CN107058353B - HIV chimeric antigen, preparation method and application - Google Patents

Abstract

The present invention discloses an HIV chimeric Gag antigen, a preparation method and an application, and specifically discloses a bivalent HIV chimeric Gag antigen, which comprises a Gag chimeric sequence 1, a Gag chimeric sequence 2 and a soluble PD1 sequence, and also discloses a method for preparing the antigen and its application in preventing and treating HIV diseases and in reagents for diagnosing HIV diseases. The bivalent HIV chimeric Gag antigen of the present invention is designed based on hundreds of HIV-1 B/B', C/CRF07/08_BC and CRF01_AE Gag P41 sequences in China, which contain the most conserved T cell antigen-determining epitopes. The present invention also designs a corresponding vaccine based on the research results of the PD1-based vaccine, which has the effect of inducing broad-spectrum HIV-specific T cell immunity.

Description

HIV chimeric antigen, preparation method and application

technical field

The present invention relates to HIV antigen, preparation method and application. Specifically, the present invention relates to an HIV chimeric Gag antigen, preparation method and application.

Background technique

For the past 30 years, human immunodeficiency virus type I (HIV-1) has been one of the most destructive infectious disease microorganisms in existence globally. By 2014, HIV-1 had infected nearly 780,000 people, half of whom died from acquired immunodeficiency syndrome (AIDS). Although life-long maintenance therapy can prolong the lives of HIV patients, the economic burden and the emergence of drug-resistant strains bring suffering to patients. It is because of the above challenges that great efforts have been made to develop an effective HIV vaccine over the past three decades, yet in the most recent clinical trial of RV144 in Thailand, the protection rate was only 31%. In addition, the genetic diversity of HIV-1 presents another challenge for global vaccine development. Ideally, an effective vaccine should elicit an immune response in the host to suppress and control infection, but so far, antigens derived from natural HIV-1 sequences have been limited in nonhuman primate research and clinical trials. Triggers a limited cellular immune response. Chimeric antigens were designed by computer algorithms to match and maximize the fusion of natural HIV-1 strain sequences. The multivalent chimeric antigen is an optimized immunogen capable of increasing cellular immune coverage against HIV-1 diversity, and the chimeric vaccine approach is a potential solution. However, the design of chimeric immunogens against HIV-1B/B', C/CRF07/08_BC and CRF01_AE subtypes circulating in China has not been reported.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide an HIV chimeric Gag antigen, a preparation method and an application.

A first aspect of the present invention provides a nucleic acid, the sequence of which is selected from:

1) comprising the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2; or

2) Homologous sequences of nucleic acids, which are at least 70%-99% identical to the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2; or

3) a variant of a nucleic acid having at least one nucleotide substitution, deletion and/or addition compared to the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2; or

4) A derivative of a nucleic acid, which is a variant of the sequence of the nucleic acid described in 1)-3).

In a preferred embodiment, the nucleic acid further comprises a soluble PD1 sequence.

In a preferred embodiment, the soluble PD1 sequence is a human soluble PD1 sequence.

In a preferred embodiment, the human soluble PD1 sequence is the nucleotide sequence shown in SEQ ID NO:6.

In a preferred embodiment, the nucleic acid sequence further comprises a linker sequence as a linker.

In a preferred embodiment, the linker sequence is located between the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2.

In a preferred embodiment, the linker sequence is located between the nucleotide sequences shown in SEQ ID NO:6 and SEQ ID NO:1.

In a preferred embodiment, the linker sequence is the nucleotide sequence shown in SEQ ID NO:7.

In a preferred example, the linker sequence located between the nucleotide sequences shown in SEQ ID NO: 6 and SEQ ID NO: 1 and SEQ ID NO: 6 contains a first enzyme cleavage site sequence.

In a preferred embodiment, the first restriction enzyme cleavage site sequence is an EcoRI restriction enzyme sequence.

In a preferred embodiment, the nucleic acid sequence further comprises a second restriction enzyme cleavage site sequence.

In a preferred embodiment, the second restriction enzyme cleavage site sequence is linked to SEQ ID NO:6.

In a preferred embodiment, the second restriction enzyme cleavage site sequence is a BamHI restriction enzyme sequence.

In a preferred embodiment, the nucleic acid sequence further comprises a signal peptide sequence and an initiation codon for tissue plasminogen activator protein expression and release.

In a preferred embodiment, the BamHI enzyme cleavage sequence is sequentially linked with the signal peptide sequence and the initiation codon of tissue plasminogen activator protein expression and release.

In a preferred embodiment, the nucleic acid sequence further comprises a third restriction enzyme cleavage site sequence and a fourth restriction enzyme cleavage site sequence.

In a preferred example, the nucleotide sequence shown in SEQ ID NO: 2 is sequentially connected with a stop codon, a third restriction enzyme cleavage site sequence and a fourth restriction enzyme cleavage site sequence.

In a preferred embodiment, the third restriction enzyme cleavage site sequence and the fourth restriction restriction restriction site sequence are XhoI restriction enzyme restriction sequence and PmeI restriction restriction sequence, respectively.

In a preferred embodiment, the nucleic acid sequence is the sequence of SEQ ID NO:5.

The second aspect of the present invention provides an amino acid encoded by the above-mentioned nucleic acid sequence.

A third aspect of the present invention provides a vector containing the above-mentioned nucleic acid sequence.

The fourth aspect of the present invention provides a host cell transformed or transfected with the above-mentioned vector.

A fifth aspect of the present invention provides a vaccine, comprising the above-mentioned nucleic acid sequence, or the above-mentioned amino acid sequence, or the above-mentioned vector, or the above-mentioned host cell.

The vaccine also includes other HIV vaccines.

The included vaccine is a PD1-based vaccine; preferably an adjuvant is also included.

A sixth aspect of the present invention provides a method for preparing a vaccine, which comprises the steps required for preparing the above-mentioned vaccine in the claims.

The seventh aspect of the present invention provides the application of the above-mentioned nucleic acid sequence, or the above-mentioned amino acid sequence, or the above-mentioned vector, or the above-mentioned host cell in preparing a medicine for preventing and treating HIV disease.

The eighth aspect of the present invention provides the use of the above-mentioned nucleic acid sequence, or the above-mentioned amino acid sequence, or the above-mentioned vector, or the above-mentioned host cell in the preparation of HIV disease diagnostic reagents.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology, and immunology laboratory operation steps used in this paper are all routine steps widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitopes" are also known in the art as "antigenic determinants". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrate or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "or conformational". See, eg, EpitopeMapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all points of interaction between a protein and an interacting molecule (eg, an antibody) exist linearly along the protein's primary amino acid sequence. In conformational epitopes, points of interaction exist across protein amino acid residues that are separated from each other.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the elements of genetic material it carries are amplified and/or expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; bacteriophages; cosmids and the like.

As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-ASynthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

The invention specifically discloses a bivalent HIV chimeric Gag antigen, the antigen comprises Gag chimeric sequence 1, Gag chimeric sequence 2 and soluble PD1 sequence, and also discloses a preparation method of the antigen and its application in the prevention and treatment of HIV disease and in the application of reagents for the diagnosis of HIV disease. The bivalent HIV chimeric Gag antigen of the present invention is designed based on the sequences of hundreds of strains of HIV-1B/B', C/CRF07/08_BC and CRF01_AE Gag P41 in China, and it contains the most conserved T cell epitope. The invention also designs a corresponding vaccine based on the research results of the PD1-based vaccine, which has the effect of inducing broad-spectrum HIV-specific T cell immunity.

Description of drawings

Figure 1: Nucleic acid sequences of Gag p41mosaic1 and Gag p41mosaic2 encoding bivalent HIV chimeric Gag antigens.

Figure 2: Amino acid sequence corresponding to the nucleic acid sequence of Figure 1 .

Figure 3: T-cell epitope coverage analysis of the amino acid sequences of two HIV-1 Gag chimeric antigens.

Figure 4: Structure diagram of PD1-based bivalent HIV chimeric Gag antigen.

Figure 5: Western blot plot: Bivalent HIV chimeric Gag antigen expression and release from 293T cells.

