An expression vector of a target protein, a method for expressing a target protein

Abstract

The application belongs to the technical field of biology, and discloses an expression vector of a target protein, which comprises a recombinant vector pXJ40-TAT and a recombinant vector pXJ40-TAR. The method for expressing viral proteins in eukaryotic cells by constructing a mammalian cell high-efficiency expression vector using the RNA sequence TAR of HIV-1 virus and its transcription activator TAT and a monkey virus SV40 Ori is helpful for efficient expression of antigens, and further helps to develop an effective vaccine for preventing viral infection. Meanwhile, the application also discloses an expression method of the target protein.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an expression vector for a target protein and a method for expressing the target protein. Background Technology

[0002] Currently available mammalian expression vectors have low expression efficiency. For some large viral structural proteins, such as the structural protein S of IBV, the expression is completely undetectable, leading to problems such as poor antigen stability, low efficacy, and poor protection.

[0003] The technical problem this case addresses is: how to efficiently express the target protein. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an expression vector for a target protein.

[0005] This invention provides three carriers, the specific design concept of which is as follows:

[0006] By constructing a method for expressing viral proteins in eukaryotic cells using the HIV-1 virus RNA sequence TAR and its transcription activator TAT, as well as a mammalian cell-efficient expression vector constructed from simian virus SV40Ori, it is possible to facilitate the efficient expression of antigens and thus develop effective vaccines to prevent viral infections.

[0007] More specifically, when TAR, TAT, and the target protein reside on the same vector, an internal ribosome entry site sequence (IRES) is added, linking the IRES to the TAT. The IRES recruits ribosomes to translate mRNA; it fuses with exogenous cDNA and can independently initiate translation. The TAT is translated, binds to TAR, promotes intracellular RNA replication, and improves expression efficiency. The SV40 large T antigen is a multifunctional protein that regulates early and late transcription of viral genes, initiates DNA replication, and facilitates cell transformation. The T antigen binds to the replicon (SV40 Ori) of the SV40 promoter, promoting transcriptional regulation and DNA synthesis. The SV40 large T antigen is also present in 293T cells. Adding the SV40 Ori element to the vector and transfecting it allows the SV40 Ori to bind to the intracellular large T antigen, initiating DNA replication and promoting plasmid replication within the cell.

[0008] In addition, the present invention also provides a method for expressing the target protein.

[0009] To achieve this objective, the present invention employs the following technical solution: an expression vector for a target protein, comprising recombinant vector pXJ40-TAT and recombinant vector pXJ40-TAR;

[0010] The recombinant vector pXJ40-TAT is prepared by constructing the transcription activator TAT into the pXJ40 vector with the T7 promoter removed, thereby obtaining the recombinant vector pXJ40-TAT.

[0011] The recombinant vector pXJ40-TAR is prepared by constructing the cDNA sequence of the target protein into the pXJ40 vector containing the TAR fragment and removing the T7 promoter to obtain the recombinant vector pXJ40-TAR.

[0012] The sequence of the pXJ40 vector with the T7 promoter removed is shown in SEQ ID NO.1;

[0013] The sequence of the transcription activator TAT is shown in SEQ ID NO.2;

[0014] The sequence of the TAR fragment is shown in SEQ ID NO.3.

[0015] An expression vector for a target protein, wherein the pXJ40 vector with the T7 promoter removed is prepared by: using the pXJ40 vector as a template, amplifying it with primers noT7-CloF1 and noT7-CloR1 to obtain fragment A;

[0016] Using the pXJ40 vector as a template, fragment B was obtained by amplification with primers noT7-CloF2 and noT7-CloR2;

[0017] Fragment A and fragment B were homologously ligated by homologous recombination enzyme to obtain the pXJ40 vector with the T7 promoter removed.

[0018] The sequence of the primer noT7-CloF1 is shown in SEQ ID NO.6;

[0019] The sequence of the primer noT7-CloR1 is shown in SEQ ID NO.7;

[0020] The sequence of the primer noT7-CloF2 is shown in SEQ ID NO.8;

[0021] The sequence of the primer noT7-CloR2 is shown in SEQ ID NO.9.

