Construction and use of Tac promoter-based plasmid expression vectors

Abstract

The present invention relates to a method for modifying and constructing a plasmid expression vector based on the Tac promoter, in which the SD sequence has been replaced and the tag sequence has been knocked out, and to the use of the expression vector constructed in this way in the expression of recombinant foreign proteins, particularly in the expression of human papillomavirus L1 antigen protein and the use of the L1 antigen protein expression product in the prevention of cervical cancer.

Description

[Technical Field]

[0001] The present invention relates to a method for modifying and constructing a Tac promoter-based plasmid expression vector, and the use of the expression vector constructed by this method in expressing recombinant proteins, particularly the expression of human papillomavirus L1 antigen protein and the use of the expression product of the L1 antigen protein in the prevention of cervical cancer. [Background technology]

[0002] Plasmid vectors have a relatively small relative molecular mass and are independent of circular DNA other than chromosomal DNA (generally 1-200 kb in size, where kb equals 1,000 base pairs). A given bacterium may have one plasmid vector, and a given bacterium may have multiple plasmids. Plasmids can be transferred from one bacterium to another by bacterial conjugation, and they can replicate independently or can be integrated into bacterial chromosomal DNA and replicate along with the replication of the chromosomal DNA.

[0003] Escherichia coli (commonly known as E. coli) expression vectors are most commonly plasmid vectors. As an expression vector, it must first meet the basic requirements of a cloning vector, i.e., be able to carry foreign genes into E. coli cells. An expression vector is constructed by cloning the basic vector backbone and then adding expression elements. The differences between various expression vectors are often reflected in the differences in their expression elements. Plasmid expression vectors for E. coli mainly include expression vectors for expressing fusion proteins and expression vectors for non-fusion proteins.

[0004] A fusion protein expression vector is a vector that expresses a target protein in the form of a fusion protein, allowing for isolation and purification by utilizing the special properties of the protein or polypeptide encoded by the vector. The fusion protein portion expressed together with the target protein is also called a tag protein or tag polypeptide (tag), and commonly includes glutathione S-transferase (GST), hexapolyhistidine peptide (poly His-6), protein A, and maltose binding protein (MBP).

[0005] One of the key issues in the vector construction process for fusion protein expression is the linker sequence (i.e., the connecting peptide) between the two proteins, and its length is crucial for protein folding and stability. A linker sequence that is too short may affect the high-level structural folding of the two proteins, resulting in interference and failure to form the correct spatial structure. A linker sequence that is too long may lead to immunogenicity issues, as the linker sequence itself may represent a new antigen. Furthermore, the use of fusion protein expression in protein drug or vaccine products may introduce new foreign proteins or polypeptides (e.g., tag proteins or residual cleavage enzymes), potentially increasing drug safety risks.

[0006] The fusion protein expression vector pGEX was constructed by Smith and Johnson in 1987. It contains a 26 kDa glutathione sulfotransferase (GST) gene. Compared to other fusion vectors, it has milder purification conditions, simpler steps, no need for denaturants, and maximizes the maintenance of the spatial conformation and immunogenicity of the purified protein. This makes it highly valuable for practical use. However, the GST fusion protein tag encoded by the pGEX vector may increase the potential safety risks of medicinal protein products.

[0007] Key regulatory elements of prokaryotic expression vectors include promoters, SD sequences, and transcription terminators. A promoter is a sequence on a DNA strand that allows RNA polymerase to bind and synthesize initiator RNA. It is an essential regulatory sequence for gene expression. Without a promoter, a gene cannot be transcribed. Because bacterial RNA polymerase cannot recognize eukaryotic gene promoters, promoters used in prokaryotic expression vectors must be prokaryotic. Regulatory promoters commonly used in prokaryotic expression systems include Lac (lactose promoter), Trp (tryptophan promoter), Tac (heterozygous promoter for lactose and tryptophan), lPL (l phage leftward promoter), and the T7 phage promoter.

[0008] The promoter of the pGEX fusion protein expression vector is the Tac promoter. This Tac promoter is a heterologous promoter constructed artificially from the Lac and Trp promoters. It is negatively regulated by the Lac repressor protein and has a stronger activation ability (higher expression levels) than Lac and Trp. Tac1 consists of a 46-bp DNA fragment (including a Pribnow box) integrated into the -35 region of the Trp promoter and an operon gene in the Lac operon. Tac2 consists of the -35 region of the Trp promoter and the -10 region of the Lac promoter fused with an operon gene portion and an SD sequence in the Lac operon. The Tac promoter is induced by IPTG. Compared to the T7 promoter, the Tac promoter has moderate transcription strength, slower protein expression rate, and slower protein folding rate, potentially resulting in better solubility of the expressed target protein.

[0009] The Shine-Dalgarno sequence (SD sequence), first discovered by Shine and Dalgarno in 1974, is a ribosome-binding site in mRNA. It is a sequence of approximately 3-9 bp located 3-10 bp upstream of the AUG sequence. This sequence is rich in purine nucleotides and is complementary to the pyrimidine-rich sequence at the 3' end of 16S rRNA, serving as the recognition and binding site for ribosomal RNA. Subsequently, people named this sequence the Shine-Dalgarno sequence, abbreviated as SD sequence. The distance between different SD sequences and the AUG initiation codon is an important factor affecting mRNA transcription and translation into protein. The binding of a protein to the SD sequence affects the binding of mRNA to ribosomes, which in turn affects protein translation and, therefore, the expression level of the recombinant foreign gene.

[0010] The pGEX vector is a commercially available vector from Universal Genetics (GE) in the United States, and its promoter is the Tac promoter and its SD sequence is AGGAAACAGTA.

[0011] Typically, the HPV-L1 protein, a human papillomavirus (HPV) vaccine antigen, is difficult to efficiently express as soluble HPV-L1 protein in Escherichia coli, and is prone to forming inclusion bodies during recombinant expression. Alternatively, the protein can be purified from inclusion bodies and conjugated to obtain HPV VLPs (Kelsall, SR and JK Kulski. Expression of the major capsid protein of human papillomavirus type 16 in Escherichia coli. 1995. J Virol Methods 53(1):75-90). However, the conjugation process results in significant protein loss and low yield, making large-scale production difficult. HPV L1 protein can also be expressed in a soluble form in Escherichia coli with the correct conformation and dissolved in the cell digestion supernatant. However, the expression level is low, and the supernatant contains a large number of heterologous proteins, making it extremely difficult to purify the target protein from them. The GST fusion expression method can increase the expression level of L1 protein in the supernatant and facilitate the purification of the target protein. As described in Chinese patent application CN201410683185, the fusion protein pGEX expression vector can efficiently express human papillomavirus L1 protein, and the tag protein can be removed and purified to obtain the L1 pentamer, which can be directly used to produce human papillomavirus pentamer vaccines. After highly purified, the pentamer can be assembled in vitro to form virus-like particles (VLPs), which can then be used to prepare adult human papillomavirus VLP vaccines.

