CN106497884A - A kind of restructuring HEK 293T cells and its secretion zika virus non-structural protein 1 - Google Patents

Abstract

The present invention relates to a recombinant HEK-293T cell, which is characterized in that the recombinant HEK-293T cell is obtained by transfecting an exogenous gene with HEK-293T cells as a host, wherein the nucleic acid sequence of the exogenous gene is as shown in SEQ.ID .NO.1 shown. The recombinant HEK-293T cells of the present invention can secrete non-structural protein 1 for Zika virus antibody detection.

Description

A recombinant HEK-293T cell and its secretion of Zika virus nonstructural protein 1

technical field

The present invention relates to cells modified by introducing foreign genetic material, in particular to recombinant HEK-293T cells.

Background technique

Zika virus (ZIKA virus) is a virus belonging to the Flavivirus genus of the Flaviviridae family. It is mainly transmitted through mosquito bites and sexual contact. In addition, the virus can pass through the placenta. In 1947, virologists discovered ZIKA virus in Uganda. The patients mainly presented with fever, maculopapular rash, and joint pain. After that, only sporadic cases and small-scale epidemics appeared. Until the outbreak of Yap Island in the South Pacific in 2007, there were successive cases of Kabuya virus infection in Africa, Southeast Asia and South America. The National Health and Family Planning Commission of China notified on February 9, 2016 that an imported case of Zika virus infection was confirmed, and so far there have been 10 imported cases of Zika virus infection. Clinical studies have confirmed that ZIKA virus can pass through the placenta and cause fetal microcephaly. Guillaume Carteaux reported a case of meningoencephalitis caused by ZIKA virus infection in an adult. ZIKA virus infection has become a serious worldwide public health problem, and at the same time caused serious economic losses to the countries and regions where the outbreak occurred. According to the latest data released by the World Bank, in Zika virus-infected countries or countries or regions where the epidemic is expected to break out on a large scale, the number of local international tourists is expected to drop sharply, and the tourism industry will lose US$63.9 billion. Our country has a vast territory and mosquitoes breed in summer, creating favorable conditions for the spread of the disease.

The ZIKA virus nucleic acid is a single-stranded positive-strand RNA with a length of 10.8 kb, encoding a precursor protein, which is cleaved into 3 structural proteins (C, prM/M and E) and 7 non-structural proteins (NS1, NS2A) by viral and host proteases. , NS2B, NS3, NS4A, NS4B, and NS5). ZIKA virus and dengue virus belong to the Flaviviridae virus, and the nucleic acid and protein homology of the two are relatively high. At present, there are many research reports on dengue virus at home and abroad. Beth-Ann G.Coller et al. used insect cells to express dengue NS1 protein. Both vaccinated mice and non-human primates showed good immunogenicity, and the challenge test showed that the protein has good protection and has the potential to become a dengue virus subunit vaccine. In addition, ZIKA virus non-structural proteins, such as NS1, NS3 and NS5, play an important role in the process of virus adhesion, invasion and replication.

Zika virus infection has become an important public health factor affecting people's health. At present, there is a lack of effective serological diagnostic methods. Some scholars expressed dengue fever NS1 protein and established a dengue fever antibody ELISA detection method. This method can differentially diagnose dengue fever and other viruses of the Flaviviridae family. . In addition, the NS1 protein is the earliest protein released after cleavage of the precursor protein encoded by the Zika virus ORF. Therefore, the establishment of a detection method for Zika virus NS1 antibody can effectively shorten the detection window period.

Contents of the invention

The technical problem to be solved by the present invention is to provide a recombinant HEK-293T cell that can stably secrete Zika virus nonstructural protein 1 (NS1).

The technical scheme that the present invention solves the above problems is as follows:

A recombinant HEK-293T cell, characterized in that the recombinant HEK-293T cell is obtained by transfecting an exogenous gene with the HEK-293T cell as a host, wherein the nucleic acid sequence of the exogenous gene is as shown in SEQ.ID.NO. 1.