Figure 6: The bivalent HIV chimeric Gag antigen (mosaic) vaccine of the present invention induces T cell responses against three Chinese subtypes (HIV-1B/B', C/CRF07/08_BC and CRF01_AE) in a mouse model ELISPOT result graph. The abscissa in the figure is the detection items, including B subtype polypeptide, BC subtype polypeptide and AE subtype polypeptide, and the ordinate is the number of ELISPOT per million spleen cells. Labels on the right: PBS refers to negative control, p24Fc is short for pVAX-P24Fc, msPD1-p24Fc is short for pVAX-msPD1-p24Fc, mosaic is short for pVAX-mosaic, and huPD1-mosaic is short for pVAX-huPD1-mosaic.

Fig. 7: ELISPOT result graph of the bivalent HIV chimeric Gag antigen vaccine of the present invention inducing T cell response in a rhesus monkey model. Figure 7A shows the time schedule of the monkey vaccine immunization test, and the numbers on the horizontal axis represent the immunization time/sampling time; Figure 7B shows the 28 weeks, different time points, four monkeys against the B subtype P24 protein three T cell responses that do not cross peptide pools; Figure 7C shows T cell responses of four monkeys against four non-crossing peptide pools of the three isoforms of P17 and P24 proteins over twenty-eight weeks; Figure 7D shows the twentieth Over eight weeks, T cell responses of four monkeys to single peptides of AE subtype P17 and P24 proteins, numbers are the number of single peptides.

Figure 8: ELISPOT results of detecting T cell responses in Chinese naturally infected HIV patients using a peptide pool of bivalent HIV chimeric Gag antigens divided into Mosaic p41-1 (top) and Mosaic p41-2 (bottom).

Detailed ways

Unless otherwise specified, terms used in the present invention have their ordinary meanings in the art to which the present invention belongs.

The present invention will be described below with reference to specific embodiments and accompanying drawings. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention. If no specific technology or conditions are indicated in the examples, conventional experimental conditions or conditions suggested in the manufacturer's instructions are used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example 1: Bivalent HIV Chimeric Gag Antigen Design

1. Antigen sequence selection:

Ideally, co-conserved epitopes between variants and branches of HIV-1 are common, thus further enhancing protection against diverse natural strains. In this example, through homologous epitope analysis (http://www.hiv.lanl.gov/content/sequence/MOSAIC/), a novel HIV-1B/B', C Sequences of strains of /CRF07/08_BC and CRF01_AE subtypes, including bivalent HIV chimeric Gag antigens for the most common conserved epitopes.

In the design of bivalent HIV chimeric Gag antigen in this example, the chimeric DNA of bivalent HIV Gag p41 gene was selected to fuse with the soluble domain of human PD-1 (also known as PDCD1 or CD279), the purpose of which is to improve the efficacy of the vaccine. reactivity. The soluble domain of the human PD-1 represents the soluble PD1 component after gene codon optimization, that is, the part of the original PD1 expressed in the extracellular after gene codon optimization, excluding the transmembrane and intracellular part, the gene The purpose of codon optimization is to enhance protein expression in human cells.

The selected chimeric DNA of the bivalent HIV Gag p41 gene included a nucleic acid sequence encoding a chimeric 1 antigen (Gagp41mosaic1) and a nucleic acid sequence encoding a chimeric 2 antigen (Gag p41mosaic 2).

The nucleic acid sequence encoding the chimeric 1 antigen is:

ATGGGGGCAAGAGCCTCCGTGCTGTCTGGCGGGAAACTGGACGCCTGGGAGAAGATCCGGCTGAGACCAGGAGGCAAGAAAAAGTACCGCCTGAAGCACATCGTGTGGGCATCCCGCGAACTGGAGCGATTCGCCCTGAACCCAGGACTGCTGGAAACCGCAGAGGGATGCCAGCAGATCATTGAGCAGCTGCAGTCTACACTGAAAACTGGCTCCGAGGAACTGAAGTCTCTGTTTAACACCATCGCTGTGCTGTGGTGCGTGCATCAGCGCATTGACGTGAAGGATACAAAAGAGGCCCTGGACAAGATCGAGGAAGTGCAGAACAAGTCACAGCAGAAGACTCAGCAGGCCGCTGCAGGAACCGGAAGCTCCTCTAAGGTGAGCCAGAACTATCCCATTGTCCAGAATGCACAGGGACAGATGGTGCACCAGCCACTGAGCCCTCGGACCCTGAACGCATGGGTGAAAGTGGTCGAGGAAAAGGGCTTCAATCCTGAAGTCATCCCAATGTTTAGTGCACTGTCAGAGGGGGCCACACCTCAGGATCTGAACATGATGCTGAATATCGTGGGGGGACATCAGGCCGCTATGCAGATGCTGAAGGAAACTATTAATGAGGAAGCAGCAGAGTGGGACCGAGTGCACCCAGTCCATGCAGGACCAATCCCACCTGGACAGATTCGAGAACCACGAGGATCCGATATCGCCGGCACCACATCTACTCTGCAGGAGCAGATTGGGTGGATGACCAACAATCCACCCATCCCTGTGGGAGACATCTACAAACGCTGGATCATTCTGGGCCTGAACAAGATCGTGCGAATGTATAGCCCAGTCTCCATCCTGGATATTCGGCAGGGACCAAAAGAGCCCTTCAGGGACTACGTGGATCGCTTTTATAAGACACTGAGAGCAGAACAGGCCACTCAGGAGGTGAAAAATTGGATGACAGAGACTCTGCTGGTCCAGAACGCCAATCCTGACTGCAAATCTATTC TGAAGGCTCTGGGGACCGGAGCAACACTGGAGGAAATGATGACCGCTTGTCAGGGAGTGGGAGGACCAGGACACAAGGCAAGGGTCCTG (SEQ ID NO: 1)

The nucleic acid sequence encoding the chimeric 2 antigen is:

ATGGGCGCCCGAGCCAGCATCCTGCGGGGAGGCAAGCTGGATAAATGGGAGAAGATTAGGCTGCGCCCCGGGGGAAAAAAGCACTACATGCTGAAGCATCTGGTGTGGGCTTCTCGGGAACTGGAGAGATTCGCAGTCAACCCAGGCCTGCTGGAAACCAGTGAGGGGTGCAAACAGATCATTAAGCAGCTGCAGCCCGCTCTGCAGACCGGAACAGAGGAACTGCGCAGTCTGTTTAACACTGTGGCCACCCTGTACTGCGTGCACCAGCGAATCGAGATCAAGGACACAAAGGAGGCCCTGGATAAAATCGAGGAAGAGCAGAATAAGTCCAAAAAGAAAGCTCAGCAGACAGCTGCAGATACTGGAAACAATTCTCAGGTGAGTCAGAACTATCCAATCGTCCAGAATCTGCAGGGCCAGATGGTGCACCAGCCTATTAGCCCAAGAACCCTGAACGCCTGGGTGAAAGTGGTCGAAGAGAAGGCTTTCAGCCCCGAAGTCATCCCTATGTTTACCGCCCTGTCCGAGGGAGCTACACCTCAGGACCTGAACACCATGCTGAATACAGTGGGCGGGCACCAGGCTGCTATGCAGATCCTGAAGGACACTATTAATGAAGAGGCAGCCGAGTGGGATAGGCTGCACCCAGTGCATGCAGGACCAGTCGCTCCTGGACAGATGAGAGAACCTAGGGGAAGTGATATCGCCGGCACTACCTCAAACCTGCAGGAGCAGATTGGCTGGATGACAAGCAATCCTCCAATCCCCGTGGGGGAAATCTACAAAAGATGGATCATTCTGGGACTGAACAAGATCGTGAGGATGTATTCACCTACTAGCATCCTGGACATCAAGCAGGGGCCAAAGGAGCCCTTCAGAGACTATGTGGATAGGTTCTTTAAGACCCTGAGAGCTGAACAGGCATCCCAGGACGTGAAAAATTGGATGACTGATACCCTGCTGGTCCAGAACGCAAATCCTGATTGCAAAACAATCC TGAAGGCCCTGGGCCCAGCTGCAACTCTGGAGGAGATGATGACCGCTTGCCAGGGCGTGGGAGGACCTTCACATAAAGCCAGAGTGCTG (SEQ ID NO:2)

The amino acid sequence corresponding to the nucleic acid sequence encoding the chimeric 1 antigen is:

MGARASVLSGGKLDAWEKIRLRPGGKKKYRLKHIVWASRELERFALNPGLLETAEGCQQIIEQLQSTLKTGSEELKSLFNTIAVLWCVHQRIDVKDTKEALDKIEEVQNKSQQKTQQAAAGTGSSSKVSQNYPIVQNAQGQMVHQPLSPRTLNAWVKVVEEKGFNPEVIPMFSALSEGATPQDLNMMLNIVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIPPGQIREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQATQEVKNWMTETLLVQNANPDCKSILKALGTGATLEEMMTACQGVGGPGHKARVL((SEQ IDNO:3)

The amino acid sequence corresponding to the nucleic acid sequence encoding the chimeric 2 antigen is:

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFAVNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQTAADTGNNSQVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVVEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQILKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIGWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQASQDVKNWMTDTLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPSHKARVL(SEQ IDNO:4)

The comparison results of the nucleic acid sequence encoding the chimeric 1 antigen and the nucleic acid sequence encoding the chimeric 2 antigen are shown in FIG. 1 .

