CN100425700C - scFvC6.5-sTrail fusion gene and protein, and its preparation - Google Patents

Abstract

The present invention relates to targeted antineoplastic drugs and gene recombination technology, specifically to scFvC6.5-sTrail fusion gene and protein and its preparation. The scFvC6.5-sTrail fusion gene has the base sequence in Sequence Table 1. The scFvC6.5-sTrail fusion gene was expressed, denatured, refolded and purified to obtain the immunotoxin scFvC6.5-sTrail. In vitro experiments have confirmed that the fusion protein has obvious killing effect on tumor cells with high expression of HER2/neu, but has no killing effect on HER2/neu negative cells, showing strong specificity of tumor killing. This immunotoxin shows a potential application prospect for the targeted therapy of tumors overexpressing HER2/neu on the cell surface.

Description

scFvC6.5-sTrail fusion gene and protein and its preparation

technical field

The invention relates to targeted antineoplastic drugs and gene recombination technology, specifically scFvC6.5-sTrail fusion gene and protein and its preparation, especially cytokine immunotoxin scFvC6.5-sTrail and its preparation and application.

Background technique

Immunotoxin is a combined molecule produced by linking antibody molecules targeting tumor cells with cytotoxic effector molecules, and is mainly used for targeted therapy of tumors or autoimmune diseases. The development of immunotoxin has gone through three generations. Recombinant immunotoxin (recombinantimmunotoxin) is considered as the third generation immunotoxin, which is produced by fusion expression of antibody functional region or growth factor gene and mutant form of toxin gene through genetic engineering. Recombinant immunotoxin has the advantages of being suitable for industrialization, low production cost, high permeability to solid tumors, and low immunogenicity.

The modification of genetic engineering and protein engineering for recombinant immunotoxins is called engineering of immunotoxins. The transformation of immunotoxin can be divided into two aspects: antibody part and toxin part. The purpose of modifying immunotoxin is to minimize immunogenicity, increase specific killing effect, ensure reasonable half-life, and reduce non-specific side effects.

Her2/neu is an oncogene with tyrosine protein kinase activity, encoding a single-chain transmembrane glycoprotein kinase with a molecular weight of 185KDa, called p185 or HER2/neu. This protein is highly homologous to the epidermal growth factor receptor and belongs to a member of the epidermal growth factor family. This family also includes EGFR (HER1/ErbB1), HER2/neu (c-neu/erbB2), HER3 (ErbB3) and HER4 (tyro2/ErbB4). The proteins encoded by them all have intrinsic tyrosine kinase activity and belong to the receptor tyrosine kinase superfamily. Her2/neu is overexpressed in a variety of human tumor tissues, such as gastric cancer, colon cancer, lung cancer, breast cancer and ovarian cancer, and has become the target molecule of these tumors. It also makes it a marker for the occurrence and development of a subtype of tumors, and research targeting HER2/neu has become a research hotspot in recent years. In addition, clinical studies have found that cancer cells with overexpression of HER2/neu are not sensitive to the effect of sTrail, which promotes tumorigenesis by resisting the cytotoxic effects of the two, allowing tumor cells to escape the host defense mechanism.

Trail (TNF-related apoptosis-induced ligand) is a new member of the tumor necrosis factor family with anti-tumor activity discovered in recent years, it mediates tumor cell apoptosis through the Trail receptor effect. Trail has attracted extensive attention in the field of tumor therapy since its inception. Existing studies have confirmed that Trail can inhibit the growth of most of the malignant tumors from the stomach, ovary, colon, lung, breast, kidney, prostate, brain and skin. and cell killing effect, while normal human cells except liver cells are resistant to Trail-induced apoptosis. Both the membrane form of Trail (memberaneTrail, mTrail) and soluble Trail (soluble Trail, sTrail) have anti-tumor activity. At present, the soluble form of Trail protein has been expressed in both prokaryotic and eukaryotic pathways. Biological tests in vitro and in vivo Both proved its specific antitumor activity.

The research on sTrail-related immunotoxins has just started, and there are only two such toxins successfully constructed in the world.