The above-mentioned recombinant HEK-293T cells were prepared by the following method:

(1) Obtain the ZIKA virus NS1 gene sequence (accession number is KU955594.1) from GenBank and then carry out codon optimization to obtain the sequence shown in SEQ.ID.NO.1; The start and stop codons were digested separately, and then cloned into the vector pMD19-T to obtain the recombinant vector named pMD19-NS1;

(2) First take the FUGW plasmid and digest it with PacⅠ, fill it up, and then use BamHI to recover the large fragment of the vector. Then take the pCDNA3. The Rocheligation kit connects the recovered large fragment of the vector with the CMV promoter to obtain the lentiviral expression plasmid pLV-CMV-EGFP;

(3) The lentiviral expression plasmid pLV-CMV-EGFP was double-enzymatically digested to recover a large fragment, and then pMD19-NS1 was double-enzymatically digested to recover the ZIKA virus gene NS1 whose nucleic acid sequence was shown in SEQ.ID.NO.1, and then the The recovered large fragment was ligated with the NS1 sequence under the action of T4 DNA ligase, transformed into stab3 competent cells, spread the LB plate containing ampicillin, cultured overnight, picked a single colony to amplify and cultured, and extracted to obtain the lentiviral recombinant vector pLV-CMV- EGFP-ZIKA-NS1;

(4) Co-transfect HEK-293T cells with the lentiviral recombinant vector pLV-CMV-EGFP-ZIKA-NS1 and the lentiviral packaging helper plasmid, and collect the lentiviruses to infect the HEK-293T cells to obtain recombinant HEK-293T cells.

The recombinant HEK-293T cells can stably secrete and express Zika virus nonstructural protein 1 (NS1), and the amino acid sequence of the Zika virus nonstructural protein 1 is shown in SEQ ID NO.2. The non-structural protein NS1 has good antigenicity and can be used to detect whether a patient is infected with Zika virus.

The present invention has the following beneficial effects:

1. Most of the existing technologies use prokaryotic expression or yeast expression of proteins. There is a big difference between the protein prepared by the above method and the natural protein in the advanced structure method. The expression system selected in the present invention is HEK-293T cells, and the obtained recombinant cell line HEK -293T-Zika-NS1 has biological characteristics similar to parental cells, which is conducive to the large-scale production of antigenic proteins; the expressed protein can obtain a natural conformation and modification process close to the viral protein in the expressing cell, and has good antigenicity; recombinant cells can be used for transgenic Bottle high-density fermentation culture, easy to produce in large quantities;

2. On the basis of the partial tropism of Zika virus gene mammalian cell codons, the present invention expresses NS1 protein by lentiviral expression system for the first time. In addition, the sequence also contains a histidine tag, which is beneficial to later purification;

3. The antigen expression amount of the present invention is high, and the yield can reach 120 mg/L by using ordinary cell culture flasks for culture.

Description of drawings

Figure 1 is a schematic diagram of the construction of the lentiviral recombinant vector pLV-CMV-EGFP-Zika-NS1.

Figure 2 is the electrophoresis diagram of the enzyme digestion identification of the lentiviral recombinant vector pLV-CMV-EGFP-Zika-NS1, in which M is a marker and 1 is a selected clone.

Fig. 3 is the electrophoresis diagram of the expression of Zika virus NS1 protein in different positive clonal cells identified by Western blot; in the figure, bands 1, 2, 3, 4, and 5 are five recombinant positive clonal cells selected at random.

Figure 4 is the Western Blot electrophoresis diagram of target protein expression in different generations of recombinant cell line HEK-293T-Zika-NS1; in the figure, band 1 is the negative control, and bands 2-9 are the 1st, 5th, 10th and 15th respectively , 20, 25, 30, 35 generation cell culture supernatant Western Blot electrophoresis results.

Fig. 5 is the absorbance profile of Zika virus nonstructural protein (NS1) purification according to the present invention.