The comparison result of the amino acid sequence corresponding to the nucleic acid sequence encoding the chimeric 1 antigen and the amino acid sequence corresponding to the nucleic acid sequence encoding the chimeric 2 antigen is shown in FIG. 2 .

2. Coverage analysis:

Sequences for HIV-1 B/B' (176 strains), HIV-1 01_AE (236 strains) and HIV-1 C/BC (127 strains) subtypes circulating in China (from Genbank, corresponding IDs include AB078678-AB078687; AB078689-AB078690；AB078692-AB078704；AB078709；AB078711；AB213667-AB213689；AB213692；AB746342；AF286226；AF286229；AF286230；AF503396；AY008714；AY008716；AY180905；AY275555-AY275557；DD033495；DQ833405-DQ833436；DQ859178-DQ859180；EF036527- EF036536；EF122502-EF122505；EF122507-EF122511；EF122513-EF122522；EF122524-EF122526；EF122528-EF122545；EF122559；EF368370-EF368372；EF394231-EF394236；EF420986；FJ441290-FJ531390；FJ531405-FJ531445；GQ845124-GQ845126；GU177863；GU564221- GU564225；GU564227-GU564230；HM067748；HQ197984-HQ197989；HQ215552-HQ215556；HQ215568-HQ215577；JF719819；JF932468-JF932500；JQ028170；JQ028172-JQ028173；JQ028179；JQ028187-JQ028188；JQ028194-JQ028195；JQ028208-JQ028211；JQ028213-JQ028214； JQ028217；JQ028220-JQ028221；JQ028223；JQ028228-JQ028231；JQ028236；JQ028239；JQ028241-JQ028242；JQ028244；JQ028246-JQ028256；JQ028271；JQ028285；JQ028288-JQ028289；JQ028291；JQ028293；JQ028295；JQ028299；JQ028617-JQ028618；JQ028624；JQ028633； J Q028638；JQ028646-JQ028649；JQ028653-JQ028661；JQ234979-JQ235007；JQ423923；JQ898186；JQ898208；JQ898220；JQ900844-JQ900942；JX140658；JX392347-JX392362；JX392378-JX392384；JX960597-JX960634；KC596061-KC596066；U71182.)，分析编码The T cell epitope coverage of the amino acid sequence corresponding to the nucleic acid sequence of the chimeric 1 antigen and the amino acid sequence corresponding to the nucleic acid sequence encoding the chimeric 2 antigen (i.e. SEQ ID NO: 3 and SEQ ID NO: 4), The analysis results are shown in Figure 3 (AE in Figure 3 corresponds to HIV-1 CRF01_AE, B corresponds to HIV-1 B/B', BC corresponds to HIV-1 C, CRF07_BC and CRF08_BC).

The results show that if the nine amino acid sequences in the T cell nonapeptide epitope of the whole and each subtype are completely identical (Exact match, 9/9 match), the coverage rate is about 70%; Eight amino acid sequences in the epitope are completely identical (8/9match), and the coverage rate is about 90%; if there are seven amino acid sequences in the T cell nonapeptide epitope (7/9match), the coverage rate is all to around 95%. Among them, the HIV-101_AE subtype virus, which is mainly sexually transmitted in China, has the highest coverage.

Example 2: Construction of PD1-based bivalent HIV chimeric Gag antigen

The bivalent HIV chimeric Gag antigen structure is shown in Figure 4, in which CMV represents the CMV promoter, tPA represents the signal peptide sequence of tissue plasminogen activator protein expression and release, and sPD1 represents the soluble PD1 component, which is the corresponding nuclear The nucleotide or amino acid sequence only contains the sequence of PD1 protein located in the extracellular region, each linker (linker) contains (G4S) ₃ amino acid sequence, and the (G4S) ₃ amino acid sequence represents the repeated amino acid sequence of three GGGGS.

The more specific sequence composition of the PD1-based bivalent HIV chimeric Gag antigen is as follows:

START -tPA-BamHI-opt-huPD1-EcoRI- linker -Gag p41mosaic1- linker -Gagp41mosaic 2- STOP -XhoI-PmeI

START represents the gene initiation codon ATG, tPA represents the signal peptide sequence of tissue plasminogen activator protein expression and release, BamHI, EcoRI, XhoI and PmeI represent the corresponding restriction endonuclease cleavage sites, opt-huPD1 Represents the soluble PD1 component after gene codon optimization, that is, the part of the original PD1 expression after gene codon optimization in the extracellular, excluding the transmembrane region and the intracellular part, STOP represents the gene stop codon, each linker (Linker) contains (G4S) ₃ amino acid sequence, (G4S) ₃ represents the repeated amino acid sequence of three GGGGS.

The specific nucleic acid sequence corresponding to START -tPA- BamHI -opt-huPD1- EcoRI -linker-Gag p41mosaic1- linker -Gagp41mosaic 2- STOP -XhoI-PmeI is as follows:

ATG GACGCCATGCTGCGCGGACTGTGCTGCGTGCTGCTACTGTGCGGCGCCGTGTTCGTGAGCCCCAGCCAGGAGATCCACGCCCGATTCAGGAGAGGAGCCAGAGGA GGATCC ATGCAGATTCCTCAGGCTCCATGGCCTGTGGTGTGGGCAGTGCTGCAGCTGGGATGGAGACCAGGATGGTTCCTGGACTCCCCTGACAGACCATGGAATCCCCCTACATTTTCTCCTGCACTGCTGGTGGTGACTGAGGGCGATAACGCCACCTTCACATGCAGCTTTTCCAACACTTCTGAAAGTTTCGTCCTGAATTGGTACAGGATGTCACCCAGCAACCAGACTGACAAGCTGGCCGCTTTTCCCGAAGACCGCTCCCAGCCTGGGCAAGATTGCCGATTCCGGGTGACACAGCTGCCTAATGGAAGGGACTTTCACATGAGTGTGGTCCGCGCTCGGAGAAACGATTCAGGAACCTATCTGTGTGGCGCAATCAGCCTGGCCCCTAAGACACAGATCAAGGAGAGCCTGAGAGCCGAACTGAGGGTGACTGAGAGGCGCGCTGAAGTCCCAACCGCACATCCTTCCCCATCTCCCCGACCAGCAGGACAG GAATTC CGG GGAGGCGGGGGAAGTGGAGGAGGAGGATCCGGAGGAGGAGGAAGC GGAGGCGGGGGAAGTGGAGGAGGAGGATCCGGAGGAGGAGGAAGC TGATAA CCG CTCGAG CGGCCGGCGCGCC GTTTAAAC AAAGCT(SEQ ID NO:5)

The codon-optimized nucleic acid (opt-huPD1) sequence of the human soluble PD1 gene is:

ATGCAGATTCCTCAGGCTCCATGGCCTGTGGTGTGGGCAGTGCTGCAGCTGGGATGGAGACCAGGATGGTTCCTGGACTCCCCTGACAGACCATGGAATCCCCCTACATTTTCTCCTGCACTGCTGGTGGTGACTGAGGGCGATAACGCCACCTTCACATGCAGCTTTTCCAACACTTCTGAAAGTTTCGTCCTGAATTGGTACAGGATGTCACCCAGCAACCAGACTGACAAGCTGGCCGCTTTTCCCGAAGACCGCTCCCAGCCTGGGCAAGATTGCCGATTCCGGGTGACACAGCTGCCTAATGGAAGGGACTTTCACATGAGTGTGGTCCGCGCTCGGAGAAACGATTCAGGAACCTATCTGTGTGGCGCAATCAGCCTGGCCCCTAAGACACAGATCAAGGAGAGCCTGAGAGCCGAACTGAGGGTGACTGAGAGGCGCGCTGAAGTCCCAACCGCACATCCTTCCCCATCTCCCCGACCAGCAGGACAG(SEQ ID NO:6)