[0022] In the above-mentioned expression vector for the target protein, the specific method for preparing the recombinant vector pXJ40-TAT is as follows:

[0023] The transcription activator TAT fragment was amplified using primers TAT-CloF and TAT-CloR to obtain fragment C; the pXJ40 vector with the T7 promoter removed was digested into linear fragments using BamHI and HindIII to obtain fragment D; fragment C and fragment D were homologously ligated using a homologous recombination enzyme to obtain the recombinant vector pXJ40-TAT.

[0024] The sequence of the primer TAT-CloF is shown in SEQ ID NO.10;

[0025] The sequence of the primer TAT-CloR is shown in SEQ ID NO.11.

[0026] In the above-mentioned expression vector for the target protein, the preparation method of the recombinant vector pXJ40-TAR is as follows: using the pXJ40 vector with the T7 promoter removed as a template, fragment E is obtained by amplification using primers TAR-CloF1 and TAR-CloR1; using the pXJ40 vector with the T7 promoter removed as a template, fragment F is obtained by amplification using primers TAR-CloF2 and TAR-CloR2; fragment E and fragment F are homologously ligated by homologous recombination enzyme to obtain the recombinant vector pXJ40-TAR;

[0027] The sequence of primer TAR-CloF1 is shown in SEQ ID NO.12;

[0028] The sequence of primer TAR-CloR1 is shown in SEQ ID NO.13;

[0029] The sequence of primer TAR-CloF2 is shown in SEQ ID NO.14;

[0030] The sequence of primer TAR-CloR2 is shown in SEQ ID NO.15.

[0031] In the above-mentioned expression vector of the target protein, the expression vector is the recombinant vector pXJ40-TAR-TAT or the recombinant vector pXJ40-TAR-TAT-Ori(SV40);

[0032] The recombinant vector pXJ40-TAR-TAT was prepared by constructing the transcription activator TAT and IRES into the recombinant vector pXJ40-TAR.

[0033] Specifically, the steps for preparing the recombinant vector pXJ40-TAR-TAT are as follows: Fragment G is obtained by amplifying the transcription activator TAT fragment (as shown in SEQ ID NO.3) using primers IRES-TAT-CloF and IRES-TAT-CloR; fragment H is obtained by amplifying the IRES fragment (as shown in SEQ ID NO.4) using primers IRES-CloF and IRES-CloR; fragment I is obtained by double digestion with NotI and KpnI; fragment G, fragment H, and fragment I are homologously ligated using a homologous recombinase to obtain the recombinant vector pXJ40-TAR-TAT.

[0034] IRES-TAT-CloF: SEQ ID NO.16

[0035] IRES-TAT-CloR: SEQ ID NO.17;

[0036] IRES-CloF: SEQ ID NO.18

[0037] IRES-CloR: SEQ ID NO.19;

[0038] The method for preparing the recombinant vector pXJ40-TAR-TAT-Ori(SV40) is as follows: the replicon of the SV40 virus is constructed into the recombinant vector pXJ40-TAR-TAT to obtain the recombinant vector pXJ40-TAR-TAT-Ori(SV40);

[0039] The preparation method of the recombinant vector pXJ40-TAR-TAT-Ori(SV40) is as follows:

[0040] The replicant fragment of SV40 virus, as shown in SEQ ID NO.5, was amplified using primers SV40 Ori-CloF and SV40 Ori-CloR to obtain fragment J; the vector was digested into linear fragments using XbaI to obtain fragment K; fragment J and fragment K were homologously ligated using a homologous recombinase to obtain the recombinant vector pXJ40-TAR-TAT-Ori(SV40);

[0041] SV40 Ori-CloF: SEQ ID NO.20;

[0042] SV40 Ori-CloR: SEQ ID NO.21;

[0043] The recombinant vector pXJ40-TAR was prepared by constructing the cDNA sequence of the target protein into the pXJ40 vector containing the TAR fragment and removing the T7 promoter, thus obtaining the recombinant vector pXJ40-TAR.