Fig. 6 is the electrophoresis figure of Zika virus non-structural protein (NS1) of the present invention purifying each step of retaining sample; Among the figure, band 1 is flow-through peak sample; Band 2 is sample before loading; Band 3 is Elution peak sample.

detailed description

The materials involved in the embodiments and their sources are as follows:

HEK-293T cells are preserved by Guangzhou Berniz Biotechnology Co., Ltd.;

Stab3 competent cells were purchased from Guangzhou Funeng Gene Co., Ltd.;

DL 2000Marker, DL 3000Marker, DL 5000Marker, DL10000Marker, Premix Ex Taq and dephosphorylation kit were purchased from Takara Company; restriction enzymes Sal I, Xho I, BamH I, NheI, MluI and T4 DNA ligase were all from NEB Company Products; pre-stained protein markers were purchased from SunShineBio; bovine serum albumin and agarose were purchased from Amresco; diaminobenzidine was purchased from Shanghai Merrill Chemical Technology Co., Ltd.; DNA agarose gel recovery kit was a product of Axygen; Plasmid mini-extraction kit was purchased from OMEGA Company; dimethyl sulfoxide and polybrene were purchased from Sigma Company; tryptone and yeast extract were from Oxiod Company; 0.25% trypsin, DMEM medium and South American fetal bovine serum were purchased from Products of Hyclon Company, Zika polyclonal antibody were purchased from Kerafast Company of the United States.

Embodiment 1 (construction and detection of recombinant cell line)

1. Design and preparation of the Zika virus NS1 gene sequence

1.1 Sequence Design of Zika Virus NS1 Gene

Sequence optimization and expression design were carried out on the basis of Zika virus Haiti strain gene (accession number is KU955594.1), and Kozak sequence, Nhe I restriction site and protective base were added before the start codon, and NS1 protein A terminator, MluI restriction site and protective bases are added to the coding end of the gene; the nucleic acid sequence of the obtained NS1 protein is shown in SEQ ID NO.1, and SEQ ID NO.2 is the translated amino acid of SEQ ID NO.1 sequence.

1.2 Synthesis of gene sequence encoding Zika virus NS1 protein

The Zika virus NS1 protein gene sequence was synthesized by Zhongmei Taihe Biotechnology Co., Ltd. and named pMD19-Zika-NS1.

The Zika virus NS1 gene sequence was synthesized by Zhongmei Taihe Biotechnology Co., Ltd. The specific method is as follows, the nucleotide sequence of the Zika virus NS1 (accession number is KU955594.1) was obtained from GenBank, and the sequence was expressed in mammalian cells using JCat software. Codon optimization, get SEQ.ID.NO.1, chemical synthesis method to get the above sequence, add Nhe I enzyme cleavage site before the start codon at the 5' end of the sequence, add Mlu I enzyme after the stop codon at the 3' end The cutting site was cloned into the commercially available vector pMD19-T (Takara, lot No.D104A), and the recombinant vector was named pMD19-NS1.

2. Construction and detection of lentiviral recombinant vector

Obtaining the lentiviral expression plasmid pLV-CMV-EGFP: the FUGW plasmid was digested with Pac Ⅰ and filled in, and then digested with BamHI to recover the large vector fragment; the pCDNA3.0 plasmid was first digested with Bgl Ⅱ, filled in, and then BamH Ⅰ Recover the CMV promoter after digestion, and then use the Roche ligation kit to connect the large fragment of the recovered vector with the CMV promoter to obtain the lentiviral expression plasmid pLV-CMV-EGFP.

The above-mentioned lentiviral expression plasmid pLV-CMV-EGFP was digested with Nhe I and Mlu I to recover a large fragment, and then ligated with the Zika-NS1 gene recovered by Nhe I and Mlu I digestion under the action of T4 DNA ligase to transform stab3 After the competent cells were coated with ampicillin-containing LB plates, after overnight culture, single colonies were picked to amplify and culture and extract plasmids, which were identified by colony PCR and Nhe I and Mlu I enzyme digestion. Example of the required lentiviral recombinant vector, named pLV-CMV-EGFP-Zika-NS1.

The above-mentioned FUGW plasmid, pCDNA3.0 plasmid and Roche ligation kit are all commercially available.

For the above LB plate containing ampicillin, the medium formula is 100mL: tryptone 1g, yeast extract 0.5g, sodium chloride 1g, agar 1.5g, add distilled water to dissolve, autoclave for 20min, wait for the liquid to cool to 55°C and add For ampicillin, pour the plate immediately after shaking well; the concentration of ampicillin is 100mg/mL, and the addition amount is 100ul of ampicillin per 100mL of medium.