Example 3: Construction of bivalent HIV chimeric Gag antigen expression pVAX1 expression plasmid

The construction of the bivalent HIV chimeric Gag antigen expressing pVAX1 expression plasmid in this example includes the following steps:

(1) Preparation of PCR amplification products: After analyzing the sequences of 539 strains of HIV (namely, the above-mentioned HIV-1B/B' (176 strains), HIV-1CRF01_AE (236 strains) and HIV-1C/CRF07/08_BC (127 strains) strain) subtype sequence), take the conserved sequence, and synthesize the above-mentioned nucleic acid sequence encoding chimeric 1 antigen (SEQ ID NO: 1)-linker-encoding chimeric 2 antigen nucleic acid sequence (SEQ ID NO: 2) in vitro, Dilute the synthesized sequence to 10uM concentration, take 5ul as template for PCR amplification, PCR reaction system (PrimeSTAR HS DNA Polymerase, Takara product number R010A): a total of 25ul reaction system, including 5ul template, 5μl of 5x buffer, 4μl of 2.5 mM dNTP, 1 μl each of 20pM forward primer and reverse primer, 1 μl Taq enzyme (2.5units/μl), 3 μl water. PCR reaction conditions: 92°C for 2 minutes; [92°C for 10 seconds; 55°C for 15 seconds; 68°C for 1.5 minutes] 33 cycles; 68°C for 7 minutes; 4°C for 5 minutes. The sequence of the forward primer (p41-1 with linker) is: 5'-CCGGAATTCCGGGGAGGC GGGGAAGT GGAGGAGGA GGATCCGGA GGAGGAGGA AGCATGGGG GCAAGAGCC TCC-3' (SEQ ID NO: 8); the sequence of the reverse primer (p41-2) is : 5'-CCGGCGCGC CGTTTAAAC AAAGCTCCGCTCGAGCGG TTATCACAG CACTCTGGC TTTATG-3' (SEQ ID NO: 9). The PCR amplification product is obtained by carrying out the PCR amplification reaction through the above reaction system and reaction conditions.

(2) Preparation of pVAX1-huPD1 expression vector: first synthesize the above-mentioned human soluble PD1 gene codon-optimized nucleic acid (opt-huPD1, namely SEQ ID NO: 6), and then connect opt-huPD1 to express through nucleic acid ligase In the plasmid vector pVAX1 (Invitrogen, Carlsbad, CA Cat. No. V26020), a human soluble pVAX1-huPD1 expression vector containing sequence optimization was formed.

(3) Preparation of pVAX-huPD1-mosaic expression plasmid: The human soluble pVAX1-huPD1 expression vector containing sequence optimization was then double-enzyme digested with EcoR1/XhoI (New England Biosystems) to cut off redundant fragments, and then use its restriction site Connect the above-mentioned PCR amplification products that are also digested by this double enzyme, and the connected products are then transfected into DH5alpha competent bacteria (GenScript, Cat. No. M00086). The above operations can be performed according to the corresponding reagent instructions or existing conventional methods. was carried out, thereby preparing an expression plasmid of pVAX-huPD1-mosaic.

Then, the expression plasmid pVAX-mosaic without soluble PD1 was prepared as a control (that is, according to the above steps but not connected to opt-huPD1), and the corresponding mouse soluble PD1 (GenBank #NM_008798) was also prepared according to the above steps. Plasmids pVAX-msPD1-mosaic and rhesus soluble PD1 (GenBank #NM_001114358) pVAX-rhPD1-mosaic.

The four pVAX1 expression plasmids constructed above were transfected into 293T cells (ATCC CRL-1573 ^™ , the same below) by polyethyleneimine (PEI, polysciences, Inc. Cat: 23966), and the expression supernatant was obtained and detected by Western blot.

The specific steps are: 1) Inoculate cells

Twenty-four hours before transfection, 0.5-2×10 ⁵ 293T cells (ATCC catalog number CRL-11268) were seeded in each well of a 24-well plate to make the transfection density 60-80%. The cells seeded in each well were cultured in 0.5ml-1ml of medium (DMEM (LifeScience, Cat. No. 111995073), respectively. The medium contained 10% fetal bovine serum (FBS, Life science, Cat. No. 110270106) and 1% antibiotics (Life science, Item No. 1 15140122).

2) Preparation of transfection complex, cell transfection

Preparation of transfection complex: Dilute 1 μg of each of the four pVAX1 expression plasmids constructed above in 50 μl optiMEM solution (Life science, product number 31985070, the same below), and mix gently to obtain 50 μl DNA dilution; 4 μl PEI (1 mg/ ml, polysciences, Inc. Cat. No. 23966) was diluted in 50 μl opti MEM solution, mixed gently, and then 50 μl PEI diluted solution was taken out; then added dropwise to 50 μl DNA dilution solution, mixed gently, and incubated at room temperature for 10- In 15 minutes, 100 μl of PEI/DNA complex was obtained.

Cell transfection: The above antibiotic-containing medium in which cells were seeded per well was replaced with fresh antibiotic-free medium containing 10% fetal bovine serum (Life science, Cat. No. 110270106). Then, 100 μl of PEI/DNA complex was slowly added to each well and shaken gently to mix evenly, then placed in a _CO2 incubator at 37°C for 6-8 hours and replaced with fresh 1% antibiotics culture medium.

3) Western blot detection

72 hours after the complex was added to the cells for transfection, the culture medium of the transfected cells was collected and the expression products were detected by Western blot to verify whether the expression products were released.

Sample diluent for transfected cell culture medium (sample diluent contains: 50 mM of Tris-HCl [pH 8.0]; 137 mM of NaCl; 2 mM of EDTA; 0.5% NP-40 lysate; 10% glycerol; 1 mg/mL of Pepsin inhibitor (pepstatin), 1mg/mL leupeptin (leupeptin), 1mg/mL trypsin inhibitor (pefabloc) diluted (transfected cell culture medium and sample dilution volume ratio is 1:5), then After heating at 95°C for 10 minutes, it was added to acrylamide gel at a concentration of 10%. The electrophoresis conditions were set at 80 volts for 30 minutes, and then raised to 130 volts for 1 hour. After electrophoresis, protein T The strips were transferred to PVDF membrane semi-dry (13 volts for less than 1 hour, Bio-Rad's Semi-Dry Transfer Cell Trans-Blot SD). The PVDF membrane was washed three times with PBS, then in blocking solution (i.e. PBS containing 5 % nonfat dry milk and 0.5% bovine serum albumin, the same below) for 1 hour at room temperature or overnight at 4°C. The primary antibody (Anti-HIV-1p24Hybridoma (183-H12-5C) (NIH product number 1513)) was diluted with blocking solution (volume The ratio of 1:5000) was added to the PVDF membrane, reacted at room temperature for 2 hours, and then washed thoroughly with PBS. The secondary antibody (fluorescent-labeled anti-mouse, rabbit and human antibody Lico, Cat. No. P/N 926-68078) was blocked with After dilution (volume ratio is 1:10000), it was added to the PVDF membrane and reacted at room temperature for 1 hour. The PVDF membrane was washed with PBS and then dried naturally, and then the protein bands were scanned with a LI-COR scanner. The test included four vaccines Antigen expression comparison: mosaic, mouse msPD1-mosaic, human huPD1-mosaic and monkey rhPD1-mosaic, mosaic is pVAX-mosaic supernatant.

The results are shown in Figure 5. It can be seen from Figure 5 that the bivalent HIV chimeric Gag antigen (mosaic) itself cannot be effectively released, but the bivalent HIV chimeric Gag antigen is associated with mouse (msPD1-mosaic), human (huPD1-mosaic) ) and monkey (rhPD1-mosaic) soluble PD1 fusion proteins can be efficiently expressed and released into the extracellular space.