[0012] However, cleavage of the fusion protein often requires expensive enzymes, and the introduction of new foreign proteins or polypeptides (tag proteins or residual proteases) may increase safety risks. Therefore, there remains a need in the art for HPV virus-like particle (VLP) vaccine production technologies with lower cost, higher yield, and better safety. Summary of the Invention

[0013] The inventors' unexpected findings revealed that the SD sequence inherent in pGEX plasmid vectors does not allow for efficient, non-fusion, soluble expression of foreign genes such as HPV16L1 or HPV18L1, and even if expression does occur, the expression levels are low (Figure 2). By modifying the SD sequence based on the pGEX plasmid vector, new non-fusion SD sequence expression vectors were constructed, which enabled efficient expression of soluble foreign proteins after transformation of E. coli. In particular, the expression level and yield of the human papillomavirus L1 antigen protein was 5-10 times higher than that of fusion protein expression techniques (Figure 3), enabling large-scale industrial production of cervical cancer vaccines. The present invention is based on the above findings.

[0014] Therefore, a first aspect of the present invention relates to the modification of expression vectors, particularly plasmid expression vectors based on the Tac promoter. More specifically, the promoter of the pGEX series products is Tac. The pGEX series products include pGEX-1λT, pGEX-2T, pGEX-2TK, pGEX-3X, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pGEX-6P-1, pGEX-6P-2, pGEX-6P-3, and recombinant vectors modified from the above pGEX series products. These vectors are obtained by deleting the GST fusion protein coding sequence in the pGEX vector and modifying the SD sequence. The resulting pKL1 expression vector is shown in Figure 1.

[0015] The pGEX vectors of the present invention include pGEX series products, such as pGEX-1λT, pGEX-2T, pGEX-2TK, pGEX-3X, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pGEX-6P-1, pGEX-6P-2, pGEX-6P-3, and recombinant vectors modified from the above pGEX series products.

[0016] In the present invention, the original SD sequence of the pGEX vector is AGGAAACAGTA.

[0017] In one example, the SD sequence of the pGEX-6P-2 vector was converted to AGGAGATATA, and the resulting vector was designated pKL1; the SD sequence of the pGEX-4T-1 vector was converted to AGGAAACAGTA, and the resulting vector was designated pKL10; the SD sequence of the pGEX-2TK vector was converted to AGGAGGAATAA, and the resulting vector was designated pKL20; and the SD sequence of the pGEX-5X-2 vector was converted to AGGAGGAATTA, and the resulting vector was designated pKL30.

[0018] In our research, we also attempted to modify the vector in other ways. For example, the modified SD sequence was AGGAAACAGCT, which was named pEZSeq-Kan; the modified SD sequence was AGGAAGCTAAA, which was named pDNR-Dual; the modified SD sequence was AGGAAGACT, which was named pBR322; the modified SD sequence was AGGAGATATACAT, which was named pRSET A; the modified SD sequence was AGGGGGTGTT, which was named pSTBlue; and the modified SD sequence was AGGAGGTTGT, which was named pEX3. However, the results were unsatisfactory, and these attempts were discontinued.

[0019] A second aspect of the present invention relates to a recombinant plasmid, which comprises the expression vector and a foreign gene encoding a foreign protein inserted into the expression vector, and the foreign gene may be a gene encoding an antigen protein or polypeptide of a microorganism such as a virus or bacterium, or a gene encoding a tumor antigen. For example, the foreign gene may be a DNA sequence of human papillomavirus L1 protein (HPVL1), a gene encoding human rhinovirus 3C protease (HPV 3C; abbreviated as 3C enzyme), a human enterovirus type 71 (EV71) capsid protein gene, or a gene encoding norovirus (NoV) capsid protein.

[0020] A third aspect of the present invention relates to an expression method, which comprises a host cell and the expression vector of the present invention or the recombinant plasmid of the present invention, wherein the host cell is Escherichia coli, including, but not limited to, DH5α, GI698, ER2566, BL21(DE3), XA90, B834(DE3), or BLR(DE3).

[0021] A fourth aspect of the present invention relates to a method for constructing an expression vector, which is a genetic recombination method and includes the following steps: S1: Design mutagenic primers based on the SD sequence. S2: Mutation PCR is performed using the pGEX vector as a template to introduce an NdeI cleavage site into the original vector. S3: The GST gene sequence is removed by double digestion with NdeI / BamHI enzymes. S4: Fill in the sticky ends with the Klenow fragment of DNA polymerase I (i.e., large (Klenow) fragment). The Klenow fragment is a C-terminal fragment of DNA polymerase I. S5: Ligated with T4 DNA ligase to obtain a closed circular plasmid. S6: Primers are designed and PCR is performed to newly introduce the original cleavage site into the plasmid. S7: Expand and propagate clones with the correct sequence, preserve the strain, extract the plasmid, preserve the plasmid, and obtain the vector of the present invention.

[0022] A fifth aspect of the present invention relates to the use of the novel SD expression vectors of the present invention for the highly efficient expression of multiple pharmaceutical recombinant proteins, including the L1 proteins and virus-like particles (VLPs) of human papillomavirus vaccine antigens HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68, human rhinovirus 3C protease, human enterovirus type 71 (EV71) capsid protein and VLPs, norovirus (NoV) capsid protein and VLPs, poliovirus capsid protein and VLPs, hepatitis A virus (HAV) capsid protein and VLPs, and hepatitis B virus (HBV) capsid protein and VLPs. Prior to the implementation of the present invention, these foreign proteins could not be solublely expressed in E. coli cells or the soluble expression levels were very low.

[0023] The beneficial effects of the present invention are as follows: Among prokaryotic expression systems, the E. coli expression system has the advantages of low culture costs and high expression yields. However, HPVL1 protein expressed in E. coli expression systems usually loses its correct native conformation and precipitates as inclusion bodies. Currently, restoring proteins expressed in inclusion bodies remains a global challenge. Due to the difficulty of complexation and low efficiency, it is difficult to produce VLPs with the correct conformation from inclusion bodies in large-scale production, limiting it to small-scale laboratory research. HPVL1 can also be expressed in a soluble form with the correct conformation in E. coli digestion supernatant, but the expression yield is low and purifying HPVL1 protein from E. coli digestion supernatant is very difficult. Purification often requires methods such as fusion expression and affinity chromatography, which often require expensive enzymes and poses drug safety concerns.

[0024] The present invention utilizes a novel SD plasmid expression vector to efficiently express HPV L1 protein in an E. coli expression system. The purification method employed does not require the use of expensive enzymes and is unrelated to tag proteins, eliminating potential drug safety risks associated with tag proteins. The highly purified HPV L1 protein obtained through further purification assembles into virus-like particles, exhibiting excellent immunogenicity and capable of inducing high-titer neutralizing antibodies against HPV, making it an effective vaccine for preventing HPV infection in humans.

[0025] Furthermore, the present inventors unexpectedly discovered that the novel SD sequence expression vector constructed by the above-described technique, when transformed into Escherichia coli, can express human papillomavirus L1 antigen protein at levels and yields 5 to 10 times higher than those achieved by fusion protein expression techniques, thereby enabling low-cost, large-scale industrial production of cervical cancer vaccines, such as human papillomavirus vaccine L1 and VLP antigen proteins for preparing HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68.

[0026] Furthermore, the novel SD plasmid expression vector constructed in this invention can be used in conjunction with appropriate host cells to achieve efficient soluble expression of various foreign proteins, including the expression of human rhinovirus 3C protease, human enterovirus type 71 (EV71) capsid protein and VLP, norovirus (NoV) capsid protein and VLP, poliovirus capsid protein and VLP, hepatitis A virus (HAV) capsid protein and VLP, and hepatitis B virus (HBV) capsid protein and VLP.