[0044] More specifically, using the pXJ40 vector with the T7 promoter removed as a template, fragment E was obtained by amplification using primers TAR-CloF1 and TAR-CloR1; using the pXJ40 vector with the T7 promoter removed as a template, fragment F was obtained by amplification using primers TAR-CloF2 and TAR-CloR2; fragment E and fragment F were then homologously ligated using a homologous recombinase to obtain the recombinant vector pXJ40-TAR.

[0045] The sequence of the pXJ40 vector with the T7 promoter removed is shown in SEQ ID NO.1;

[0046] The sequence of the transcription activator TAT is shown in SEQ ID NO.2;

[0047] The sequence of the TAR fragment is shown in SEQ ID NO.3;

[0048] The sequence of the internal ribosome entry site IRES is shown in SEQ ID NO.4;

[0049] The sequence of the SV40 virus replicon is shown in SEQ ID NO.5;

[0050] The sequence of primer TAR-CloF1 is shown in SEQ ID NO.12;

[0051] The sequence of primer TAR-CloR1 is shown in SEQ ID NO.13;

[0052] The sequence of primer TAR-CloF2 is shown in SEQ ID NO.14;

[0053] The sequence of primer TAR-CloR2 is shown in SEQ ID NO.15.

[0054] Meanwhile, the present invention also discloses a method for expressing a target protein, wherein the expression vector described above is transfected into a host eukaryotic cell for expression to obtain the target protein.

[0055] In the above-described method for expressing a target protein, the target protein is a fluorescent protein or a viral protein; preferably, the viral protein is a viral structural protein; the fluorescent protein is green fluorescent protein, and more preferably, the viral structural protein is an IBVS structural protein.

[0056] In the above method for expressing the target protein, the mass ratio of recombinant vector pXJ40-TAT to recombinant vector pXJ40-TAR is 1:5.

[0057] In the above-described method for expressing the target protein, the host eukaryotic cell is the human kidney epithelial cell line 293T.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] In this invention, we constructed a TAR-TAT-Ori (SV40) eukaryotic cell expression system for viral proteins. This system relies on two components: 1) the trans-acting factor TAT and the corresponding cis-acting element TAR of human immunodeficiency virus (HIV); 2) the replicon of the SV40 promoter (SV40 Ori). TAT, a transcription activator, is an RNA-binding protein that activates HIV-1 transcription by binding to the stem-loop structure of its cis-acting response element TAR RNA, resulting in intracellular RNA transcription elongation and improved expression efficiency. The replicon of the SV40 promoter (SV40 Ori) binds to the SV40 large T antigen contained in 293T cells, promoting transcriptional regulation and DNA synthesis, and facilitating plasmid replication within the cell. Previous mammalian expression vectors have low expression efficiency; for example, the expression of the IBV structural protein S is completely undetectable, leading to problems such as poor antigen stability, low potency, and inadequate protection. However, the mammalian cell high-efficiency expression vector system constructed using the HIV-1 virus RNA sequence TAR and its transcription activator TAT, along with simian virus SV40 Ori, in this invention enables good transient expression efficiency of the IBV S protein in the human kidney epithelial cell line 293T. Furthermore, the entire vector is only 5670 bp, smaller than most mammalian high-efficiency expression systems. Therefore, this system is helpful in improving the efficient expression of antigens and developing effective vaccines to prevent viral infections, representing a valuable scientific research tool. Attached Figure Description

[0060] Figure 1A This is a spectrum of plasmid pXJ40-TAT;

[0061] Figure 1B This is a map of plasmid pXJ40-TAR, showing the position of the target protein between the β-globin intron and SV40 poly(A)signal.

[0062] Figure 1C This is a map of plasmid pXJ40-TAR-TAT, showing the position of the target protein between the β-globin intron and IRES.

[0063] Figure 1DThis is a map of plasmid pXJ40-TAR-TAT-Ori(SV40), showing the position of the target protein between the β-globin intron and IRES.

[0064] Figure 2A The results of fluorescence measurement were obtained after expressing the fluorescent protein GFP in 293T cells.