Example 4: Mouse Experiment

In this example, ELISPOT was used to detect the ability of spleen CD4+ and CD8+ T lymphocytes to secrete IFN-γ stimulated by Gagp17 and Gag p24 after Balb/c mice were electroporated with 100ug DNA vaccine. This example was carried out in a standard sterile room of the Laboratory Animal Center of the University of Hong Kong, and the experiment using live animals was approved by the University Committee. Live animals use six- to eight-week-old female BALB/c mice, and their daily care is as follows: six- to eight-week-old female BALB/c mice are housed in cages under conventional standard temperature and humidity, food and water The animals were fed ad libitum and were under daily care by the full-time staff of the Laboratory Animal Center of the University of Hong Kong.

The specific plans are as follows:

(1) Immunotherapy

Through intramuscular injection, each group of mice received 100 microliters of pVAX-huPD1-mosaic (that is, the expression plasmid of pVAX-huPD1-mosaic prepared in Example 3), pVAX-mosaic (that is, the expression plasmid of Example 3 as a control), respectively. The expression plasmid pVAX-mosaic) and the reported pVAX-P24Fc and pVAX-msPD1-p24Fc (Zhou, et al. PD1-based DNA vaccine amplifies HIV-1GAG-specific CD8 ⁺ T cells inmice. Journal of Clinical Investigation 2013, 123 : 2629-2642.) electroshock (EP60V) immunization injection. The time of 3 times of immunization injection was week 0, week 3 and week 6, respectively, and the doses of 3 times of immunization for each mouse were 4 μg, 20 ug, and 100 ug (ie, the actual DNA content). Two weeks after each immunization, blood samples were collected for serological testing. Two weeks (standard short-term) or 30 weeks (long-term) after the last immunization, mice were sacrificed, and plasma, spleen, and peripheral blood mononuclear cells were collected or prepared for immunoreactivity analysis.

(2) Obtain target cells

The spleen of the sacrificed mice was separated with 3 ml of lymphocyte separation buffer (Daktronics Biotechnology Co., Ltd., product number DKW33-R0100), and then the separated cells were passed through a 70 micrometer (μm) filter (BD Company) to obtain single cells. Carefully suspend the single cells in 1ml RPMI1640 medium (Life Technologies Limited, Cat. No. 21870092), transfer to a 15ml centrifuge tube and centrifuge at 800g for 30 minutes without braking, carefully collect the intermediate layer, and then wash three times with RPMI1640 medium to obtain the target cell.

(3) Assessment of HIV-1gag-specific T cell responses

Dilute the target cells obtained in step (2) with 100 μL RPMI1640 medium to an appropriate concentration (usually 2×10 ⁵ cells/100 μL per well), and add HIV- 1gag (p17 and p24) B subtype polypeptide, C subtype polypeptide (all provided by the National Institutes of Health, catalogue No. 8117 and No. 8118) and AE subtype polypeptide (both synthesized by Gill Biochemical Shanghai Co., Ltd.), In vitro stimulation of splenocytes was performed.

Positive control: The difference is that the target cells are stimulated with 500 ng/ml of phorbol 12-mystrate 13-acetate (PMA; Sigma-Aldrich) plus 1 μg/ml of calcionin.

Negative control: The difference is that the target cells do not add peptide stimuli, but only contain cell culture medium (ie, RPMI1640 medium).

The aforementioned target cells, positive control and negative control cells were respectively incubated at 37°C, 5% CO ₂ , and 100% humidity for 20 hours, and then detected by ELISPOT (using Diaclone's Murine IFN-γ ELISpotkit kit, The specific steps are carried out according to the operation method of the kit) the ability of spleen CD4+ and CD8+ T lymphocytes to secrete IFN-γ, the spots formed by the cells are developed and scanned with an immunospot tester, and then an image analyzer (ThermoScientific) is used to analyze the experimental data. analyze.

Sources of the three viral subtype peptide libraries in Figure 6: HIV Consensus B Gag peptides Set (Cat#8117, Lot#10080196, NIH AIDS Reagent Program), HIV Consensus C Gag peptides (Cat#8118, Lot#3, NIH AIDS Reagent Program), and HIV Consensus 01_AE Gag peptides (according to the complete sequence of 01_AE Gag, derived from Chinese infected persons, determined by analysis as the following amino acid sequence:

HIV 01_AE Gag P17 (SEQ ID NO: 12):

MGARASVLSGGKLDAWEKIRLRPGGKKKYRMKHLVWASRELERFALNPGLLETAEGCQQGCQQLQSTLKTGSEELKSLFNTVATLWCVHQRIDVKDTKEALDKIEEVQNKSQQKTQQAAAGTGSSSKVSQNYPIV;

and HIV 01_AE Gag P24 (SEQ ID NO: 13):

QNAQGQMVHQPVSPRTLNAWVKVVEEKGFNPEVIPMFSALSEGATPQDLNMMLNIVGGHQAAMQMLKETINEEAADWDRTWDRTAGPIPPGQIREPRGSDIAGTTSTLQEQIAWMTNNPPIPVGDIYKRWIILGLNKIVRMYSPPVSILDIRQGPKEPFRDYVDRFYKTLRAEQATQEVKNWMTETLLVQNANPDCKSILKALGTGATLEEMMTARQGVGGPS. The peptides of 15 amino acids were synthesized sequentially from the beginning to the end, and the next one had an overlap of 11 amino acids with the previous one, that is, the conventional method of 15-mer overlap by 11). In BALB/c mice, the B-subtype P24 single peptide GAG [GHAQAAMQMLKETINE] (SEQ ID NO: 14) is specific for mouse CD8+ T cells, while the B-subtype P24 single peptide GAG [TNNPPIPVGEIYKRWIILGLN] ( SEQ ID NO: 15), for specific mouse CD4+ T cells; C/BC subtype P24 single peptide GAG [GGHQAAMQMLKDTIN] (SEQ ID NO: 16), for specific mouse CD8+ T cells, The C/BC subtype P24 single peptide GAG [TSNPPVPVGDIYKRWIILGL] (SEQ ID NO: 17) is directed against specific mouse CD4+ T cells; the AE subtype P24 single peptide GAG [GHQAAMQMLKETINE] (SEQ ID NO: 18) It is directed against specific mouse CD8+ T cells, while the AE subtype P24 single peptide GAG [TNNPPIPVGDIYKRWIILGLN] (SEQ ID NO: 19) is directed against specific mouse CD4+ T cells. These peptide pools or single peptides differ from GAG mosaic 1,2 or bivalent HIV chimeric Gag antigens (mosaic) and are not 100% identical, thus reflecting that the vaccine of the present invention indeed induces broad-spectrum targeting of the three T-cell immune responses to viral subtypes.

The results are shown in Figure 6, showing that the bivalent HIV chimeric Gag antigen (mosaic) vaccine (pVAX-huPD1-mosaic) induced high-level, broad-spectrum targeting of three Chinese subtypes (AE, B, C) T cell responses while demonstrating that soluble PD1 enhances the immunogenicity of the bivalent HIV chimeric Gag antigen. The sources of the peptide libraries for the three viral subtypes in the figure are as described above.

Example 5: Rhesus macaque experiment

The live animal experiments used in this example were approved by the Ethics Committee of Veterinary Laboratory Animals of Foshan Institute of Science and Technology. Four rhesus monkeys were used in the immunization experiment, and they were immunized at 0, 6, 12, and 25 weeks (Fig. 7A), each immunization was anesthetized with 10-20 mg/kg ketamine, followed by a total of 4-needle electrode arrays. 2 mg of the bivalent pVAX-huPD1-mosaic nucleic acid vaccine (ie, the expression plasmid of pVAX-huPD1-mosaic prepared in Example 3) was inoculated by intramuscular electroporation.

Blood sampling was performed every two weeks after animals were anesthetized with a mixture of ketamine and xylazine. PBMC and plasma were separated from blood, stored at -80°C, and then used MABTECH's kit according to its instructions to detect vaccine immune response (ELIspot Assay for Monkey Interferon-γ, MABTECH, 3421M-2A).

Result analysis:

(1) When rhesus monkeys were immunized for the first time (ie, the 0th day of immunization), no specific CD8+ T cell responses against Gag were detected.