[0027] These and other inventive and novel features of the present invention will become apparent upon reference to the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0028] [Figure 1] Schematic diagram of the expression vector pKL1. The schematic diagrams of the expression vectors pKL10, pKL20, and pKL30 are similar to pKL1. [Figure 2] Electrophoretic detection of HPV16 L1 protein expression before and after SD sequence substitution. No L1 protein expression was observed. Shown are the results of two experiments. [Figure 3] Electrophoretic detection of HPV16 L1 protein expression after substitution and modification of the SD sequence. L1 protein was expressed in large amounts. Shown are the results of two experiments. [Figure 4] FIG. 10 shows the results of electrophoretic detection of large-scale soluble expression of HRV 3C protease in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides expression vectors containing all the expression elements that can direct non-fusion protein expression for a variety of foreign genes.

[0030] Example 1: Construction of pKL series vectors 1.1 Introduce an NdeI cleavage site into the pGEX-6P-2 plasmid by mutagenic PCR. The names and sequences of the PCR primers are as follows: Forward primer: 6p1-NdeImut-F (5'to3') ATTTCA CACAGG AAACAG TACATA TGTCCC CTATAC TAGGTT ATTGGAAAATTA AG Reverse primer: 6p1-NdeImut-R sequence (5' to 3') ATAACC TAGTAT AGGGGA CATATG TACTGT TTCCTG TGTGAA ATTGTT ATCC

[0031] PCR reaction system: 5×phusion HF buffer 10μL, ddH2O 30.5μL, 10mM dNTP 2μL, 6PNE-SDm-F 1μL, 6PNE-SDm-R 1μL, pGEX-6P-2 (20 times diluted) 5μL, Phusion HF Enzyme 0.5μL.

[0032] PCR reaction process: 95℃ 3 min; 95℃ 1 min, 55℃ 1 min, 72℃ 10 min; 20 cycles; 72℃ 15 min.

[0033] The PCR product was digested with DpnI and transformed into E. coli DH5α. After overnight incubation, monoclonal colonies were obtained. The monoclonal colonies were expanded and then the vector sequences within them were sequenced by a specialized gene sequencing company. The clones with correct sequencing results were selected and then expanded. The plasmid was extracted, and a vector with the NdeI cleavage site successfully introduced was obtained.

[0034] 1.2 Design mutation PCR primers to replace the SD sequence, and replace the SD sequence of the original vector by PCR. Primer Information: 6PNE-SDm-F(5'to3'):CAATTTCACACAGGAGATATACATATGTCCCCTATACTAGG 6PNE-SDm-R(5'to3'):GTATAGGGGACATATGTATATCTCCTGTGTGAAATTGTTATCC PCR reaction system: 10 μL of 5x Phusion HF buffer, 30.5 μL of ddH2O, 2 μL of 10 mM dNTP, 1 μL of 6PNE-SDm-F, 1 μL of 6PNE-SDm-R, 5 μL of the plasmid obtained in step 1.1, and 0.5 μL of Phusion HF Enzyme. PCR reaction process: 95℃ 3 min; 95℃ 1 min, 55℃ 1 min, 72℃ 10 min; 20 cycles; 72℃ 15 min.

[0035] The PCR product was digested with DpnI to form template DNA, which was then transformed into E. coli DH5α. After overnight incubation, monoclonal colonies were obtained. The monoclonal colonies were expanded and then sequenced by a specialized gene sequencing company. Correct clones were selected and propagated, and the plasmid was extracted to obtain a vector with the SD sequence successfully replaced.

[0036] 1.3 The vector is double digested with NdeI and BamHI to remove the GST gene. Enzyme digestion system: Cutsmart buffer 3 μl, ddH2O 3 μl, vector obtained in step 1.2 20 μl, NdeI 2 μl, BamHI 2 μl.

[0037] Enzymatic digestion at 37°C for 2 hours; 0.8% agarose gel electrophoresis at 120V for 1 hour; gel excision to obtain the electrophoretic band corresponding to the vector fragment from which the GST gene had been removed, and storage at 4°C.

[0038] The vector fragment was recovered using an agarose gel recovery kit, and 3 μl of the resulting vector fragment was electrophoresed to detect the recovery results. The sticky ends of the double-cleaved enzyme product were then supplemented with DNA polymerase I. The reaction mixture consisted of 2.5 μl of 10X T4 DNA Ligase buffer, 1.8 μl of ddH2O, 20 μl of the enzyme-cleaved vector fragment recovered from the gel, 0.2 μl of 10 mM dNTPs, and 0.5 μl of DNA polymerase I. The reaction was then incubated at 25°C for 15 minutes, followed by the addition of EDTA (final EDTA concentration: 10 mM) and heating to 75°C for 20 minutes to terminate the reaction.

[0039] The enzyme-digested vector with the completed ends was religated and circularized. Ligation system: 2 μl of T4 DNA Ligase buffer, 16 μl of linearized blunt-ended vector fragment, and 2 μl of T4 DNA ligase. Ligation was carried out at 16°C for 4 hours.

[0040] The ligation product was digested and transformed into E. coli DH5α. After overnight incubation, monoclonal colonies were obtained. The monoclonal colonies were expanded and then the vector sequences within them were sequenced by a specialized gene sequencing company. Correct clones were selected and then expanded. The plasmids were extracted and the SD sequence was successfully replaced to obtain vectors that removed the GST gene.

[0041] In the PCR amplified plasmid, NdeI and BamHI cleavage sites were newly introduced.

[0042] PCR primers: 6PNE-SDm-noG-F(5'to3'):CAGGAGATATACATATGGGATCCCCGGAATTCCCG 6PNE-SDm-noG-R(5'to3'):GAATTCCGGGGATCCCATATGTATATCTCCTGTGTG PCR reaction system: 5x Phusion HF buffer 10 μL, ddH2O 30.5 μL, 10 mM dNTP 2 μL, 6PNE-SDm-noG-F 1 μL, 6PNE-SDm-noG-R 1 μL, template plasmid 5 μL, Phusion HF Enzyme 0.5 μL. PCR reaction process: 95℃ 3 min; 95℃ 1 min, 55℃ 1 min, 72℃ 10 min; 20 cycles; 72℃ 15 min.

[0043] The PCR product was digested with DpnI to form template DNA, which was then transformed into E. coli DH5α and grown overnight to yield monoclonal colonies. The monoclonal colonies were expanded and then sequenced by a specialized gene sequencing company. Correct clones were selected and propagated, and the plasmid was extracted to obtain a vector in which the SD sequence had been successfully replaced, the GST gene had been removed, and NdeI and BamHI had been reintroduced. This completed the construction of vector pKL1.

[0044] Referring to the above experimental steps, pGEX-2TK was used as the starting vector instead of pGEX-6P-2 in the above experimental steps, and pKL20 was constructed as a new SD sequence substituting SEQ ID NO: 3. The SD sequence mutation primers were designed as follows: Forward primer (5' to 3'): CAATTTCACACAGGAAGGAGGAATAACATATGCCGTCTGAAGCTAC Reverse primer (5'to3'): GACGGCATATGTTATTCCTCCTTCCTGTGTGAAATTGTTATCC Other than the starting vector and SD sequence mutation primers, other steps and methods of vector construction are similar to the construction of pKL1.