Contents of the invention

In order to increase the sensitivity of tumor cells overexpressing HER2/neu to sTrail and improve the tumor killing effect of HER2/neu antibody, the applicant constructed a new humanized anti-HER2/neu single chain antibody (scFvC6.5) and The immunotoxin scFvC6.5-sTrail composed of human sTrail, and its immunospecificity and biological activity were identified; the results showed that the recombinant immunotoxin could specifically recognize and kill HER2/neu-positive ovarian cancer cells SKOV-3 and breast cancer cells MCF-7 , has no effect on HER2/neu-negative melanoma cells A375; suggesting that it has potential application value in anti-HER2/neu overexpression tumor targeted therapy.

The purpose of the present invention is to provide a cytokine-like immunotoxin with strong specificity, high penetrability, and ability to target and kill tumor cells with overexpression of HER2/neu, scFvC6.5-sTrail fusion gene and protein and its preparation.

To achieve the above object, the technical solution adopted in the present invention is:

A scFvC6.5-sTrail fusion gene has the base sequence in the sequence table SEQ ID NO: 1; its encoded protein has the amino acid sequence in the sequence table SEQ ID NO: 2.

Preparation of scFvC6.5-sTrail fusion gene: Use humanized single-chain antibody scFvC6.5 and the 114-281 base fragment of human soluble TNF-related apoptosis-inducing ligand sTrail gene as templates for PCR to amplify the product After being recovered by agarose electrophoresis, it was inserted into the expression vector pET28a after double enzyme digestion, and subjected to enzyme ligation reaction to obtain pET28a-scFvC6.5-sTrail;

Preparation of scFvC6.5-sTrail fusion protein: pET28a-scFvC6.5-sTrail was transformed into Escherichia coli BL21(DE3) for expression; SDS-PAGE proved that the protein scFvC6.5-sTrail mainly existed in the bacteria in the form of inclusion bodies ;Using the principle of the combination of histidine residues and Ni+, the inclusion body protein was denatured, refolded and purified by gradient renaturation of metal chelation column chromatography, and the purified scFvC6.5-sTrail fusion protein was obtained.

The encoded protein of the scFvC6.5-sTrail fusion gene can be used in the preparation of a drug with a strong specific killing effect on tumor cells highly expressing HER2/neu, and can induce apoptosis of tumor cells induced by scFvC6.5-sTrail.

The present invention has the following advantages:

1. The scFvC6.5-sTrail fusion gene is expressed, denatured, and refolded to obtain the immunotoxin scFvC6.5-sTrail; the scFvC6.5 in the scFvC6.5-sTrail fusion protein is a humanized antibody, and sTrail is a human antibody Cytokines avoid possible immunogenicity in practical applications.

2. The scFvC6.5-sTrail gene is a new fusion gene prepared artificially, which is the first gene produced. The expression of this gene can be used in the research of targeted therapy against tumors with high expression of HER2/neu. In vitro experiments have confirmed that the fusion protein has obvious killing effect on tumor cells with high expression of HER2/neu, but has no killing effect on HER2/neu negative cells, showing strong specificity of tumor killing. The immunotoxin shows the potential application prospect for the tumor targeting therapy of overexpressing HER2/neu on the cell surface; the fusion toxin scFvC6.5-sTrail has a molecular weight of 47KDa and has the ability to penetrate solid tumors.

3. The present invention provides a kind of processing method for preparing fusion toxin scFvC6.5-sTrail, according to which method can make analytically pure scFvC6.5-sTrail protein; application of the present invention can further provide directly used for producing fusion toxin scFvC6 .5-sTrail engineered strains.

Description of drawings

Figure 1 is the expression electrophoresis of scFvC6.5-sTrail fusion gene in Escherichia coli: among them: 1 is the medium molecular weight protein standard, 2 is the pure fusion protein, 3 is the periplasmic protein after induction, and 4 is the cell bacterium after induction Protein, 5 is uninduced protein of engineered bacteria (negative control).

Detailed ways

Example 1

A scFvC6.5-sTrail fusion gene has the base sequence of SEQ ID NO: 1 in the sequence table.