(2) However, two weeks after the second immunization (the eighth week), the pVAX-huPD1-mosaic nucleic acid vaccine was found to induce a high level of three non-overlapping (no-overlapping) antibodies against subtype B HIV-Gag P24. ) specific T cell responses of subpeptide pools PP1 (peptide pool 24-1), PP2 (peptide pool 24-2) and PP3 (peptide pool 24-3) (Cat#8117, Lot#10080196, NIH AIDS ReagentProgram) ( Figure 7B), the ordinate shows that the secreted puncta can be increased to about ^2000/106 PBMCs (peripheral blood mononuclear cells), the abscissa represents each monkey (1 to 4), at different times after immunization (0,8 , 12, 14, 16, 25, 28 weeks) for testing. The results demonstrate the induction of a broad spectrum of specific T cell responses within the B subtype.

(3) Subsequently, at the 14th and 28th weeks, the bivalent pVAX-huPD1-mosaic nucleic acid vaccine immunization induced a high level of three subpeptide pools PP1, T cell responses to PP2 and PP3, demonstrating that immune memory has also been established.

(4) In the 28th week after the booster immunization in the 25th week, the peptide libraries of AE subtype, B and BC subtypes were tested at the same time, including a peptide library of HIV-Gag P17 and B subtype of P24. The three subpeptide pools of PP1, PP2 and PP3, the results detected T cell memory immune responses to each subtype (Figure 7C), where the ordinate represents 10 ⁶ PBMC (peripheral blood mononuclear cells) secreting IFN-γ T cells The number of spots, the abscissa represents each monkey (1 to 4), the response to the AE, B or BC subtype polypeptide pool was determined, and the results proved that the bivalent pVAX-huPD1-mosaic vaccine antigen of the present invention can also be used in primates. A broad spectrum of specific T cell responses across the three subtypes were induced in vivo.

(5) Next, the peptide library decomposition analysis of HIV-Gag AE subtype was further used (Fig. 7D). The ordinate shows the number of spots of IFN-γ T cells secreting IFN-γ in 10 ⁶ PBMCs (peripheral blood mononuclear cells), and the abscissa represents each T-cell responses of monkeys (1 to 4) to single peptides (eg 75, 76 etc. represent the numbers of single peptides of the AE subtype in our peptide library). It can be seen from the figure that the single-peptide T cell responses of each monkey are different, suggesting that the bivalent huPD1-mosaic vaccine antigen of the present invention conforms to the actual situation of individual differences in human use. The amino acid sequence of the AE subtype single peptide is as follows:

Gag38: PRTLNAWVKVVEEKG (SEQ ID NO: 20)

Gag41: EKGFNPEVIPMFSAL (SEQ ID NO: 21)

Gag42: NPEVIPMFSALSEGA (SEQ ID NO: 22)

Gag49: GHQAAMQMLKETINE (SEQ ID NO: 23)

Gag50: AMQMLKETINEEAAD (SEQ ID NO: 24)

Gag66: IYKRWIILGLNKIVR (SEQ ID NO: 25)

Gag68: GLNKIVRMYSPVSIL (SEQ ID NO: 26)

Gag75: VDRFYKTLRAEQATQ (SEQ ID NO: 27)

Gag76: YKTLRAEQATQEVKN (SEQ ID NO: 28)

Example 6: Clinical Sample Experiment

In this example, the actual reaction performance of the bivalent HIV chimeric Gag antigen in HIV natural infection was studied, and the peptide libraries Mosaic p41-1 (SEQ ID NO: 3) and Mosaic p41-2 ( SEQ ID NO: 4) were respectively used to detect the T cell ELISPOT response of Chinese naturally infected HIV patients. The detection was carried out using the Human IFN-g ELISpot ^BASIC (ALP) detection kit with the product number 3420-2A of MABTECH Company, and the steps were carried out according to the instructions of the supporting kit. The number of PBMC (peripheral blood mononuclear cells) samples of patients receiving antiviral combined drug treatment was n=9, and the number of PBMC (peripheral blood mononuclear cells) samples of control patients who did not receive treatment was n=17.

The results are shown in Figure 8. The abscissas 1-26 represent each patient, of which 1-17 represent control patients who did not receive treatment, 18-26 represent patients who received antiviral combination therapy, and the ordinate represents bivalent HIV Two peptide pools of chimeric Gag antigens, Mosaic p41-1 and Mosaic p41-2, the number of spots of IFN-γ secreting T cells in 10 ⁶ PBMCs (peripheral blood mononuclear cells).

Result analysis:

(1) There was a broad ELISPOT response against the Mosaic p41-1 peptide pool (13/17, 76%) and the Mosaic p41-2 peptide pool (7/17, 41%) in untreated control patients (in SFU >200 counts) (SFU: spot-forming unit, spot-forming unit), while the overall response rate (positive for Mosaic p41-1 or Mosaic p41-2) reached 94.1% (16/17), so based on the present invention's two Vaccines with chimeric HIV chimeric Gag antigens can be candidates for HIV prevention and clinical treatment.

(2) In patients who received antiviral combination therapy, there was no detectable virus in the body, and the T cell ELISPOT response rate was lower than that of control patients who did not receive treatment, and the overall response rate was only 22% (2/9). Thus, it can be seen that the bivalent HIV chimeric Gag antigen vaccine of the present invention can be used to enhance the broad-spectrum T cell immune response against HIV in the treated patient to facilitate immunotherapy.

The above content is a further detailed description of the present application in conjunction with specific embodiments, and it cannot be considered that the specific implementation of the present application is limited to these descriptions. For those of ordinary skill in the technical field of the present application, without departing from the concept of the present application, some simple deductions or substitutions can be made, which should be regarded as belonging to the protection scope of the present application.

SEQUENCE LISTING

<110> Acker Immune Healing Co., Ltd.

<120> HIV chimeric antigen, preparation method and application

<130> 16PA0176CN.B1

<160> 28

<170> PatentIn version 3.3

<210> 1

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atgggggcaa gagcctccgt gctgtctggc gggaaactgg acgcctggga gaagatccgg 60

ctgagaccag gaggcaagaa aaagtaccgc ctgaagcaca tcgtgtgggc atcccgcgaa 120

ctggagcgat tcgccctgaa cccaggactg ctggaaaccg cagagggatg ccagcagatc 180

attgagcagc tgcagtctac actgaaaact ggctccgagg aactgaagtc tctgtttaac 240

accatcgctg tgctgtggtg cgtgcatcag cgcattgacg tgaaggatac aaaagaggcc 300

ctggacaaga tcgaggaagt gcagaacaag tcacagcaga agactcagca ggccgctgca 360

ggaaccggaa gctcctctaa ggtgagccag aactatccca ttgtccagaa tgcacaggga 420

cagatggtgc accagccact gagccctcgg accctgaacg catgggtgaa agtggtcgag 480

gaaaagggct tcaatcctga agtcatccca atgtttagtg cactgtcaga gggggccaca 540

cctcaggatc tgaacatgat gctgaatatc gtggggggac atcaggccgc tatgcagatg 600

ctgaaggaaa ctattaatga ggaagcagca gagtgggacc gagtgcaccc agtccatgca 660

ggaccaatcc cacctggaca gattcgagaa ccacgaggat ccgatatcgc cggcaccaca 720

tctactctgc aggagcagat tgggtggatg accaacaatc cacccatccc tgtgggagac 780

atctacaaac gctggatcat tctgggcctg aacaagatcg tgcgaatgta tagcccagtc 840

tccatcctgg atattcggca gggaccaaaa gagcccttca gggactacgt ggatcgcttt 900

tataagacac tgagagcaga acaggccact caggaggtga aaaattggat gacagagact 960

ctgctggtcc agaacgccaa tcctgactgc aaatctattc tgaaggctct ggggaccgga 1020

gcaacactgg aggaaatgat gaccgcttgt cagggagtgg gaggaccagg acacaaggca 1080

agggtcctg 1089

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atgggcgccc gagccagcat cctgcgggga ggcaagctgg ataaatggga gaagattagg 60