[0045] Referring to the above experimental steps, pKL30 was constructed using pGEX-5X-2 as the starting vector and a new SD sequence substituted for SEQ ID NO: 4. The SD sequence mutation primers were designed as follows: Forward primer (5' to 3'): CAATTTCACACAGGA AGGAGGAATTA CATATGCCGTCTGAAGCTAC Reverse primer (5'to3'): GACGGCATATGTAATTCCTCCT TCCTGTGTGAAATTGTTATCC Other than the starting vector and SD sequence mutation primers, other steps and methods of vector construction are similar to the construction of pKL1.

[0046] Referring to the above experimental steps, pGEX-4T-1 was used as the starting vector to construct pKL10 as a new SD sequence replacing SEQ ID NO: 2. The SD sequence mutation primers were designed as follows: Forward primer (5' to 3'): CAATTTCACACAGGA AGGAAACAGTA CATATGCCGTCTGAAGCTAC Reverse primer (5'to3'): GACGGCATATGTACTGTTTCCT TCCTGTGTGAAATTGTTATCC Other than the starting vector and SD sequence mutation primers, other steps and methods of vector construction are similar to the construction of pKL1.

[0047] Thus, expression vectors containing four different SD sequences (e.g., elimination of tags) were successfully constructed: the SD sequence in vector pKL1 was AGGAGATATA, the SD sequence in pKL10 was AGGAAACAGTA, the SD sequence in pKL20 was AGGAGGAATAA, and the SD sequence in pKL30 was AGGAGGAATTA.

[0048] Example 2: Construction of various HPV L1 proteins based on vectors pKL1 and pKL30 The capsid protein L1 genes of the following types of human papillomavirus were artificially synthesized, and the sequences are shown in the sequence listing. The names corresponding to the specific sequence numbers are as follows: The sequence shown in SEQ ID NO: 1 is an HPV6L1 N / C-terminal truncation (469 aa, Theoretical pI / Mw: 5.94 / 52079.77 Da, the N-terminus is truncated by 2 amino acids and the C-terminus is truncated by 29 amino acids). The sequence shown in SEQ ID NO: 2 is HPV11L1 N / C-terminal truncation (470 aa, Theoretical pI / Mw: 6.08 / 52274.02 Da, the N-terminus is truncated by 3 amino acids and the C-terminus is truncated by 29 amino acids). The sequence shown in SEQ ID NO: 3 is HPV16L1 N / C-terminal truncation (472 aa, Theoretical pI / Mw: 6.08 / 52548.71 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 29 amino acids). The sequence shown in SEQ ID NO: 4 is HPV18L1 N / C-terminal truncation (473 aa, Theoretical pI / Mw: 5.94 / 52722.42 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 30 amino acids). The sequence shown in SEQ ID NO: 5 is HPV31L1 N / C-terminal truncation (473 aa, Theoretical pI / Mw: 6.27 / 52869.89 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 27 amino acids). The sequence shown in SEQ ID NO: 6 is an HPV33L1 N / C-terminal truncation (471 aa, Theoretical pI / Mw: 5.88 / 52765.94 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 24 amino acids). The sequence shown in SEQ ID NO: 7 is HPV35L1 N / C-terminal truncation (470 aa, Theoretical pI / Mw: 6.10 / 52494.45 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 28 amino acids). The sequence shown in SEQ ID NO: 8 is HPV39L1N9 N / C-terminal truncation (467 aa, Theoretical pI / Mw: 6.16 / 52393.22 Da, the N-terminus is truncated by 9 amino acids and the C-terminus is truncated by 29 amino acids). The sequence shown in SEQ ID NO: 9 is HPV45L1 N / C-terminal truncation (476 aa, Theoretical pI / Mw: 5.94 / 53244.30 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 30 amino acids). The sequence shown in SEQ ID NO: 10 is an HPV51L1t N / C-terminal truncation (472 aa, Theoretical pI / Mw: 5.88 / 52769.56 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 28 amino acids). The sequence shown in SEQ ID NO: 11 is HPV52L1 N / C-terminal truncation (476 aa, Theoretical pI / Mw: 5.88 / 53176.26 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 23 amino acids). The sequence shown in SEQ ID NO: 12 is HPV56L1t N / C-terminal truncation (470 aa, Theoretical pI / Mw: 6.11 / 52796.70 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 25 amino acids). The sequence shown in SEQ ID NO: 13 is HPV58L1 N / C-terminal truncation (471 aa, Theoretical pI / Mw: 5.80 / 52994.97 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 23 amino acids). The sequence shown in SEQ ID NO: 14 is HPV59L1 N / C-terminal truncation (473 aa, Theoretical pI / Mw: 6.08 / 52891.89 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 31 amino acids). The sequence shown in SEQ ID NO: 15 is HPV68L1a N / C-terminal truncation (473 aa, Theoretical pI / Mw: 5.75 / 53032.99 Da, the N-terminus is truncated by 4 amino acids and the C-terminus is truncated by 28 amino acids).

[0049] First, the DNA fragment of HPV L1 was amplified by PCR, and the primer information used is shown in Table 1 below.

[0050] Table 1: Primer information [Table 1]

[0051] The PCR fragment of the L1 gene containing NdeI and XhoI sites and the recombinant vector pKL1 or pKL30 were each double-cleaved with NdeI and XhoI enzymes, and the recovered gene fragment was then ligated with pKL1 or pKL30 containing the corresponding sticky ends using T4 DNA ligase (16°C, 10-15 hours). Ligation system: 6 μl of pKL1 or pKL30 vector fragment, 2 μl of L1 gene fragment, 1 μl of T4 DNA ligase, 1 μl of T4 DNA ligase buffer.

[0052] After the ligation reaction, the ligation product was transformed into E. coli DH5α and recombinants were screened. The screened monoclonal colonies were expanded and the plasmids were extracted. After sequencing, the recombinant expression vectors pKL1-HPVL1 and pKL30-HPVL1 were obtained.

[0053] The recombinant vector with correct sequencing results was transformed into E. coli XA90 host cells to produce the HPVL1 protein. The activation medium for the engineered strain was LB medium (10 g / L tryptone; 5 g / L yeast powder; 10 g / L NaCl), and the expression medium was 2YT medium (16 g / L tryptone; 10 g / L yeast powder; 5 g / L NaCl).

[0054] The engineered strain was inoculated into LB medium (Amp+) at a 0.05% inoculum concentration and cultured at 37°C and 220 rpm for 16 hours for activation. The activated bacterial solution was inoculated into 2YT medium at a 0.5% inoculum concentration and cultured at 30°C and 220 rpm for 7 hours. After that, IPTG was added to a final concentration of 0.2 mM and cultured at 30°C and 220 rpm for 16 hours for induction. After fermentation was terminated, the cells were collected by centrifugation for expression level detection and purification experiments.

[0055] Using HPV16 L1 protein expression as an example, Figure 2 shows the electrophoretic detection results for HPV16 L1 protein expression before the SD sequence substitution modification (no L1 protein expression observed), and Figure 3 shows the electrophoretic detection results for HPV16 L1 protein expression after the SD sequence substitution modification (high L1 protein expression). Quantified protein expression is shown in the table below.