[0065] Figure 2B The results of Western blot analysis were obtained after expressing the fluorescent protein GFP and the viral structural protein IBVS on 293T cells.

[0066] Figure 3A The fluorescence detection results of three different expression methods of the TAR-TAT-Ori(SV40) high-efficiency expression vector on 293T cells;

[0067] Figure 3B The results of Western blot analysis of the expression of proteins in 293T cells using three different expression methods of the TAR-TAT-Ori(SV40) high-efficiency expression vector.

[0068] Figure 4 The image shows the results of Western blot analysis of the expression of the viral structural protein IBVS on 293T cells using three different expression methods of the TAR-TAT-Ori(SV40) high-efficiency expression vector. Detailed Implementation

[0069] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0070] Example 1: Construction of plasmid pXJ40 (No T7 pro)

[0071] Using the pXJ40 vector as a template (this vector has been disclosed in the literature: "Cloning, Expression, and Transcriptional Properties of the Human Enhancer Factor TEF-1, Cell. 1991: P551-568"), primers noT7-CloF1 and noT7-CloR1 were designed for amplification to obtain fragment A; using the pXJ40 vector as a template, primers noT7-CloF2 and noT7-CloR2 with the same homologous arms as fragment A were designed for amplification of fragment B. The PCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2. Fragment A and fragment B were homologously ligated using homologous recombination enzymes. The ligation reaction system is shown in Table 3, and the reaction conditions are shown in Table 4. The ligation product was transformed into E. coli DH5α by calcium chloride chemical transformation. In competent cells, an appropriate amount of transformation product was plated and cultured on LB solid medium containing 100 μg / ml ampicillin. Several single colonies were then selected for PCR to verify successful cloning. The primers used in this PCR were the upstream primer RBGI-F and the downstream primer noT7-CloR2. Several single colonies were then selected for plasmid extraction. Digestion with EcoRI and XbaI yielded two fragments of 210 bp and 4077 bp as target plasmids. Further verification of plasmid success was conducted by selecting positive plasmids and sending them to a company for sequencing verification. The positive plasmid pXJ40 (No T7 pro) was confirmed, and its sequence is shown in SEQ ID NO.1.

[0072] noT7-CloF1: 5'-CTCGCGCGTTTCGGTGATGA-3';

[0073] noT7-CloR1: 5'-GAATTCGCCAATTCTTTGCCAAAATGATGAGACAGC-3';

[0074] noT7-CloF2: 5'-GGCAAAGAATTGGCGAATTCGGATCCAAG-3';

[0075] noT7-CloR2: 5'-CACCGTCATCACCGAAACGC-3';

[0076] RBGI-F: 5'-GCAACGTGCTGGTTATTGTG-3'.

[0077] Table 1 PCR reaction system

[0078]

[0079] Table 2 PCR reaction conditions

[0080]

[0081] Table 3 Homologous recombination reaction system

[0082]

[0083] Table 4. Conditions for homologous recombination reaction

[0084]

[0085] Example 2: Construction of plasmid pXJ40-TAT

[0086] pXJ40 (No T7 pro) was digested with BamHI and HindIII to divide the vector into linear fragments. Using the TAT gene fragment synthesized by Genewiz Biotechnology Co., Ltd. (sequence shown in SEQ ID NO.2) as a template, a linearized vector pXJ40 (No T7 pro) was designed. Primers TAT-CloF and TAT-CloR, representing the homologous arms of the pro) fragment, were used to amplify the TAT fragment. The PCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2. The linearized vector and the TAT fragment were ligated using homologous recombination enzymes. The ligation reaction system is shown in Table 3, and the reaction conditions are shown in Table 4. The ligation product was transformed into *E. coli* DH5α competent cells using calcium chloride chemical transformation. An appropriate amount of the transformation product was plated and cultured on LB solid medium containing 100 μg / ml ampicillin. Several single colonies were selected for PCR to verify successful cloning. The primers used in this PCR were the upstream primer RBGI-F and the downstream primer TAT-CloR. Several single colonies were then selected for plasmid extraction. Digestion with EcoRI and XhoI yielded two fragments of 279 bp and 4269 bp as target plasmids. Further verification of plasmid success was performed by sending positive plasmids to the company for sequencing verification. The positive plasmid pXJ40-TAT was confirmed, and the constructed positive plasmid map is shown below. Figure 1A As shown.