The schematic diagram of the construction of the lentiviral recombinant vector in this example is shown in Figure 1. After the extracted recombinant plasmid was digested with NheI and MluI, it was analyzed by agarose gel electrophoresis, and two bands were found. The results were consistent (see Figure 2).

3. Lentivirus packaging and titer detection

3.1. Cell plating

Cell plating adopts routine operations of those skilled in the art, and the specific steps are as follows:

Pour off the culture supernatant in the HEK-293T cell culture flask, wash it once with 1-3ml PBS, and aspirate it; add 1ml 0.25% trypsin, and let it react at room temperature for 1-2min; when the cells are all round, add 2-3ml DMEM Blow down the cells with complete medium, collect them in a centrifuge tube, centrifuge at 1000rpm for 5 minutes, and discard the supernatant; fully resuspend the cell pellet with DMEM complete medium to make the cells form a single-cell suspension;

Add 10ml DMEM complete medium to a 100mm cell culture dish, count the cells with a cell counting plate, add approximately ⁶ ×106 cells to each dish, and culture overnight in a 37°C, 5% _CO2 incubator.

The formulation of DMEM complete medium is: DMEM complete medium (Corning cellgro, USA, lot number 10-013-CVR-500ml) containing 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

The cell state is crucial for virus packaging, and the state of good cell growth is conducive to virus packaging, so it is necessary to ensure a good cell state.

3.2 Lentiviral Packaging and Infection

The lentiviral packaging is to co-transfect the human embryonic kidney cell HEK-293T with the previously prepared lentiviral recombinant vector pLV-CMV-EGFP-Zika-NS1 and the lentiviral packaging helper plasmid, and the transfection method adopts PEI (polyethyleneimine ) method, the specific steps are as follows.

3.2.1 Transfection system

The PEI transfection method is a routine operation of those skilled in the art, and its transfection system is divided into A liquid and B liquid, specifically as follows:

Liquid A

PEI storage solution 24μl;

1×HBS to make up to 1ml;

Liquid B

pLV-CMV-EGFP-Zika-NS13 μg;

The formula of the above PEI stock solution is: weigh 1.25 mg of PEI powder and dissolve it in 50 ml×HBS (pH 7.4), filter it through a 0.2 μm filter membrane, and store it at 4° C. for later use.

The formula for the above 1×HBS is: dissolve 8.76g NaCl in 900ml ultrapure water, add 20ml 1M HEPES, adjust the pH to 7.4, dilute to 1L, filter through a 0.2μm membrane and store at 4°C for later use.

The above-mentioned lentiviral packaging system plasmids gag/pol, Rev, and VSVG were purchased from Invitrogen (Cat. No.: K4975-00). These packaging plasmids provide the structural proteins, polymerases and envelope proteins necessary for the packaging of viral genome mRNA into complete virions .

3.2.2 Transfection

On the second day after cell plating, when the cell confluence reaches 70%-90%, add liquid A of the transfection system to liquid B, mix well, let stand at room temperature for 20 minutes, and then gently add it to the cells in a 100mm dish In the culture supernatant, gently shake the culture dish to mix well, put it in a 37°C, 5% CO ₂ incubator for 48 hours and collect the supernatant, then add 10ml DMEM complete medium to the cell culture dish, continue to culture for 48 hours and collect the supernatant The supernatant was combined twice, and then centrifuged at 3000rpm for 5min to remove cell debris to obtain the desired virus solution.

100 μl of the virus solution was used to detect the virus titer, and the rest was aliquoted and stored in a -80°C refrigerator or used for subsequent cell infection.

3.3 Detection of virus titer

Virus titer detection adopts RT-QPCR to measure virus titer, and its operation method can adopt the routine operation of those skilled in the art and the steps described in the kit used. The test result shows that the virus titer is 1×10 ⁸ copies/ml, indicating that the virus Successfully packaged, it can infect cells and insert the gene into the cell genome.