[0056] Table 2: L1 protein expression levels before and after vector modification [Table 2]

[0057] As can be seen from the results, the vector showed little expression before modification, while the protein expression level of the modified vector reached at least 0.2 mg / g wet cells, and could reach a maximum of 6.77 mg / g wet cells.

[0058] Example 3: Expression of HPV16 L1 antigen protein using different pKL series vectors, the HPV16L1 expression vector The recombinant vector-HPV16L1 with correct sequencing results from Example 2 was transformed into E. coli XA90 host cells, and the resulting engineered strain was used to express the HPVL1 protein. A 0.05% inoculum was inoculated into LB medium (Amp+) and cultured at 37°C and 220 rpm for 16 hours for activation. The activated bacterial solution was inoculated into 2YT medium at a 0.5% inoculum and cultured at 30°C and 220 rpm for 7 hours. IPTG was then added to a final concentration of 0.2 mM, and induction culture was continued at 30°C and 220 rpm for 16 hours. Fermentation was then terminated, and the cells were collected by centrifugation for expression level detection and purification experiments.

[0059] The results are shown in Table 3. As can be seen from the results, two of the SD sequences showed a large amount of expression, while the other two SD sequences showed almost no expression.

[0060] Table 3: Comparison of target protein expression levels by HPV16L1 engineered strains containing different SD sequences. [Table 3]

[0061] Example 4: Expression of HPV59 L1 antigen protein using different pKL series vectors, the HPV59L1 expression vector The recombinant vector HPV59L1, which showed correct sequencing results in Example 2, was transformed into E. coli XA90 host cells, and the resulting engineered strain was used to express the HPVL1 protein. A 0.05% inoculum was inoculated into LB medium (Amp+) and cultured at 37°C and 220 rpm for 16 hours for activation. The activated bacterial solution was inoculated into 2YT medium at a 0.5% inoculum and cultured at 30°C and 220 rpm for 7 hours. IPTG was then added to a final concentration of 0.2 mM, and induction culture was continued at 30°C and 220 rpm for 16 hours. Fermentation was then terminated, and the cells were collected by centrifugation for expression level detection and purification experiments.

[0062] The results are shown in Table 4. As can be seen from the results, one of the SD sequences showed a large amount of expression, while the other three SD sequences showed almost no expression.

[0063] Table 4: Comparison of target protein expression levels by HPV59L1 engineered strains containing different SD sequences. [Table 4]

[0064] Example 5: Expression of HPV68L1 antigen protein using different pKL series vectors, HPV68L1 expression vectors The recombinant vector-HPV68L1, which showed correct sequencing results in Example 2, was transformed into E. coli XA90 host cells, and the resulting engineered strain was used to express the HPVL1 protein. A 0.05% inoculum was inoculated into LB medium (Amp+) and cultured at 37°C and 220 rpm for 16 hours for activation. The activated bacterial solution was inoculated into 2YT medium at a 0.5% inoculum and cultured at 30°C and 220 rpm for 7 hours. IPTG was then added to a final concentration of 0.2 mM, and induction culture was continued at 30°C and 220 rpm for 16 hours. Fermentation was then terminated, and the cells were collected by centrifugation for expression level detection and purification experiments.

[0065] The results are shown in Table 5. As can be seen from the results, one of the SD sequences showed a large amount of expression, while the other three SD sequences showed almost no expression.

[0066] Table 5: Comparison of target protein expression levels by HPV68L1a engineered strains containing different SD sequences. [Table 5]

[0067] Example 6: Construction of human rhinovirus 3C protease expression vector pKL1-HRV3C The human rhinovirus 3C protease (HRV 3C) gene was artificially synthesized, and its specific sequence is shown in SEQ ID NO: 16. First, the HRV 3C DNA fragment was amplified by PCR, and the primer information was as follows (the cleavage sites were NdeI and XhoI): Forward primer P3C-NdeI: GGAATTCCATATGGGACCAAACACAGAATTTGCAC Reverse primer P3C-XhoI:GGTCTCGAGTTATTGTTTCTCTACAAAATATTG The L1 gene PCR fragment containing NdeI and Xho1 cleavage sites and the modified expression vector pKL1 were each treated with NdeI / Xho1 double enzyme cleavage, and the recovered gene fragment was then ligated with pKL1 containing the corresponding sticky end using mT4 DNA ligase (16°C, 10-15 hours).

[0068] Ligation system: pKL1 vector fragment 6 μl, 16L1 gene fragment 2 μl, T4 DNA ligase 1 μl, T4 DNA ligase buffer 1 μl.

[0069] Retransformation and identification: After ligation, the ligation product was transformed into E. coli DH5α and recombinants were screened. The screened monoclonal colonies were expanded and the plasmids were extracted. After sequencing verification, the recombinant expression vector pKL1-HRV3C was obtained.

[0070] The following steps are followed to detect the expression and results of the recombinant HRV 3C protease. (1) The expression vector pKL1-3C was transformed into the host strain E. coli, and the empty vector pKL1 was transformed into the host strain as a negative control strain. (2) Streaking and inoculation of single clones of expression-engineered bacteria: Three clones were selected from a plasmid transformation plate obtained by transforming pKL1-3C into the host bacteria and streaked using a streak pin. The clones were then selected and subcultured. The streaked pin was then placed in 40 ml of LB liquid medium containing 100 μg / ml Amp. For the control group, a single colony was selected from a transformation plate obtained by transforming the host bacteria with the empty vector pKL1, streaked, and inoculated into 40 ml of LB medium containing 100 μg / ml Amp. The culture conditions were 37°C, 220 rpm, and shaking overnight. (3) Lactose induction: The overnight cultured bacterial solution was transferred to 2YT fermentation medium at 0.5% and cultured at 37°C and 220 rpm until the OD600 of the bacterial solution reached 1.5-2.0. Lactose was then added to a final concentration of 2 g / L, and the mixture was induced overnight at 30°C and 220 rpm. (4) After the cells were disrupted by ultrasonication, the expression results were analyzed by SDS-PAGE electrophoresis to determine whether the target protein was expressed and whether it was soluble. The results are shown in Figure 4 (parallel experiments using three single-colony engineered strains 1#-3#), demonstrating that the method of the present invention can achieve large-scale soluble expression of HRV 3C protease.