ctgcgccccg ggggaaaaaa gcactacatg ctgaagcatc tggtgtgggc ttctcgggaa 120

ctggagagat tcgcagtcaa cccaggcctg ctggaaacca gtgaggggtg caaacagatc 180

attaagcagc tgcagcccgc tctgcagacc ggaacagagg aactgcgcag tctgtttaac 240

actgtggcca ccctgtactg cgtgcaccag cgaatcgaga tcaaggacac aaaggaggcc 300

ctggataaaa tcgaggaaga gcagaataag tccaaaaaga aagctcagca gacagctgca 360

gatactggaa acaattctca ggtgagtcag aactatccaa tcgtccagaa tctgcagggc 420

cagatggtgc accagcctat tagcccaaga accctgaacg cctgggtgaa agtggtcgaa 480

gagaaggctt tcagccccga agtcatccct atgtttaccg ccctgtccga gggagctaca 540

cctcaggacc tgaacaccat gctgaataca gtgggcgggc accaggctgc tatgcagatc 600

ctgaaggaca ctattaatga agaggcagcc gagtgggata ggctgcaccc agtgcatgca 660

ggaccagtcg ctcctggaca gatgagagaa cctaggggaa gtgatatcgc cggcactacc 720

tcaaacctgc aggagcagat tggctggatg acaagcaatc ctccaatccc cgtgggggaa 780

atctacaaaa gatggatcat tctgggactg aacaagatcg tgaggatgta ttcacctact 840

agcatcctgg acatcaagca ggggccaaag gagcccttca gagactatgt ggataggttc 900

tttaagaccc tgagagctga acaggcatcc caggacgtga aaaattggat gactgatacc 960

ctgctggtcc agaacgcaaa tcctgattgc aaaacaatcc tgaaggccct gggcccagct 1020

gcaactctgg aggagatgat gaccgcttgc cagggcgtgg gaggaccttc acataaagcc 1080

agagtgctg 1089

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Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp

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Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys

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His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro

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Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Ile Glu Gln Leu

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Gln Ser Thr Leu Lys Thr Gly Ser Glu Glu Leu Lys Ser Leu Phe Asn

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Thr Ile Ala Val Leu Trp Cys Val His Gln Arg Ile Asp Val Lys Asp

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Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Val Gln Asn Lys Ser Gln

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Gln Lys Thr Gln Gln Ala Ala Ala Gly Thr Gly Ser Ser Ser Lys Val

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Ser Gln Asn Tyr Pro Ile Val Gln Asn Ala Gln Gly Gln Met Val His

130 135 140

Gln Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu

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Glu Lys Gly Phe Asn Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser

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Glu Gly Ala Thr Pro Gln Asp Leu Asn Met Met Leu Asn Ile Val Gly

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Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu

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Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Pro

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Pro Gly Gln Ile Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr

225 230 235 240

Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile

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Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys

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Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly

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Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu

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Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Glu Thr

305 310 315 320

Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Ser Ile Leu Lys Ala

325 330 335

Leu Gly Thr Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly

340 345 350

Val Gly Gly Pro Gly His Lys Ala Arg Val Leu

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Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys His Tyr Met Leu Lys

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

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Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Lys Gln Leu

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Gln Pro Ala Leu Gln Thr Gly Thr Glu Glu Leu Arg Ser Leu Phe Asn

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Thr Val Ala Thr Leu Tyr Cys Val His Gln Arg Ile Glu Ile Lys Asp

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Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys

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Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His

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Gln Pro Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu

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Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser

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Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly

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Gly His Gln Ala Ala Met Gln Ile Leu Lys Asp Thr Ile Asn Glu Glu

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atggacgcca tgctgcgcgg actgtgctgc gtgctgctac tgtgcggcgc cgtgttcgtg 60