3.4. Cell infection

Infect HEK-293T cells with the virus liquid obtained above, the specific steps are as follows:

HEK-293T cells in good growth state were digested and counted, diluted with DMEM complete medium to 1×10 ⁵ /mL, added to a 24-well plate, 500 μL/well, prepared 2 duplicate wells, placed in 37°C, 5% CO ₂ Cultivate in the incubator for 24 hours;

Add polybrene (polybrene) in DMEM complete medium, prepare the medium containing polybrene (the final concentration of Polybrene in the medium is 6-8 μ g/mL);

Add 10 μl of the virus solution prepared in 3.2.2 above to the above-mentioned polybrene-containing medium to make the final volume 300 μl and gently blow and mix to prepare the virus-containing medium;

Pour out the old culture medium of HEK-293T cells cultured for 24 hours in step 1, add 300 μl virus-containing medium to each well, and culture in a 37°C, 5% CO ₂ incubator;

After culturing for 24 hours, pour out the medium in the well, replace it with 500 μl DMEM complete medium, and place it in a 37°C, 5% CO ₂ incubator for cultivation;

4. Establishment of stable gene secretion and expression cell lines

After culturing for the 3rd to 5th day, the cells that overgrown the culture well were digested with trypsin, and then passaged in a 96-well plate by the limiting dilution method to continue the culture. After 15 days, the number of clones of cells in each well was observed under an inverted microscope; For the well of 1 cell colony (i.e. 1 cell clump), culture the cloned cells in a 24-well plate for 3 days, collect the supernatant, concentrate it 10 times, add electrophoresis loading buffer, centrifuge and draw the supernatant for SDS -PAGE electrophoresis, after transfer membrane, use 5% skimmed milk (BioRad company) to block for 2h, use anti-Zika virus polyclonal antibody to incubate overnight (U.S. Kerafast company, 1:10000 dilution), wash 3 times with TBST, each 10min, incubate HRP Labeled rabbit anti-human secondary antibody (Tiangen Biochemical Technology (Beijing) Co., Ltd., 1:10000 dilution), 37 ° C for 1 hour, and then exposed.

The results of Western blotting are shown in Figure 3.

The results showed that the 5 clones we screened could all express the Zika virus NS1 protein, but the expression levels were different. We selected the No. 1 cell clone with the highest expression level to continue to expand and store it, and named it HEK-293T-Zika- NS1, and its stability test.

5. Stability detection of the recombinant cell line of the present invention

5.1 Western blotting identification of different passages of cloned cells

The recombinant cell line HEK-293T-Zika-NS1 obtained from the above screening was normally passaged and cell culture supernatants of different passages (1st, 5th, 10th, 15th, 20th, 25th, 30th, 35th passages) were collected for Western blotting identification. The identification results are shown in Figure 4, and the results show that the cells of different passages can stably express the target protein.

It can be seen from the above test results that the recombinant cell line HEK-293T-Zika-NS1 capable of stably expressing the Zika NS1 protein was indeed obtained in this example.

Embodiment 2. (purification of recombinant protein)

1. Zika virus NS1 protein purification

The cell culture supernatant was filtered through a 0.45 μm filter membrane, and after the HisTrap ^TM HP column was correctly connected to the BioLogic LP protein purification instrument, the sample buffer (20mM pH7.4 phosphate buffer, 0.5M NaCl) was used for 3 times the column bed volume , equilibrate the column at 1ml/min, load the pretreated sample at a speed of 1ml/min, and wash it with loading buffer after the end, 1ml/min, 5 times the column bed volume, and then use 20mM pH7.4 phosphate The buffer (containing 200mM imidazole) eluted the recombinant protein, while monitoring with BioLogic LP, when the baseline was observed to rise, that is, the elution peak was collected. Collect the eluate until the elution peak returns to the baseline, continue to equilibrate 3-5 times the column bed volume with loading buffer, and adjust the flow rate to 1ml/min. Equilibrate 5 times the column bed volume with 20% ethanol.

2. Protein purification analysis

The reserved samples of each step of protein purification were collected for SDS-PAGE and Western blotting analysis, and the method was the same as that in Example 1.