[0071] The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art may still modify the technical solutions described in the above various embodiments or substitute some of the technical features therein with equivalents. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0072] Sequence Listing SEQ ID NO: 1 HPV6L1 N / C-terminal truncation (469 aa, Theoretical pI / Mw: 5.94 / 52079.77 Da, the N-terminus was truncated by 2 amino acids, and the C-terminus was truncated by 29 amino acids) 1 MPSDSTVYVP PPNPVSKVVA TDAYVTRTNI FYHASSSRLL AVGHPYFSIK 51 RANKTVVPKV SGYQYRVFKV VLPDPNKFAL PDSSLFDPTT QRLVWACTGL 101 EVGRGQPLGV GVSGHPFLNK YDDVENSGSG GNPGQDNRVN VGMDYKQTQL 151 CMVGCAPPLG EHWGKGKQCT NTPVQAGDCP PLELITSVIQ DGDMVDTGFG 201 AMNFADLQTN KSDVPIDICG TTCKYPDYLQ MAADPYGDRL FFFLRKEQMF 251 ARHFFNRAGE VGEPVPDTLI IKGSGNRTSV GSSIYVNTPS GSLVSSEAQL 301 FNKPYWLQKA QGHNNGICWG NQLFVTVVDT TRSTNMTLCA SVTTSSTYTN 351 SDYKEYMRHV EEYDLQFIFQ LCSITLSAEV MAYIHTMNPS VLEDWNFGLS 401 PPPNGTLEDT YRYVQSQAIT CQKPTPEKEK PDPYKNLSFW EVNLKEKFSS 451 ELDQYPLGRK FLLQSGYRG SEQ ID NO: 2 HPV11L1 N / C-terminal truncation (470 aa, Theoretical pI / Mw: 6.08 / 52274.02 Da, 3 amino acids were truncated at the N-terminus and 29 amino acids were truncated at the C-terminus) 1 MPSDSTVYVP PPNPVSKVVA TDAYVKRTNI FYHASSSRLL AVGHPYYSIK 51 KVNKTVVPKV SGYQYRVFKV VLPDPNKFAL PDSSLFDPTT QRLVWACTGL 101 EVGRGQPLGV GVSGHPLLNK YDDVENSGGY GGNPGQDNRV NVGMDYKQTQ 151 LCMVGCAPPL GEHWGKGTQC SNTSVQNGDC PPLELITSVI QDGDMVDTGF 201 GAMNFADLQT NKSDVPLDIC GTVCKYPDYL QMAADPYGDR LFFYLRKEQM 251 FARHFFNRAG TVGEPVPDDL LVKGGNNRSS VASSIYVHTP SGSLVSSEAQ 301 LFNKPYWLQK AQGHNNGICW GNHLFVTVVD TTRSTNMTLC ASVSKSATYT 351 NSDYKEYMRH VEEFDLQFIF QLCSITLSAE VMAYIHTMNP SVLEDWNFGL 401 SPPPNGTLED TYRYVQSQAI TCQKPTPEKE KQDPYKDMSF WEVNLKEKFS 451 SELDQFPLGR KFLLQSGYRG SEQ ID NO: 3 HPV16L1 N / C-terminal truncation (472 aa, Theoretical pI / Mw: 6.08 / 52548.71 Da, 4 amino acids were truncated at the N-terminus and 29 amino acids were truncated at the C-terminus) 1 MPSEATVYLP PVPVSKVVST DEYVARTNIY YHAGTSRLLA VGHPYFPIKK 51 PNNNKILVPK VSGLQYRVFR IHLPDPNKFG FPDTSFYNPD TQRLVWACVG 101 VEVGRGQPLG VGISGPLLN KLDDTENASA YAANAGVDNR ECISMDYKQT 151 QLCLIGCKPP IGEHWGKGSP CTNVAVNPGD CPPLELINTV IQDGDMVDTG 201 FGAMDFTTLQ ANKSEVPLDI CTSICKYPDY IKMVSEPYGD SLFFYLRREQ 251 MFVRHLFNRA GAVGENVPDD LYIKGSGSTA NLASSNYFPT PSGSMVTSDA 301 QIFNKPYWLQ RAQGHNNGIC WGNQLFVTVV DTTRSTNMSL CAAISTSETT 351 YKNTNFKEYL RHGEEYDLQF IFQLCKITLT ADVMTYIHSM NSTILEDWNF 401 GLQPPPGGTL EDTYRFVTSQ AIACQKHTPP APKEDPLKKY TFWEVNLKEK 451 FSADLDQFPL GRKFLLQAGL KA SEQ ID NO: 4 HPV18L1 N / C-terminal truncation (473 aa, Theoretical pI / Mw: 5.94 / 52722.42 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 30 amino acids) 1 MPSDNTVYLP PPSVARVVNT DDYVTRTSIF YHAGSSRLLT VGNPYFRVPA 51 GGGNKQDIPK VSAYQYRVFR VQLPDPNKFG LPDNSIYNPE TQRLVWACAG 101 VEIGRGQPLG VGLSGHPFYN KLDDTESHA ATSNVSEDVR DNVSVDYKQT 151 QLCILGCAPA IGEHWAKGTA CKSRPLSQGD CPPLELKNTV LEDGDMVDTG 201 YGAMDFSTLQ DTKCEVPLDI CQSICKYPDY LQMSADPYGD SMFFCLRREQ 251 LFARHFWNRA GTMGDTVPQS LYIKGTGMRA SPGSCVYSPS PSGSIVTSDS 301 QLFNKPYWLH KAQGHNNGVC WHNQLFVTVV DTTRSTNLTI CASTQSPVPG 351 QYDATKFKQY SRHVEEYDLQ FIFQLCTITL TADVMSYIHS MNSSILEDWN 401 FGVPPPPTTS LVDTYRFVQS VAITCQKDAA PAENKDPYDK LKFWNVDLKE 451 KFSLDLDQYP LGRKFLVQAG LRR SEQ ID NO: 5 HPV31L1 N / C-terminal truncation (473 aa, Theoretical pI / Mw: 6.27 / 52869.89 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 27 amino acids) 1 MPSEATVYLP PVPVSKVVST DEYVTRTNIY YHAGSARLLT VGHPYYSIPK 51 SDNPKKIVVP KVSGLQYRVF RVRLPDPNKF GFPDTSFYNP ETQRLVWACV 101 GLEVGRGQPL GVGISGHPLL NKFDDTENSN RYAGGPGTDN RECISMDYKQ 151 TQLCLLGCKP PIGEHWGKGS PCSNNAITPG DCPPLELKNS VIQDGDMVDT 201 GFGAMDFTAL QDTKSNVPLD ICNSICKYPD YLKMVAEPYG DTLFFYLRRE 251 QMFVRHFFNR SGTVGESVPT DLYIKGSGST ATLANSTYFP TPSGSMVTSD 301 AQIFNKPYWM QRAQGHNNGI CWGNQLFVTV VDTTRSTNMS VCAAIANSDT 351 TFKSSNFKEY LRHGEEFDLQ FIFQLCKITL SADIMTYIHS MNPAILEDWN 401 FGLTTPPSGS LEDTYRFVTS QAITCQKTAP QKPKEDPFKD YVFWEVNLKE 451 KFSADLDQFP LGRKFLLQAG YRA SEQ ID NO: 6 HPV33L1 N / C-terminal