 140

Glu Leu Ala Ala Ala Met Glu Leu Ala Lys Gln Ala Gly Ala Ala Ala

145 150 155 160

Gly Ile Lys Ala Gly His Leu Ala Gly Thr Lys Val Val Ile Asp Gln

165 170 175

Leu His Thr Leu Gly Ile Tyr Phe Val Gly Asn Lys Gln Leu Glu Thr

180 185 190

Ile Ile Asp Val Thr Asn Tyr Met Asn Val Ser Val Ile Asn Asp Asn

195 200 205

Val Tyr Ser His Tyr Ile Thr Ser Cys Thr Pro Ser Val Val Asn Gly

210 215 220

Arg Pro Val Gly Thr Phe Lys Phe Ser Gly Pro Val Cys Asn Leu Val

225 230 235 240

Gln Pro Asn His Gln Gly Ile Trp Asp Arg Ser Leu Ala Gln Ala Ile

245 250 255

Ile Lys Lys Lys Val Glu Glu Ala Val Ala Glu Gly Thr Gln Ala Ala

260 265 270

Glu Thr Glu Ala Ala Arg Val Thr Ala Thr Lys Thr Ala Ala Phe Glu

275 280 285

Ala Lys Asn Ile Ala Glu Val Glu Ala Ala Thr Thr Ser Tyr Tyr Thr

290 295 300

Pro Ile Ile Ala Ser Ile Val Ala Ile Val Ile Ile Val Leu Ile Met

305 310 315 320

Val Ile Ile Tyr Lys Ile Leu Arg Tyr Arg Arg Lys Lys Lys Met Lys

325 330 335

Lys Lys Leu Gln Tyr Ile Lys Leu Leu Glu Glu

340 345

Claims

1. a Plasmodium falciparum RIFIN recombinant protein encoded by the Plasmodium falciparum RIFIN gene PF3D7_0833100, is characterized in that: contains the Plasmodium falciparum RIFIN protein, its gene sequence is as shown in SEQ ID No.1, and its amino acid sequence is as shown in SEQID No.4 shown.

2. A recombinant plasmid containing Plasmodium falciparum RIFIN gene PF3D7_0833100, characterized in that: it contains Plasmodium falciparum RIFIN gene PF3D7_0833100.

3. A protein chip, characterized in that: it contains Plasmodium falciparum RIFIN protein, and its amino acid sequence is shown in SEQ ID No.4.

4. A specific primer for PCR amplification of Plasmodium falciparum RIFIN protein gene, characterized in that: its upstream sequence is as shown in SEQ ID NO: 2; its downstream sequence is as shown in SEQ ID NO: 3.

5. a kind of preparation method of the Plasmodium falciparum RIFIN recombinant protein encoded by the Plasmodium falciparum RIFIN gene PF3D7_0833100 described in claim 1, is characterized in that comprising the steps:

1) PCR amplification of the specific primer for Plasmodium falciparum RIFIN protein gene according to claim 4, the Plasmodium falciparum RIFIN gene sequence, the gained Plasmodium falciparum RIFIN gene fragment sequence is as shown in SEQ ID No.1;

2) connect the gene fragment obtained in (1) with the carrier pEU-E01-His-N2, construct and obtain the recombinant plasmid pEU-E01-His-N2-PfRIFIN-54, and transfer the recombinant plasmid into competent Escherichia coli respectively In E.coliDH5α, the obtained recombinant strain was confirmed by PCR and agarose gel electrophoresis;

3) Inoculate the recombinant strain obtained in (2) with pEU-E01-His-N2 as a carrier in LB liquid medium, and cultivate at 37° C. and 200 rpm for 12 hours;

4) using the bacterial liquid obtained in (3) to extract plasmids with a kit to obtain a large number of recombinant plasmids pEU-E01-His-N2-PfRIFIN-54;

5) (4) was used in the wheat germ cell-free protein expression system to express the Plasmodium falciparum RIFIN protein, and the expressed protein was detected by Western blot (WB).

6. a kind of preparation method of Plasmodium falciparum RIFIN recombinant protein encoded by Plasmodium falciparum RIFIN gene PF3D7_0833100 as claimed in claim 5, is characterized in that: in step 1), design SEQ ID NO: 2 and SEQ ID NO : The nucleotide sequence shown in 3 or its complementary chain is used as a primer, and the total DNA of Plasmodium falciparum is extracted and used as a template for PCR amplification; the PCR amplification reaction conditions are pre-denaturation at 98°C for 2.0min; 98°C Denaturation for 20 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 1.0 min, 35 cycles; extension at 72 °C for 10 min, and finally stored at 4 °C.

7. a kind of preparation method of the Plasmodium falciparum RIFIN recombinant protein encoded by Plasmodium falciparum RIFIN gene PF3D7_0833100 as claimed in claim 5, is characterized in that: in step 2), by plasmid vector pEU-E01-His-N2 Double-enzyme digestion by Xho I and BamH I, and recovery and purification; In-Fusion connection was performed according to the PCR product obtained in step 1) and the concentration of the plasmid vector restriction fragment, and a recombinant plasmid pEU for prokaryotic expression of the RIFIN encoding gene of Plasmodium falciparum was constructed. -E01-His-N2-PfRIFIN-54; this recombinant plasmid was transformed into Escherichia coli DH5α competent cells by heat shock method, after culture, spread on LB agar plate containing ampicillin, randomly pick the After the colony was shaken and cultured, the bacterial liquid was identified by PCR and sequencing.

8. the preparation method of a kind of Plasmodium falciparum RIFIN recombinant protein encoded by Plasmodium falciparum RIFIN gene PF3D7_0833100 as claimed in claim 5, it is characterized in that: in step 3) in: step 2) contains pEU after sequencing is correct -E01-His-N2-PfRIFIN-54 E. coli DH5a recombinant strain was inoculated into LB liquid medium, cultured at 37°C, 200rpm for 12h, the strain was collected, and the recombinant plasmid pEU-E01-His-N2 was extracted using the kit -PfRIFIN-54, the obtained DNA was placed at -30°C or -80°C for use.

9. a kind of preparation method of the Plasmodium falciparum RIFIN recombinant protein encoded by Plasmodium falciparum RIFIN gene PF3D7_0833100 as claimed in claim 5, is characterized in that: in step 4): utilize step 3) the obtained recombinant plasmid pEU- The Plasmodium falciparum RIFIN protein PfRIFIN-54 was obtained by expressing E01-His-N2-PfRIFIN-54 and wheat germ cell-free expression system, and the protein expression results were identified by Western-blot.

10. Use of the recombinant Plasmodium falciparum RIFIN protein encoded by the Plasmodium falciparum RIFIN gene PF3D7_0833100 according to claim 1 in the preparation of a kit for detecting severe or non-severe Plasmodium falciparum-infected patients.