[0087] TAT-CloF: 5'-TTGGCGAATTCGGATCCATGGAGCCAGTAGATCCTAGAC-3';

[0088] TAT-CloR: 5'-CGCCTCGAGAAGCTTCTATTCCTTCGGGCCTGTC-3'.

[0089] Example 3: Construction of plasmid pXJ40-TAR

[0090] The HIV-1 virus RNA sequence TAR is shown in SEQ ID NO.3. The TAR fragment sequence was placed onto TAR-CloR1 and TAR-CloF2 amplification primers, retaining homologous regions. Using the pXJ40 (No T7 pro) vector as a template, primers TAR-CloF1 and TAR-CloR1 were designed for amplification, yielding fragment E. Using the pXJ40 (No T7 pro) vector as a template, primers TAR-CloF2 and TAR-CloR2, with the same homologous arms as fragment E, were designed for amplification of fragment F. The PCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2. Fragments E and F were ligated using homologous recombination enzymes. The ligation reaction system is shown in Table 3, and the reaction conditions are shown in Table 4. The ligation product was transformed into *E. coli* DH5α competent cells using calcium chloride chemical transformation. An appropriate amount of the transformation product was then added to LB solid medium containing 100 μg / ml ampicillin. After plating culture, several single colonies were selected for PCR to verify successful cloning. The primers used in this PCR were the upstream primer TAR-CloF2 and the downstream primer pXJ40-R. Several single colonies were then selected for plasmid extraction. BglII digestion yielded two fragments of 886 bp and 3460 bp, which were selected as the target plasmids. To further verify plasmid success, positive plasmids were selected and sent to a company for sequencing verification. The positive plasmid pXJ40-TAR was confirmed, and its sequence is shown below. Figure 1B As shown.

[0091] TAR-CloF1: 5'-ATCCAAGCTTCTCGAGGCG-3';

[0092] TAR-CloR1: 5'-TCCCAGGCTCAGATCTGGTCTAACCAGAGAGACCCAGCTCTGCTTATATAGACCTCC-3';

[0093] TAR-CloF2: 5'-AGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCCGTTTAGTGAACCGT-3';

[0094] TAR-CloR2: 5'-CGCCTCGAGAAGCTTGGAT-3';

[0095] pXJ40-R: 5'-AGCGGAAGAGTCTAGAGTCG-3'.

[0096] Example 4: Construction of IBV S protein into cloning vector pXJ40-TAR

[0097] Viral RNA was extracted from cell samples infected with IBV virus (i.e., chicken infectious bronchitis virus (IBV), purchased from American Type Culture Collection, ATCC), which exhibited cell adaptation in our laboratory (first adapted and passaged 3 times in 10-day-old chicken embryos, then transferred to African green monkey kidney cells (Vero, purchased from American Type Culture Collection, ATCC) for 65 consecutive passages (p65) to obtain the adapted strain IBV-p65. In this experiment, Vero cells were infected with the adapted strain IBV-p65; when all cells exhibited cytopathic effects, the cells were lysed with Trizol, and the samples were collected). The RNA was reverse transcribed into cDNA. The reverse transcription system is shown in Table 5, and the reaction conditions are shown in Table 6. Primers with the same homologous arms as the vector pXJ40-TAR were designed. Using cDNA as a template, PCR amplification of the structural protein IBV S gene fragment was performed using primers IBVS-CloF and IBVS-CloR. The reaction system is shown in Table 1, and the reaction conditions are shown in Table 2. Using pXJ40 (No...)... Using T7pro as a template, the linear vector fragment was amplified using primers noT7-CloF3 and noT7-CloR1. The two fragments were then ligated using homologous recombination enzymes to obtain pXJ40-TAR-IBV S. The plasmid construction method was the same as in Example 1. The target protein map is shown below. Figure 1B As shown.