truncation (471 aa, Theoretical pI / Mw: 5.88 / 52765.94 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 24 amino acids) 1 MPSEATVYLP PVPVSKVVST DEYVSRTSIY YYAGSSRLLA VGHPYFSIKN 51 PNNAKKLLVP KVSGLQYRVF RVRLPDPNKF GFPDTSFYNP DTQRLVWACV 101 GLEIGRGQPL GVGISGHPLL NKFDDTETSN KYPGQPGADN RECLSMDYKQ 151 TQLCLLGCKP PTGEHWGKGV ACTNAAPAND CPPLELINTI IEDGDMVDTG 201 FGCMDFKTLQ ANKSDVPIDI CGSTCKYPDY LKMTSEPYGD SLFFFLRREQ 251 MFVRHFFNRA GKLGEAVPDD LYIKGSGTTA SIQSSAFFPT PGSMSVTSES 301 QLFNKPYWLQ RAQGHNNGIC WGNQVFVTVV DTTRSTNMTL CTQVTSDSTY 351 KNENFKEYIR HVEEYDLQFV FQLCKVTLTA EVMTYIHAMN PDILEDWQFG 401 LTPPPSASLQ DTYRFVTSQA ITCQKTVPPK EKEDPLGKYT FWEVDLKEKF 451 SADLDQFPLG RKFLLQAGLK A SEQ ID NO: 7 HPV35L1 N / C-terminal truncation (470 aa, Theoretical pI / Mw: 6.10 / 52494.45 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 28 amino acids) 1 MSNEATVYLP PVSVSKVVST DEYVTRTNIY YHAGSSRLLA VGHPYYAIKK 51 QDSNKIAVPK VSGLQYRVFR VKLPDPNKFG FPDTSFYDPA SQRLVWACTG 101 VEVGRGQPLG VGISGPLLN KLDDTENSNK YVGNSGTDNR ECISMDYKQT 151 QLCLIGCRPP IGEHWGKGTP CNANQVKAGE CPPLELLNTV LQDGDMVDTG 201 FGAMDFTTLQ ANKSDVPLDI CSSICKYPDY LKMVSEPYGD MLFFYLRREQ 251 MFVRHLFNRA GTVGETVPAD LYIKGTTGTL PSTSYFPTPS GSMVTSDAQI 301 FNKPYWLQRA QGHNNGICWS NQLFVTVVDT TRSTNMSVCS AVSTSDSTYK 351 NDNFKEYLRH GEEYDLQFIF QLCKITLTAD VMTYIHSMNP SILEDWNFGL 401 TPPPSGTLED TYRYVTSQAV TCQKPSAPKP KDDPLKNYTF WEVDLKEKFS 451 ADLDQFPLGR KFLLQAGLKA SEQ ID NO: 8 HPV39L1 N9 N / C-terminal truncation (467 aa, Theoretical pI / Mw: 6.16 / 52393.22 Da, 9 amino acids were truncated at the N-terminus and 29 amino acids were truncated at the C-terminus) 1 MVYLPPSVA KVVNTDDYVT RTGIYYYAGS SRLLTVGHPY FKVGMNGGRK 51 QDIPKVSAYQ YRVFRVTLPD PNKFSIPDAS LYNPETQRLV WACVGVEVGR 101 GQPLGVGISG HPLYNRQDDT ENSPFSSTTN KDSRDNVSVD YKQTQLCIIG 151 CVPAIGEHWG KGKACKPNNV STGDCPPLEL VNTPIEDGDM IDTGYGAMDF 201 GALQETKSEV PLDICQSICK YPDYLQMSAD VYGDSMFFCL RREQLFARHF 251 WNRGGMVGDA IPAQLYIKGT DIRANPGSSV YCPSPSGSMV TSDSQLFNKP 301 YWLHKAQGHN NGICWHNQLF LTVVDTTRST NFTLSTSIES SIPSTYDPSK 351 FKEYTRHVEE YDLQFIFQLC TVTLTTDVMS YIHTMNSSIL DNWNFAVAPP 401 PSASLVDTYR YLQSAAITCQ KDAPAPEKKD PYDGLKFWNV DLREKFSLEL 451 DQFPLGRKFL LQARVRR SEQ ID NO: 9 HPV45L1 N / C-terminal truncation (476 aa, Theoretical pI / Mw: 5.94 / 53244.30 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 30 amino acids) 1 MPSDSTVYLP PPSVARVVNT DDYVSRTSIF YHAGSSRLLT VGNPYFRVVP 51 NGAGNKQAVP KVSAYQYRVF RVALPDPNKF GLPDSTIYNP ETQRLVWACV 101 GMEIGRGQPL GIGLSGHPFY NKLDDTESAH AATAVITQDV RDNVSVDYKQ 151 TQLCILGCVP AIGEHWAKGT LCKPAQLQPG DCPPLELKNT IIEDGDMVDT 201 GYGAMDFSTL QDTKCEVPLD ICQSICKYPD YLQMSADPYG DSMFFCLRRE 251 QLFARHFWNR AGVMGDTVPT DLYIKGTSAN MRETPGSCVY SPSPSGSIIT 301 SDSQLFNKPY WLHKAQGHNN GICWHNQLFV TVVDTTRSTN LTLCASTQNP 351 VPSTYDPTKF KQYSRHVEEY DLQFIFQLCT ITLTAEVMSY IHSMNSSILE 401 NWNFGVPPPP TTSLVDTYRF VQSVAVTCQK DTTPPEKQDP YDKLKFWTVD 451 LKEKFSSDLD QYPLGRKFLV QAGLRR SEQ ID NO: 10 HPV51L1t N / C-terminal truncation (472 aa, Theoretical pI / Mw: 5.88 / 52769.56 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 28 amino acids) 1 MTNDSKVYLP PAPVSRIVNT EEYITRTGIY YYAGSSRLIT LGHPYFPIPK 51 TSTRAAIPKV SAFQYRVFRV QLPDPNKFGL PDPNLYNPDT DRLVWGCVGV 101 EVGRGQPLGV GLSGHPLFNK YDDTENSRIA NGNAQQDVRD NTSVDNKQTQ 151 LCIIGCAPPI GEHWGIGTTC KNTPVPPGDC PPLELVSSVI QDGDMIDTGF 201 GAMDFAALQA TKSDVPLDIS QSVCKYPDYL KMSADTYGNS MFFHLRREQI 251 FARHYYNKLG SVGEDIPNDY YIKGSGNGRD PIESYIYSAT PSGSMITSDS 301 QIFNKPYWLH RAQGHNNGIC WNNQLFITCV DTTRSTNLTI STATAAVSPT 351 FTPSNFKQYI RHGEEYELQF IFQLCKITLT TEVMAYLHTM DPTILEQWNF 401 GLTLPPSASL EDAYRFVTNA ATSCQKDTPP QAKPDPLAKY KFWDVDLKER 451 FSLDLDQFAL GRKFLLQVGV QR SEQ ID NO: 11 HPV52L1 N / C-terminal truncation (476 aa, Theoretical pI / Mw: 5.88 / 53176.26 Da, 4 amino acids were truncated at the N-terminus and 23 amino acids were truncated at the C-terminus) 1 MPSEATVYLP PVPVSKVVST DEYVSRTSIY YYAGSSRLLT VGHPYFSIKN 51 TSSGNGKKVL VPKVSGLQYR VFRIKLPDPN KFGFPDTSFY NPETQRLVWA 101 CTGLEIGRGQ PLGVGISGHP LLNKFDDTET SNKYAGKPGI DNRECLSMDY 151 KQTQLCILGC KPPIGEHWGK GTPCNNNSGN PGDCPPLQLI NSVIQDGDMV 201 