CATATGCAGGTGCAGCTGTTGCAGTCTGGGGCAGAGTTGAAAAAACCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGGGGCTCATCTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAAGTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGAGACATGACGTGGGATATTGCAGTAGTTCCAACTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATCTATGGTCACACCAATCGGCCCGCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTGCGGCCGCAGGTGGAGGCGGATCCGTGAGAGAAAGAGGTCCTCAGAGAGTAGCAGCTCACATAACTGGGACCAGAGGAAGAAGCAACACATTGTCTTCTCCAAACTCCAAGAATGAAAAGGCTCTGGGCCGCAAAATAAACTCCTGGGAATCATCAAGGAGTGGGCATTCATTCCTGAGCAACTTGCACTTGAGGAATGGTGAACTGGTCATCCATGAAAAAGGGTTTTACTACATCTATTCCCAAACATACTTTCGATTTCAGGAGGAAATAAAAGAAAACACAAAGAACGACAAACAAATGGTCCAATATATTTACAAATACACAAGTTATCCTGACCCTATATTGTTGATGAAAAGTGCTAGAAATAGTTGTTGGTCTAAAGATGCAGAATATGGACTCTATTCCATCTATCAAGGGGGAATATTTGAGCTTAAGGAAAATGACAGAATTTTTGTTTCTGTAACAAATGAGCACTTGATAGACATGGACCATGAAGCCAGTTTTTTCGGGGCCTTTTTAGTTGGCCTCGAG

(1) Information of SEQ ID NO: 1 (see sequence listing)

(a) Sequence features:

* Length: 1305 bp

*Type: nucleic acid

* Chain type: double chain

*Topology: Linear

(b) Molecular type: cDNA

(c) Assumption: No

(d) Antisense: No

(e) Original source: scFvC6.5 cDNA and sTrail cDNA

(2) Preparation of scFvC6.5-sTrail fusion gene

In order to obtain pET28a-scFvC6.5-sTrail fusion gene, two pairs of primers were designed and synthesized: a pair of scFvC6.5 primers and a pair of sTrail primers.

scFvC6.5 Primer1:

5`-gggtttcatatgcaggtgcagctggtgc-3`, containing NdeI restriction site.

scFvC6.5 Primer2:

5`-atttgcggccgcacctaggacggtcagc-3`contains NotI restriction site.

sTrail Primer1:

5`-atttgcggccgcaggtggaggcggatccgtcaggtcatcttcac-3`contains NotI restriction site and Gly4Ser connecting peptide.

sTrail Primer2:

5`-aattctcgagcagggcaataatcccaaag-3`contains XhoI restriction site.

Use the above primers to PCR amplify the scFvC6.5 and sTrail genes respectively, the conditions are:

PCR conditions for scFvC6.5:

Denaturation at 94°C, 30sec;

Annealing at 58°C, 30sec;

Extend at 72°C for 1 min;

35 cycles;

Finally, extend at 72°C for 10 min.

sTrail PCR conditions:

Denaturation at 94°C, 30sec;

Annealing at 62°C, 30sec;

Extend at 72°C for 1 min;

35 cycles;

Finally, extend at 72°C for 10 min.

After the PCR amplification product was recovered by agarose electrophoresis, the scFvC6.5 gene was digested with NdeI and NotI, and the sTrail gene was digested with NotI and XhoI. The vector pET28a digested by NedI and XhoI was mixed with scFvC6.5 and sTrail digested fragments in a ratio of 1:3:3, and T4 DNA ligase was added to carry out the ligation reaction overnight at 16°C.

Enzyme ligation reaction system (30μl):

The mixture after enzyme ligation was transformed into Escherichia coli competent cell BL21(DE3). Preparation of competent cells, CaCl2 transformation method, extraction and identification of plasmid DNA Reference: "Molecular Cloning Experiment Guide" J. Sambrook, E.F. Felici, T. Maniartis, Science Press, 1998 edition.

Kanamycin plate screening, positive clones were picked, inoculated in 5 ml LB (50 μg/ml kanamycin) medium, cultured overnight at 37°C, 220 rpm, a small amount of plasmid was extracted, and sequenced. Sent to Shanghai United Genomics Company for sequencing; the sequencing results of the transformants showed that the DNA sequence and reading frame of scFvC6.5-sTrail were completely correct.

(3) Expression of recombinant pET28a-scFvC6.5-sTrail in Escherichia coli

The above-mentioned recombinant bacteria were expanded and cultivated in 500ml LB culture solution (50μg/ml kanamycin) at 37°C and 220rpm until OD600nm=0.5. Add IPTG to a final concentration of 1 mM, and induce expression for 4 h. The cells were collected by centrifugation at 4000 g for 20 min.