[0098] Table 5 Reverse transcription reaction system

[0099]

[0100] Table 6 Reverse transcription reaction conditions

[0101]

[0102] IBVS-CloF:

[0103] 5'-GGCAAAGAATTGGCGAATTCcaccATGTTGGTAACACCTCTTTTACTAGT-3';

[0104] IBVS-CloR: 5'-CGCCTCGAGAAGCTTTCAAACAGACTTTTTGGGTCTGTAT-3';

[0105] noT7-CloF3: 5'-AAGCTTCTCGAGGCGG-3'.

[0106] Example 5: Construction of GFP fragment into cloning vector pXJ40-TAR

[0107] Primers with homologous arms identical to those of the vector pXJ40-TAR were designed. The EGFP fragment synthesized by the company (as shown in SEQ ID NO. 6) was used as a template to amplify the EGFP fragment, with the upstream primer EGFP-CloF and the downstream primer EGFP-CloR. Using pXJ40 (No T7 pro) as a template, amplification was performed using primers noT7-CloF3 and noT7-CloR1 to obtain the linear vector fragment. The PCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2. The two fragments were homologously ligated using a homologous recombinase to obtain pXJ40-TAR-EGFP. The plasmid construction method was the same as in Example 1. The target protein map is shown below. Figure 1B As shown.

[0108] EGFP-CloF:

[0109] 5'-GGCAAAGAATTGGCGAATTCGGATCCATGGTGAGCAAGGGCGAGGA-3';

[0110] EGFP-CloR: 5'-CGCCTCGAGAAGCTTTACTTGTACAGCTCGTCCATGC-3'.

[0111] Example 6: Construction of plasmid pXJ40-TAR-GFP / IBV S-TAT

[0112] Primers with homologous arms identical to IRES and the vector pXJ40-TAR-GFP / IBV S were designed. The TAT fragment synthesized by the company (as shown in SEQ ID NO.3) was used as a template to amplify the TAT fragment. The upstream primer was IRES-TAT-CloF, and the downstream primer was IRES-TAT-CloR. Primers with homologous arms identical to the vector pXJ40-TAR-GFP / IBV S and TAT were also designed. The IRES fragment synthesized by the company (as shown in SEQ ID NO.4) was used as a template to amplify the IRES fragment. The upstream primer was IRES-CloF, and the downstream primer was IRES-CloR. The PCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2. The vector pXJ40-TAR-GFP / IBV S was digested into linear fragments by NotI and KpnI double digestion. The three fragments were homologously ligated using a homologous recombination enzyme to obtain pXJ40-TAR-GFP / IBV. The method for constructing the plasmid for S-TAT is the same as that in Example 1, and the target protein map is constructed as follows. Figure 1C As shown.

[0113] IRES-TAT-CloF:

[0114] 5'-CCACAAGGAGACGACCTTCATGGAGCCAGTAGATCCTAGAC-3';

[0115] IRES-TAT-CloR: 5'-GCTTTAATAAGATCTGGTACCCTATTCCTTCGGGCCTGTC-3';

[0116] IRES-CloF: 5'-AAGCTTCTCGAGGCGGCCGAAAAGTAAGGATCCCGCCCC-3';

[0117] IRES-CloR: 5'-GAAGGTCGCTCCTTGTGG-3'.

[0118] Example 7: Construction of plasmid pXJ40-TAR-GFP / IBV S-TAT-Ori(SV40)

[0119] Primers with homologous arms identical to those of the vector pXJ40-TAR-GFP / IBV S-TAT were designed. The SV40 Ori fragment was amplified using a synthesized SV40 virus replicon fragment (as shown in SEQ ID NO. 5) as a template. The upstream primer was SV40 Ori-CloF, and the downstream primer was SV40 Ori-CloR. The PCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2. The vector pXJ40-TAR-GFP / IBV S-TAT was digested into linear fragments using XbaI. The two fragments were then ligated using homologous recombination enzyme to obtain pXJ40-TAR-GFP / IBV S-TAT-Ori(SV40). The plasmid construction method was the same as in Example 1. The target protein map is shown below. Figure 1D As shown.