As can be seen from Figure 5, the peak monitored during sample loading is a non-binding peak (impurity protein peak), and along with the phosphate buffer is separated, the peak 280nm wavelength OD value is 0.5-0.6AU; The desired peak is dissociated from the column when eluted with 200mM imidazole eluent. The maximum OD value of this peak at 280nm wavelength is 0.75AU, which is higher than the breakthrough peak, and the separation between the two peaks is good. Western blotting and SDS-PAGE results showed that Zika virus NS1 protein purification conditions were mature, and the purification efficiency was high (see Figure 6).

SEQUENCE LISTING

<110> Southern Medical University

<120> A recombinant HEK-293T cell and its secretion of Zika virus nonstructural protein 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1182

<212> DNA

<213> Artificial sequence

<400> 1

gctagcatgg aagacagacac actcctgcta tgggtgctgc tgctctgggt tccaggttcc 60

actggtgacg tggggtgctc ggtggacttc tcaaagaagg agacgagatg cggtacaggg 120

gtgttcgtct ataacgacgt tgaagcctgg agggacaggt acaagtacca tcctgactcc 180

ccccgtagat tggcagcagc agtcaagcaa gcctgggaag atggtatctg cgggatctcc 240

tctgtttcaa gaatggaaaa catcatgtgg agatcagtag aaggggagct caacgcaatc 300

ctggaagaga atggagttca actgacggtc gttgtgggat ctgtaaaaaa ccccatgtgg 360

agaggtccac agagattgcc cgtgcctgtg aacgagctgc cccacggctg gaaggcttgg 420

gggaaatcgc acttcgtcag agcagcaaag acaaataaca gctttgtcgt ggatggtgac 480

acactgaagg aatgcccact caaacataga gcatggaaca gctttcttgt ggaggatcat 540

gggttcgggg tatttcacac tagtgtctgg ctcaaggtta gagaagatta ttcattagag 600

tgtgatccag ccgttattgg aacagctgtt aagggaaagg aggctgtaca cagtgatcta 660

ggctactgga ttgagagtga gaagaatgac acatggaggc tgaagagggc ccatctgatc 720

gagatgaaaa catgtgaatg gccaaagtcc caacacattgt ggacagatgg aatagaagag 780

agtgatctga tcatacccaa gtctttagct gggccactca gccatcacaa taccagagag 840

ggctacagga cccaaatgaa agggccatgg cacagtgaag agcttgaaat tcggtttgag 900

gaatgcccag gcactaaggt ccacgtggag gaaacatgtg gaacaagagg accatctctg 960

agatcaacca ctgcaagcgg aagggtgatc gaggaatggt gctgcaggga gtgcacaatg 1020

cccccactgt cgttccgggc taaagatggc tgttggtatg gaatggagat aaggcccagg 1080

aaagaaccag aaagcaactt agtaaggtca atggtgactg caggatcaac tgatcacatg 1140

gatcacttct cccttcatca tcaccatcac cattagacgc gt 1182

<210> 2

<211> 389

<212> PRT

<213> Artificial sequence

<400> 2

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Val Gly Cys Ser Val Asp Phe Ser Lys Lys Glu