(4) Denaturation, renaturation and purification of the fusion protein scFvC6.5-sTrail

The cells were washed once with PBS. The cells were resuspended in 50ml 50mM Tris·HCl pH8.3, and then ultrasonically disrupted. Centrifuge at 12000g for 30min to obtain supernatant and precipitate. SDS-PAGE and Western-blot identification were performed on the supernatant and the precipitate, respectively, and it was found that the scFvC6.5-sTrail protein mainly existed in the form of inclusion bodies. After washing the inclusion bodies of scFvC6.5-sTrail with 2.5M NaCl, 0.05% TritonX-100 and 2M urea in sequence, centrifuge at 10,000rpm for 30min, and dissolve the washed inclusion bodies in denaturing solution (8M urea, 50mM Tris·HCl, pH8.3), stirred overnight at 4°C, filtered through a 0.45μm micromembrane, and prepared for column purification.

The IMAC gradient renaturation method was used for the purification and renaturation of the product protein. Fill the column with 5ml Chelating Sepharose and saturate it with Ni ions, and equilibrate the column with denaturing solution. Add 10ml of denatured inclusion bodies to the column and incubate at 4°C for 5h. Wash the baseline with a denaturing solution, and then use a 200ml linear gradient to gradually transition from the denaturing solution containing 8M urea to the refolding buffer (50mM Tris·HCl, pH8.3). Then, the refolded protein was washed out of the column with an eluent (0.5M imidazole, 50mM Tris·HCl, pH8.3). Eluted samples were dialyzed overnight against dialysate (10% glycerol in PBS). Retain each eluted product, aliquot 10 μl, and perform SDS-PAGE to identify protein purification. The first antibody is anti-sTrail mouse monoclonal antibody, the second antibody is HRP-labeled goat anti-mouse antibody, and the substrate is Supersignal luminescent substrate.

(5) SDS-PAGE detection of fusion protein scFvC6.5-sTrail

The bacterial protein before and after scFvC6.5-sTrail induction was mixed with 30 μl of loading 6× loading buffer, mixed well, boiled at 100°C for 5 minutes, and 5 μl was taken for 12% SDS-PAGE. Coomassie brilliant blue staining, and the results were detected by UV gel imaging system for protein expression.

Results: Comparing the whole protein electrophoresis band of the engineered bacteria after induction and the whole protein band of the engineered bacteria before induction, it was found that the protein band at 47KDa was obviously thicker, and the protein band was here after the inclusion body was purified, which was in line with scFvC6 .5-sTrail molecular size. Referring to Fig. 1, the ultraviolet gel imaging scanning system shows that the expressed protein accounts for about 5% of the total bacterial protein. Purified protein purity is greater than 95%. The converted yield is 700μg/1L bacterial liquid.

(6) ELISA to detect the binding activity of scFvC6.5-sTrail to tumor cells expressing HER2/neu in different degrees

SKOV-3, MCF-7 and A-375 cells were cultured to the logarithmic phase, inoculated into 96-well cell culture plates at 5×104 per well, and cultured in a 37°C, 5% CO2 cell incubator for 12 hours. Discard the supernatant and fix with acetone for 15 min at 4°C. After blocking with 100 μl of 5% BSA/PBS solution at 37°C for 2 hours, 50 μl/well of purified scFvC6.5-sTrail at different concentrations were added, and incubated at 37°C for 2 hours. Wash with PBS three times, add 50 μl of mouse anti-6×His·Tag antibody (1:2000), and incubate at 37° C. for 2 hours. Wash with PBS three times, then add 50 μl of HRP-labeled goat anti-mouse antibody, and incubate at 37°C for 1 hour. Wash with PBS three times, add OPD substrate for 5min, add 12.5% sulfuric acid 50μl/well to stop the reaction. Set up a microplate reader to measure the absorbance at 492nm. The group added with PBS was the negative control. Three parallel wells were set up for each sample, and the results were the average of three repeated experiments.

ELISA experiments showed that the immunotoxin scFvC6.5-sTrail had the highest activity in binding to ovarian cancer SKOV-3 cells, and it was strengthened with the increase of the dose. 375 cells. This shows that the binding activity of the fusion protein scFvC6.5-sTrail is specific and can distinguish HER2/neu positive and negative cells.