[0120] SV40 Ori-CloF: 5'-TGTCTGGTCGACTCTAGACTTTCCAGTCGGGAAACCT-3';

[0121] SV40 Ori-CloR: 5'-GCGGAAGAGTCTAGACGAGTCAGTGAGCGAGGAA-3'.

[0122] Example 8 describes plasmid expression using a transfection experiment to determine the optimal co-transfection ratio of pXJ40-TAT plasmid and the target protein pXJ40-TAR plasmid.

[0123] The pXJ40-TAT plasmid was mixed with pXJ40-TAR-EGFP and pXJ40-TAR-IBV S plasmids at mass ratios of 1:5 and 1:1, respectively. The pXJ40-GFP and pXJ40-IBV S plasmids were mixed with the control pXJ40 plasmid at the same mass ratios. According to the company's instructions, transfection reagent (Transfection EL Transfection Reagent, FT201-2, TransGold) was added to the plasmid mixture, and the mixture was allowed to stand for 20 minutes. Then, the plasmid transfection reagent mixture was added dropwise to the previously seeded human kidney epithelial cell line 293T (American Type Culture Collection, ATCC). After 24 hours of transfection, images were taken using an inverted fluorescence microscope to preliminarily determine the expression of the target protein under different transfection ratios. Figure 2A As shown, protein samples were collected.

[0124] Example 9: Detection of protein expression by Western blotting

[0125] After initially assessing plasmid expression using an inverted fluorescence microscope, Western blotting was used to examine protein expression at different mass ratios to determine the optimal plasmid ratio for protein expression. The specific steps were as follows: Protein samples collected in Example 8 were taken and boiled in a 95°C metal bath for 10 minutes. Equal volumes of each protein sample were subjected to SDS-PAGE gel electrophoresis. The proteins were then transferred to an NC membrane using a membrane transfer assay. The NC membrane was then blocked with 5% skim milk powder. After removing the blocking solution, diluted primary antibody was added according to experimental requirements. After primary antibody incubation, a corresponding fluorescent secondary antibody was added. After incubation at room temperature, the NC membrane was washed and scanned using a membrane scanner to obtain the target protein. Figure 2B As shown, the optimal expression of the target protein was achieved when the pXJ40-TAT plasmid and the pXJ40-TAR plasmid expressing the target protein were transfected at a mass ratio of 1:5.

[0126] Example 10: Plasmid expression was performed using a transfection experiment. The effects of three expressed proteins in the TAR-TAT-Ori(SV40) mammalian high-efficiency expression system were compared, and pXJ40-TAR-TAT-Ori(SV40) showed the best expression effect.

[0127] The pXJ40-TAT plasmid was mixed with pXJ40-TAR-EGFP and pXJ40-TAR-IBV S plasmids at a mass ratio of 1:5. Plasmids pXJ40-GFP, pXJ40-IBV S, pXJ40-TAR-GFP-TAT, pXJ40-TAR-IBV S-TAT, pXJ40-TAR-GFP-TAT-Ori(SV40), and pXJ40-TAR-IBV S-TAT-Ori(SV40) were transfected with the control pXJ40 plasmid at the same mass ratio as described above to express the target protein. The expression effects of the three protein expression methods were detected using an inverted fluorescence microscope, Western blotting, and Coomassie brilliant blue assay. The inverted fluorescence microscope and Western blotting methods were the same as in Examples 8 and 9. The results are as follows. Figure 3A , Figure 3B , Figure 4 As shown, the specific steps of the Coomassie Brilliant Blue assay are as follows: The protein samples collected in Example 8 are taken out and boiled in a metal bath at 95°C for 10 minutes. An equal volume of each protein sample is then subjected to protein electrophoresis in an SDS-PAGE gel. After electrophoresis, the gel is transferred to a container, rinsed with deionized water, and BeyoBlue is added according to the company's instructions. TM Coomassie Brilliant Blue Ultrafast Staining Solution (P0017F, Beyotime) is used for staining for 10-60 minutes. After repeated replacement with deionized water, a decolorization experiment is performed to obtain an almost colorless and transparent background, which is then ready for protein detection.