20 25 30

Thr Arg Cys Gly Thr Gly Val Phe Val Tyr Asn Asp Val Glu Ala Trp

35 40 45

Arg Asp Arg Tyr Lys Tyr His Pro Asp Ser Pro Arg Arg Leu Ala Ala

50 55 60

Ala Val Lys Gln Ala Trp Glu Asp Gly Ile Cys Gly Ile Ser Ser Ser Val

65 70 75 80

Ser Arg Met Glu Asn Ile Met Trp Arg Ser Val Glu Gly Glu Leu Asn

85 90 95

Ala Ile Leu Glu Glu Asn Gly Val Gln Leu Thr Val Val Val Gly Ser

100 105 110

Val Lys Asn Pro Met Trp Arg Gly Pro Gln Arg Leu Pro Val Pro Val

115 120 125

Asn Glu Leu Pro His Gly Trp Lys Ala Trp Gly Lys Ser His Phe Val

130 135 140

Arg Ala Ala Lys Thr Asn Asn Ser Phe Val Val Asp Gly Asp Thr Leu

145 150 155 160

Lys Glu Cys Pro Leu Lys His Arg Ala Trp Asn Ser Phe Leu Val Glu

165 170 175

Asp His Gly Phe Gly Val Phe His Thr Ser Val Trp Leu Lys Val Arg

180 185 190

Glu Asp Tyr Ser Leu Glu Cys Asp Pro Ala Val Ile Gly Thr Ala Val

195 200 205

Lys Gly Lys Glu Ala Val His Ser Asp Leu Gly Tyr Trp Ile Glu Ser

210 215 220

Glu Lys Asn Asp Thr Trp Arg Leu Lys Arg Ala His Leu Ile Glu Met

225 230 235 240

Lys Thr Cys Glu Trp Pro Lys Ser His Thr Leu Trp Thr Asp Gly Ile

245 250 255

Glu Glu Ser Asp Leu Ile Ile Pro Lys Ser Leu Ala Gly Pro Leu Ser

260 265 270

His His Asn Thr Arg Glu Gly Tyr Arg Thr Gln Met Lys Gly Pro Trp

275 280 285

His Ser Glu Glu Leu Glu Ile Arg Phe Glu Glu Cys Pro Gly Thr Lys

290 295 300

Val His Val Glu Glu Thr Cys Gly Thr Arg Gly Pro Ser Leu Arg Ser

305 310 315 320

Thr Thr Ala Ser Gly Arg Val Ile Glu Glu Trp Cys Cys Arg Glu Cys

325 330 335

Thr Met Pro Pro Leu Ser Phe Arg Ala Lys Asp Gly Cys Trp Tyr Gly

340 345 350

Met Glu Ile Arg Pro Arg Lys Glu Pro Glu Ser Asn Leu Val Arg Ser

355 360 365

Met Val Thr Ala Gly Ser Thr Asp His Met Asp His Phe Ser Leu His

370 375 380

His His His His His His

385

Claims (3)

1. a kind of restructuring HEK-293T cells, it is characterised in that restructuring HEK-293T cells are with HEK-293T cells as place Main restructuring foreign gene is obtained, and the nucleotide sequence of wherein described foreign gene is as shown in SEQ.ID.NO.1.

2. a kind of restructuring HEK-293T cells according to claim 1, it is characterised in that described by following methods system ?：

(1) ZIKA virus N S1 gene orders are obtained from GenBank and then carries out codon optimization, obtain such as SEQ.ID.NO.1 Shown sequence；The initiating terminal and clearing end codon of obtained ZIKA virus N S1 genes are carried out digestion, Ran Houke respectively In the grand pMD19-T to carrier, recombinant vector life pMD19-NS1 is obtained；

(2) filling-in after first taking I digestions of FUGW plasmids Pac reclaims carrier large fragment with I digestions of BamH again, then takes pCDNA3.0 Plasmid first reclaims CMV promoter with after I digestions of BamH again with filling-in after II digestions of Bgl, then uses Roche ligation Kit will obtain slow virus expression plasmid pLV-CMV-EGFP after the carrier large fragment for being reclaimed and CMV promoter connection；

(3) slow virus expression plasmid pLV-CMV-EGFP double digestions are reclaimed large fragment, then pMD19-NS1 double digestions is reclaimed core ZIKA viral gene NS1 of the acid sequence as shown in SEQ.ID.NO.1, then by the large fragment for being reclaimed and NS1 sequences in T4DNA Connect under connection enzyme effect, convert LB flat board of the coating containing ammonia benzyl after stab3 competent cells, picking single bacterium colony after incubated overnight Amplification cultivation is simultaneously extracted and obtains slow virus recombinant vector pLV-CMV-EGFP-ZIKA-NS1；

(4) the slow virus recombinant vector pLV-CMV-EGFP-ZIKA-NS1 is packed helper plasmid cotransfection with slow virus HEK-293T cells, collect recombinant virus infection HEK-293T cells, obtain final product restructuring HEK-293T cells.

3. a kind of zika virus non-structural protein 1 by restructuring HEK-293T cells secretion described in claim 1, the zika virus The amino acid sequence of non-structural protein 1 is as shown in SEQ ID NO.2.