(7) Cell killing activity test of fusion protein scFvC6.5-sTrail

MTT method was used to detect the cytotoxic activity of different concentrations of scFvC6.5-sTrail on SKOV-3, MCF-7 and A-375 cells: SKOV-3, MCF-7 and A-375 cells cultured to the logarithmic phase, Transfer 104 cells per well into 96-well cell culture plates, and culture them in a 37°C, 5% CO2 cell culture incubator for 12 hours. Add different concentrations of scFvC6.5-sTrail50μl/well, and culture for 12h. MTT was added to a final concentration of 1 mg/ml. Incubate for 2h. Aspirate the culture medium. Add 50 μl DMSO. Shake for 5 minutes, place in a microplate reader to measure the A570 absorbance value. The group added with PBS containing 10% glycerol served as negative control. Each group was set up three times in parallel, and the average value of the three experiments was taken. A blank control was set up.

The cytotoxic activity of scFvC6.5-sTrail to SKOV-3, MCF-7 and A-375 cells at different times at 50nM was detected by MTT method: the same method as above, after adding scFvC6.5-sTrail 2, 8, 12 , 16, and 24h, respectively add MTT to a final concentration of 1mg/ml. Determination of A570 absorbance value.

The MTT test showed that scFvC6.5-sTrail had a significant inhibitory effect on the growth of SKOV-3 cells with high expression of HER2/neu in a dose-dependent manner. At a certain concentration, scFvC6.5-sTrail also had obvious cytotoxic effect on MCF-7 cells with moderate expression of HER2/neu, but was not sensitive to A-375 cells with negative expression of HER2/neu.

When the applicant selected 50nM scFvC6.5-sTrail as a fixed dose and observed the effects of different time (2-24h) on cell growth, it was found that the inhibitory effect of scFvC6.5-sTrail on cell growth began to accelerate from the 12th after incubation, and at At 24 hours, the death rate of SKOV-3 cells was close to 100%, and the death rate of MCF-7 cells was about 80%. However, A-375, a cell negative for HER2/neu expression, did not produce obvious cytotoxicity over time, indicating that scFvC6 .5-sTrail has a time-dependent killing effect on target cells.

sTrail

SEQUENCE LISTING

<110> Shenyang Institute of Applied Ecology, Chinese Academy of Sciences

<120>scFvC6.5-sTrail fusion gene and protein and its preparation

<130>

<160>2

<170>PatentIn version 3.1

<210>1

<211>1305

<212>DNA

<213> Artificial sequence

<220>

<221> CDS

<222>(1)..(1305)

<223>

<400>1

cat atg cag gtg cag ctg ttg cag tct ggg gca gag ttg aaa aaa ccc 48

His Met Gln Val Gln Leu Leu Gln Ser Gly Ala Glu Leu Lys Lys Pro

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ggg gag tct ctg aag atc tcc tgt aag ggt tct gga tac agc ttt acc 96

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

20 25 30

agc tac tgg atc gcc tgg gtg cgc cag atg ccc ggg aaa ggc ctg gag 144

Ser Tyr Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

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tac atg ggg ctc atc tat cct ggt gac tct gac acc aaa tac agc ccg 192

Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro

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Ser Phe Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Ser Thr

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gcc tac ttg caa tgg agc agt ctg aag ccc tcg gac agc gcc gtg tat 288

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser Ala Val Tyr

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ttt tgt gcg aga cat gac gtg gga tat tgc agt agt tcc aac tgc gca 336