[0128] Figures 2 and 3 Figure 4 The results show that the method of expressing proteins in eukaryotic cells using the HIV-1 virus RNA sequence TAR and its transcription activator TAT, as well as the mammalian cell high-efficiency expression vector constructed by simian virus SV40 Ori, is feasible and effective, among which pXJ40-TAR-TAT-Ori(SV40) showed the best expression effect.

[0129] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An expression vector for a target protein, characterized in that, The expression vector is the recombinant vector pXJ40-TAR-TAT-Ori (SV40). The method for preparing the recombinant vector pXJ40-TAR-TAT-Ori (SV40) is as follows: the replicon of the SV40 virus is constructed into the recombinant vector pXJ40-TAR-TAT to obtain the recombinant vector pXJ40-TAR-TAT-Ori (SV40). The recombinant vector pXJ40-TAR-TAT is prepared by constructing transcription activator TAT and IRES into the recombinant vector pXJ40-TAR to obtain the recombinant vector pXJ40-TAR-TAT. The recombinant vector pXJ40-TAR is prepared by constructing the cDNA sequence of the target protein into the pXJ40 vector containing the TAR fragment and removing the T7 promoter to obtain the recombinant vector pXJ40-TAR. The sequence of the pXJ40 vector with the T7 promoter removed is shown in SEQ ID NO.1; The sequence of the transcription activator TAT is shown in SEQ ID NO.2; The sequence of the TAR fragment is shown in SEQ ID NO.3; The sequence of the internal ribosome entry site IRES is shown in SEQ ID NO.4; The sequence of the SV40 virus replicon is shown in SEQ ID NO.

5.

2. The expression vector for the target protein according to claim 1, characterized in that, The preparation method of the pXJ40 vector with the T7 promoter removed is as follows: using the pXJ40 vector as a template, amplification is performed using primers noT7-CloF1 and noT7-CloR1 to obtain fragment A; Using the pXJ40 vector as a template, fragment B was obtained by amplification with primers noT7-CloF2 and noT7-CloR2; Fragment A and fragment B were homologously ligated by homologous recombination enzyme to obtain the pXJ40 vector with the T7 promoter removed. The sequence of the primer noT7-CloF1 is shown in SEQ ID NO.6; The sequence of the primer noT7-CloR1 is shown in SEQ ID NO.7; The sequence of the primer noT7-CloF2 is shown in SEQ ID NO.8; The sequence of the primer noT7-CloR2 is shown in SEQ ID NO.

9.

3. The expression vector for the target protein according to claim 1, characterized in that, The specific preparation method of the recombinant vector pXJ40-TAR is as follows: using the pXJ40 vector with the T7 promoter removed as a template, fragment E is obtained by amplification using primers TAR-CloF1 and TAR-CloR1; using the pXJ40 vector with the T7 promoter removed as a template, fragment F is obtained by amplification using primers TAR-CloF2 and TAR-CloR2; fragment E and fragment F are homologously ligated by homologous recombination enzyme to obtain the recombinant vector pXJ40-TAR. The sequence of primer TAR-CloF1 is shown in SEQ ID NO.12; The sequence of primer TAR-CloR1 is shown in SEQ ID NO.13; The sequence of primer TAR-CloF2 is shown in SEQ ID NO.14; The sequence of primer TAR-CloR2 is shown in SEQ ID NO.

15.

4. A method for expressing a target protein, characterized in that, The expression vector as described in claim 1 is transfected into host eukaryotic cells for expression to obtain the target protein.

5. The method for expressing the target protein according to claim 4, characterized in that, The target protein is a fluorescent protein or a viral protein.

6. The method for expressing the target protein according to claim 5, characterized in that, The viral protein is a viral structural protein; the fluorescent protein is a green fluorescent protein.

7. The method for expressing the target protein according to claim 6, characterized in that, The viral structural protein mentioned is the IBVS structural protein.

8. The method for expressing the target protein according to claim 4, characterized in that, The host eukaryotic cells are human kidney epithelial cell line 293T.

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