agccccagcc aggagatcca cgcccgattc aggagaggag ccagaggagg atccatgcag 120

attcctcagg ctccatggcc tgtggtgtgg gcagtgctgc agctgggatg gagaccagga 180

tggttcctgg actcccctga cagaccatgg aatcccccta cattttctcc tgcactgctg 240

gtggtgactg agggcgataa cgccaccttc acatgcagct tttccaacac ttctgaaagt 300

ttcgtcctga attggtacag gatgtcaccc agcaaccaga ctgacaagct ggccgctttt 360

cccgaagacc gctcccagcc tgggcaagat tgccgattcc gggtgacaca gctgcctaat 420

ggaagggact ttcacatgag tgtggtccgc gctcggagaa acgattcagg aacctatctg 480

tgtggcgcaa tcagcctggc ccctaagaca cagatcaagg agagcctgag agccgaactg 540

agggtgactg agaggcgcgc tgaagtccca accgcacatc cttccccatc tccccgacca 600

gcaggacagg aattccgggg aggcggggga agtggaggag gaggatccgg aggaggagga 660

agcatggggg caagagcctc cgtgctgtct ggcgggaaac tggacgcctg ggagaagatc 720

cggctgagac caggaggcaa gaaaaagtac cgcctgaagc acatcgtgtg ggcatcccgc 780

gaactggagc gattcgccct gaacccagga ctgctggaaa ccgcagaggg atgccagcag 840

atcattgagc agctgcagtc tacactgaaa actggctccg aggaactgaa gtctctgttt 900

aacaccatcg ctgtgctgtg gtgcgtgcat cagcgcattg acgtgaagga tacaaaagag 960

gccctggaca agatcgagga agtgcagaac aagtcacagc agaagactca gcaggccgct 1020

gcaggaaccg gaagctcctc taaggtgagc cagaactatc ccattgtcca gaatgcacag 1080

ggacagatgg tgcaccagcc actgagccct cggaccctga acgcatgggt gaaagtggtc 1140

gaggaaaagg gcttcaatcc tgaagtcatc ccaatgttta gtgcactgtc agagggggcc 1200

acacctcagg atctgaacat gatgctgaat atcgtggggg gacatcaggc cgctatgcag 1260

atgctgaagg aaactattaa tgaggaagca gcagagtggg accgagtgca cccagtccat 1320

gcaggaccaa tcccacctgg acagattcga gaaccacgag gatccgatat cgccggcacc 1380

acatctactc tgcaggagca gattgggtgg atgaccaaca atccacccat ccctgtggga 1440

gacatctaca aacgctggat cattctgggc ctgaacaaga tcgtgcgaat gtatagccca 1500

gtctccatcc tggatattcg gcagggacca aaagagccct tcagggacta cgtggatcgc 1560

ttttataaga cactgagagc agaacaggcc actcaggagg tgaaaaattg gatgacagag 1620

actctgctgg tccagaacgc caatcctgac tgcaaatcta ttctgaaggc tctggggacc 1680

ggagcaacac tggaggaaat gatgaccgct tgtcagggag tgggaggacc aggacacaag 1740

gcaagggtcc tgggaggcgg gggaagtgga ggaggaggat ccggaggagg aggaagcatg 1800

ggcgcccgag ccagcatcct gcggggaggc aagctggata aatgggagaa gattaggctg 1860

cgccccgggg gaaaaaagca ctacatgctg aagcatctgg tgtgggcttc tcgggaactg 1920

gagagattcg cagtcaaccc aggcctgctg gaaaccagtg aggggtgcaa acagatcatt 1980

aagcagctgc agcccgctct gcagaccgga aacagaggaac tgcgcagtct gtttaacact 2040

gtggccaccc tgtactgcgt gcaccagcga atcgagatca aggacacaaa ggaggccctg 2100

gataaaatcg aggaagagca gaataagtcc aaaaagaaag ctcagcagac agctgcagat 2160

actggaaaca attctcaggt gagtcagaac tatccaatcg tccagaatct gcagggccag 2220

atggtgcacc agcctattag cccaagaacc ctgaacgcct gggtgaaagt ggtcgaagag 2280

aaggctttca gccccgaagt catccctatg tttaccgccc tgtccgaggg agctacacct 2340

caggacctga acaccatgct gaatacagtg ggcgggcacc aggctgctat gcagatcctg 2400

aaggacacta ttaatgaaga ggcagccgag tgggataggc tgcacccagt gcatgcagga 2460

ccagtcgctc ctggacagat gagagaacct aggggaagtg atatcgccgg cactacctca 2520

aacctgcagg agcagattgg ctggatgaca agcaatcctc caatccccgt gggggaaatc 2580

tacaaaagat ggatcattct gggactgaac aagatcgtga ggatgtattc acctactagc 2640

atcctggaca tcaagcaggg gccaaaggag cccttcagag actatgtgga taggttcttt 2700

aagaccctga gagctgaaca ggcatcccag gacgtgaaaa attggatgac tgataccctg 2760

ctggtccaga acgcaaatcc tgattgcaaa acaatcctga aggccctggg cccagctgca 2820

actctggagg agatgatgac cgcttgccag ggcgtgggag gaccttcaca taaagccaga 2880

gtgctgtgat aaccgctcga gcggccggcg cgccgtttaa acaaagct 2928

<210> 6

<211> 495

<212> DNA

<213> human

<400> 6

atgcagattc ctcaggctcc atggcctgtg gtgtgggcag tgctgcagct gggatggaga 60

ccaggatggt tcctggactc ccctgacaga ccatggaatc cccctacatt ttctcctgca 120

ctgctggtgg tgactgaggg cgataacgcc accttcacat gcagcttttc caacacttct 180

gaaagtttcg tcctgaattg gtacaggatg tcacccagca accagactga caagctggcc 240

gcttttcccg aagaccgctc ccagcctggg caagattgcc gattccgggt gacacagctg 300

cctaatggaa gggactttca catgagtgtg gtccgcgctc ggagaaacga ttcaggaacc 360

tatctgtgtg gcgcaatcag cctggcccct aagacacaga tcaaggagag cctgagagcc 420

gaactgaggg tgactgagag gcgcgctgaa gtcccaaccg cacatccttc cccatctccc 480

cgaccagcag gacag 495

<210> 7

<211> 45

<212> DNA

<213> Artificial Sequences

<400> 7

ggaggcgggg gaagtggagg aggaggatcc ggaggaggag gaagc 45

<210> 8

<211> 75

<212> DNA

<213> Artificial Sequences

<400> 8

ccggaattcc ggggaggcgg gggaagtgga ggaggaggat ccggaggagg aggaagcatg

ggggcaagag cctcc 75

<210> 9

<211> 60

<212> DNA

<213> Artificial Sequences

<400> 9

ccggcgcgcc gtttaaacaa agctccgctc gagcggttat cacagcactc tggctttatg 60

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequences

<400> 10

Ala Met Gln Met Leu Lys Asp Thr Ile

1 5

<210> 11

<211> 20

<212> PRT

<213> Artificial Sequences

<400> 11

Thr Ser Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile

1 5 10 15

Ile Leu Gly Leu

20

<210> 12

<211> 135

<212> PRT

<213> Human

<400> 12

Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp

1 5 10 15

Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Met Lys

20 25 30

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

35 40 45

Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Gly Cys Gln Gln Leu

50 55 60

Gln Ser Thr Leu Lys Thr Gly Ser Glu Glu Leu Lys Ser Leu Phe Asn

65 70 75 80

Thr Val Ala Thr Leu Trp Cys Val His Gln Arg Ile Asp Val Lys Asp

85 90 95

Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Val Gln Asn Lys Ser Gln

100 105 110

Gln Lys Thr Gln Gln Ala Ala Ala Gly Thr Gly Ser Ser Ser Lys Val

115 120 125

Ser Gln Asn Tyr Pro Ile Val

130 135

<210> 13

<211> 229

<212> PRT

<213> human

<400> 13

Gln Asn Ala Gln Gly Gln Met Val His Gln Pro Val Ser Pro Arg Thr

1 5 10 15

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

20 25 30

Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp

35 40 45

Leu Asn Met Met Leu Asn Ile Val Gly Gly His Gln Ala Ala Met Gln

50 55 60

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

65 70 75 80

Trp Asp Arg Thr Ala Gly Pro Ile Pro Pro Gly Gln Ile Arg Glu Pro

85 90 95

Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile

100 105 110

Ala Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys

115 120 125

Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro

130 135 140

Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg

145 150 155 160

Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr

165 170 175

Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala

180 185 190

Asn Pro Asp Cys Lys Ser Ile Leu Lys Ala Leu Gly Thr Gly Ala Thr

195 200 205

Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Ser His

210 215 220

Lys Ala Arg Val Leu

225

<210> 14

<211> 16

<212> PRT

<213> Artificial Sequences

<400> 14

Gly His Ala Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu

1 5 10 15

<210> 15

<211> 21

<212> PRT

<213> Artificial Sequences

<400> 15

Thr Asn Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile

1 5 10 15

Ile Leu Gly Leu Asn

20

<210> 16

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 16

Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr Ile Asn

1 5 10 15

<210> 17

<211> 20

<212> PRT

<213> Artificial Sequences

<400> 17

Thr Ser Asn Pro Pro Val Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile

1 5 10 15

Ile Leu Gly Leu

20

<210> 18

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 18

Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu

1 5 10 15

<210> 19

<211> 21

<212> PRT

<213> Artificial Sequences

<400> 19

Thr Asn Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile

1 5 10 15

Ile Leu Gly Leu Asn

20

<210> 10

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 10

Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Gly

1 5 10 15

<210> 21

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 21

Glu Lys Gly Phe Asn Pro Glu Val Ile Pro Met Phe Ser Ala Leu

1 5 10 15

<210> 22

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 22

Asn Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala

1 5 10 15

<210> 23

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 23

Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu

1 5 10 15

<210> 24

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 24

Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Asp

1 5 10 15

<210> 25

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 25

Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg

1 5 10 15

<210> 26

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 26

Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu

1 5 10 15

<210> 27

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 27

Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln

1 5 10 15

<210> 28

<211> 15

<212> PRT

<213> Artificial Sequences

<400> 28

Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn

1 5 10 15

Claims (15)

1. a nucleic acid, is characterized in that, the sequence of described nucleic acid is the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2; further comprising a soluble PD1 sequence; The soluble PD1 sequence is a human soluble PD1 sequence; The human soluble PD1 sequence is the nucleotide sequence shown in SEQ ID NO:6; Described nucleic acid sequence further comprises the linker sequence as linker; The linker sequence is between the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2; The linker sequence is between the nucleotide sequences shown in SEQ ID NO:6 and SEQ ID NO:1; The linker sequence is the nucleotide sequence shown in SEQ ID NO:7; The linker sequence between the nucleotide sequences shown in SEQ ID NO: 6 and SEQ ID NO: 1 and SEQ ID NO: 6 contain the first enzyme cleavage site sequence; The first enzyme cleavage site sequence is EcoRI enzyme cleavage sequence; The nucleic acid sequence further comprises a second restriction enzyme cleavage site sequence; The second enzyme cleavage site sequence is linked to SEQ ID NO: 6; The second enzyme cleavage site sequence is the BamHI enzyme cleavage sequence; The nucleic acid sequence further comprises a signal peptide sequence and an initiation codon for the expression and release of tissue plasminogen activator protein. 2 . The nucleic acid according to claim 1 , wherein the BamHI enzyme cleavage sequence is sequentially connected with the signal peptide sequence and the initiation codon of tissue plasminogen activator protein expression and release. 3 . 3. The nucleic acid according to claim 2, wherein the nucleic acid sequence further comprises a third restriction site sequence and a fourth restriction restriction site sequence. 4. nucleic acid according to claim 3, is characterized in that, the nucleotide sequence shown in described SEQ ID NO:2 is successively with stop codon, the third restriction enzyme cutting site sequence and the fourth restriction enzyme cutting site sequence connected. 5. nucleic acid according to claim 4, is characterized in that, described third restriction enzyme cutting site sequence and fourth restriction enzyme cutting site sequence are XhoI restriction enzyme cutting sequence and PmeI restriction enzyme cutting sequence respectively. 6. The nucleic acid according to claim 5, wherein the nucleic acid sequence is the sequence of SEQ ID NO:5. 7. An amino acid, characterized by being encoded by the nucleic acid sequence of any one of claims 1 to 6. 8. A vector comprising the nucleic acid sequence according to any one of claims 1 to 6. 9. A host cell characterized in that the vector of claim 8 has been transformed or transfected. 10. A vaccine, characterized by comprising the nucleic acid sequence according to any one of claims 1 to 6, or the amino acid sequence according to claim 7, or the vector according to claim 8, or the nucleotide sequence according to claim 9. described host cells. 11. The vaccine of claim 10, further comprising other HIV vaccines. 12. The vaccine of claim 11, wherein the other HIV vaccine is a PD1-based vaccine. 13. The vaccine of claim 12, further comprising an adjuvant. 14. A method for preparing a vaccine, comprising the steps required for preparing the vaccine according to any one of claims 10 to 13. 15. The nucleic acid according to any one of claims 1 to 6 or the amino acid according to claim 7 or the vector according to claim 8 or the host cell according to claim 9 in the preparation of drugs for preventing and treating HIV diseases. application.

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