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115 120 125

gtc tcc tca ggt gga ggc ggt tca ggc gga ggt ggc tct ggc ggt ggc 432

sTrail

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

gga tcg cag tct gtg ttg acg cag ccg ccc tca gtg tct gcg gcc cca 480

Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro

145 150 155 160

gga cag aag gtc acc atc tcc tgc tct gga agc agc tcc aac att ggg 528

Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly

165 170 175

aat aat tat gta tcc tgg tac cag cag ctc cca gga aca gcc ccc aaa 576

Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys

180 185 190

ctc ctc atc tat ggt cac acc aat cgg ccc gca ggg gtc cct gac cga 624

Leu Leu Ile Tyr Gly His Thr Asn Arg Pro Ala Gly Val Pro Asp Arg

195 200 205

ttc tct ggc tcc aag tct ggc acc tca gcc tcc ctg gcc atc agt ggg 672

Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly

210 215 220

ttc cgg tcc gag gat gag gct gat tat tac tgt gca gca tgg gat gac 720

Phe Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp

225 230 235 240

agc ctg agt ggt tgg gtg ttc ggc gga ggg acc aag ctg acc gtc cta 768

Ser Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

245 250 255

ggt gcg gcc gca ggt gga ggc gga tcc gtg aga gaa aga ggt cct cag 816

Gly Ala Ala Ala Gly Gly Gly Gly Ser Val Arg Glu Arg Gly Pro Gln

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Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu

275 280 285

tct tct cca aac tcc aag aat gaa aag gct ctg ggc cgc aaa ata aac 912

Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn

290 295 300

tcc tgg gaa tca tca agg agt ggg cat tca ttc ctg agc aac ttg cac 960

Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His

305 310 315 320

ttg agg aat ggt gaa ctg gtc atc cat gaa aaa ggg ttt tac tac atc 1008

Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile

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tat tcc caa aca tac ttt cga ttt cag gag gaa ata aaa gaa aac aca 1056

Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr

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aag aac gac aaa caa atg gtc caa tat att tac aaa tac aca agt tat 1104

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

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Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser

370 375 380

aaa gat gca gaa tat gga ctc tat tcc atc tat caa ggg gga ata ttt 1200

Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe

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gag ctt aag gaa aat gac aga att ttt gtt tct gta aca aat gag cac 1248

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405 410 415

ttg ata gac atg gac cat gaa gcc agt ttt ttc ggg gcc ttt tta gtt 1296

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ggc ctc gag 1305

Gly Leu Glu

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sTrail

<210>2

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<213> Artificial sequence

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His Met Gln Val Gln Leu Leu Gln Ser Gly Ala Glu Leu Lys Lys Pro

1 5 10 15

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

20 25 30

Ser Tyr Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

35 40 45

Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro

50 55 60

Ser Phe Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Ser Thr

65 70 75 80

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser Ala Val Tyr

85 90 95

Phe Cys Ala Arg His Asp Val Gly Tyr Cys Ser Ser Ser Asn Cys Ala

100 105 110

Lys Trp Pro Glu Tyr Phe Gln His Trp Gly Gln Gly Thr Leu Val Thr

115 120 125

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro

145 150 155 160

Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly

165 170 175

Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys

180 185 190

Leu Leu Ile Tyr Gly His Thr Asn Arg Pro Ala Gly Val Pro Asp Arg

195 200 205

Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly

210 215 220

Phe Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp

225 230 235 240

Ser Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

245 250 255

sTrail

Gly Ala Ala Ala Gly Gly Gly Gly Ser Val Arg Glu Arg Gly Pro Gln

260 265 270

Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu

275 280 285

Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn

290 295 300

Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His

305 310 315 320

Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile

325 330 335

Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr

340 345 350

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

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Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser

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Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe

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Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His

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Claims (3)

1. A scFvC6.5-sTrail fusion gene, characterized in that: its base sequence is shown in SEQ ID NO: 1 in the sequence table. 2. A coded protein of scFvC6.5-sTrail fusion gene described in claim 1, characterized in that: its amino acid sequence is shown in the sequence table SEQ ID NO:2. 3. a preparation method of the encoded protein of scFvC6.5-sTrail fusion gene described in claim 2, is characterized in that: 1) Preparation of scFvC6.5-sTrail fusion gene: use the humanized single-chain antibody scFvC6.5 and the 114-281 base fragment of the human soluble TNF-related apoptosis-inducing ligand sTrail gene as templates, perform PCR and amplify After the product was recovered by agarose electrophoresis, it was digested with double enzymes and inserted into the expression vector pET28a for enzyme ligation reaction to obtain pET28a-scFvC6.5-sTrail; 2) Preparation of scFvC6.5-TNF-α fusion protein: transform the above pET28a-scFvC6.5-sTrail into Escherichia coli BL21 for expression; use IMAC gradient renaturation method to denature, renature and purify the expressed product, The purified product of scFvC6.5-sTrail was obtained.

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