DTGFGCMDFN TLQASKSDVP IDICSSVCKY PDYLQMASEP YGDSLFFFLR 251 REQMFVRHFF NRAGTLGDPV PGDLYIQGSN SGNTATVQSS AFFPTPSGSM 301 VTSESQLFNK PYWLQRAQGH NNGICWGNQL FVTVVDTTRS TNMTLCAEVK 351 KESTYKNENF KEYLRHGEEF DLQFIFQLCK ITLTADVMTY IHKMDATILE 401 DWQFGLTPPP SASLEDTYRF VTSTAITCQK NTPPKGKEDP LKDYMFWEVD 451 LKEKFSADLD QFPLGRKFLL QAGLQA SEQ ID NO: 12 HPV56L1t N / C-terminal truncation (470 aa, Theoretical pI / Mw: 6.11 / 52796.70 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 25 amino acids) 1 MPSENKVYLP PTPVSKVVAT DSYVKRTSIF YHAGSSRLLA VGHPYYSVTK 51 DNTKTNIPKV SAYQYRVFRV RLPDPNKFGL PDTNIYNPDQ ERLVWACVGL 101 EVGRGQPLGA GLSGHPLFNR LDDTESSNLA NNNVIEDSRD NISVDGKQTQ 151 LCIVGCTPAM GEHWTKGAVC KSTQVTTGDC PPLALINTPI EDGDMIDTGF 201 GAMDFKVLQE SKAEVPLDIV QSTCKYPDYL KMSADAYGDS MWFYLRREQL 251 FARHYFNRAG KVGETIPAEL YLKGSNGREP PPSSVYVATP SGSMITSEAQ 301 LFNKPYWLQR AQGHNNGICW GNQLFVTVVD TTRSTNMTIS TATEQLSKYD 351 ARKINQYLRH VEEYELQFVF QLCKITLSAE VMAYLHNMNA NLLEDWNIGL 401 SPPVATSLED KYRYVTSTAI TCQREQPPTE KQDPLAKYKF WDVNLQDSFS 451 TDLDQFPLGR KFLMQLGTRS SEQ ID NO: 13 HPV58L1 N / C-terminal truncation (471 aa, Theoretical pI / Mw: 5.80 / 52994.97 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 23 amino acids) 1 MPSEATVYLP PVPVSKVVST DEYVSRTSIY YYAGSSRLLA VGNPYFSIKS 51 PNNNKKVLVP KVSGLQYRVF RVRLPDPNKF GFPDTSFYNP DTQRLVWACV 101 GLEIGRGQPL GVGVSGHPYF NKFDDTETSN RYPAQPGSDN RECLSMDYKQ 151 TQLCLIGCKP PTGEHWGKGV ACNNNAAATD CPPLELFNSI IEDGDMVDTG 201 FGCMDFGTLQ ANKSDVPIDI CNSTCKYPDY LKMASEPYGD SLFFFLRREQ 251 MFVRHFFNRA GKLGEAVPDD LYIKGSGNTA VIQSSAFFPT PGSMSVTSES 301 QLFNKPYWLQ RAQGHNNGIC WGNQLFVTVV DTTRSTNMTL CTEVTKEGTY 351 KNDNFKEYVR HVEEYDLQFV FQLCKITLTA EIMTYIHTMD SNILEDWQFG 401 LTPPPSASLQ DTYRFVTSQA ITCQKTAPPK EKEDPLNKYT FWEVNLKEKF 451 SADLDQFPLG RKFLLQSGLK A SEQ ID NO: 14 HPV59L1 N / C-terminal truncation (473 aa, Theoretical pI / Mw: 6.08 / 52891.89 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 31 amino acids) 1 MSSDNKVYLP PPSVAKVVST DEYVTRTSIF YHAGSSRLLT VGHPYFKVPK 51 GGNGRQDVPK VSAYQYRVFR VKLPDPNKFG LPDNTVYDPN SQRLVWACVG 101 VEIGRGQPLG VGLSGHPLYN KLDDTENSHV ASAVDTKDTR DNVSVDYKQT 151 QLCIIGCVPA IGEHWTKGTA CKPTTVVQGD CPPLELINTP IEDGDMVDTG 201 YGAMDFKLLQ DNKSEVPLDI CQSICKYPDY LQMSADAYGD SMFFCLRREQ 251 VFARHFWNRS GTMGDQLPES LYIKGTDIRA NPGSYLYSPS PSGSVVTSDS 301 QLFNKPYWLH KAQGLNNGIC WHNQLFLTVV DTTRSTNLSV CASTTSSIPN 351 VYTPTSFKEY ARHVEEFDLQ FIFQLCKITL TTEVMSYIHN MNTTILEDWN 401 FGVTPPPTAS LVDTYRFVQS AAVTCQKDTA PPVKQDPYDK LKFWPVDLKE 451 RFSADLDQFP LGRKFLLQLG ARP SEQ ID NO: 15 HPV68L1a N / C-terminal truncation (473 aa, Theoretical pI / Mw: 5.75 / 53032.99 Da, the N-terminus was truncated by 4 amino acids and the C-terminus was truncated by 28 amino acids) 1 MASDNMVYLP PPSVAKVVNT DDYVTRTGMY YYAGTSRLLT VGHPYFKVPM 51 SGGRKQGIPK VSAYQYRVFR VTLPDPNKFS VPESTLYNPD TQRMVWACVG 101 VEIGRGQPLG VGLSGHPLYN RLDDTENSPF SSNKNPKDSR DNVAVDCKQT 151 QLCIIGCVPA IGEHWAKGKS CKPTNVQQGD CPPLELVNTP IEDGDMIDTG 201 YGAMDFGTLQ ETKSEVPLDI CQSVCKYPDY LQMSADVYGD SMFFCLRREQ 251 LFARHFWNRG GMVGDTIPTD MYIKGTDIRE TPSSYVYAPS PSGSMVSSDS 301 QLFNKPYWLH KAQGHNNGIC WHNQLFLTVV DTTRSTNFTL STTTDSTVPA 351 VYDSNKFKEY VRHVEEYDLQ FIFQLCTITL STDVMSYIHT MNPAILDDWN 401 FGVAPPPSAS LVDTYRYLQS AAITCQKDAP APVKKDPYDG LNFWNVDLKE 451 KFSSELDQFP LGRKFLLQAG VRR Sequence number 16 SEQ (HRV 3C gene, 552bp): 1 ATGGGACCAA ACACAGAATT TGCACTATCC CTGTTAAGGA AAAACATAAT 51 GACTATAACA ACCTCAAAGG GAGAGTTCAC AGGGTTAGGC ATACATGATC 101 GTGTCTGTGT GATACCCACA CACGCACAGC CTGGTGATGA TGTACTAGTG 151 AATGGTCAGA AAATTAGAGT TAAGGATAAG TACAAATTAG TAGATCCAGA 201 GAACATTAAT CTAGAGCTTA CAGTGTTGAC TTTAGATAGA AATGAAAAAT 251 TCAGAGATAT CAGGGGATTT ATATCAGAAG ATCTAGAAGG TGTGGATGCC 301 ACTTTGGTAG TACATTCAAA TAACTTTACC AACACTATCT TAGAAGTTGG 351 CCCTGTAACA ATGGCAGGAC TTATTAATTT GAGTAGCACC CCCACTAACA 401 GAATGATTCG TTATGATTAT GCAACAAAA CTGGGCAGTG TGGAGGTGTG 451 CTGTGTGCTA CTGGTAAGAT CTTTGGTATT CATGTTGGCG GTAATGGAAG 501 ACAAGGATTT TCAGCTCAAC TTAAAAAACA ATATTTTGTA GAGAAACAAT 551 AA

Claims

1. A Tac promoter-based plasmid expression vector, characterized in that the SD sequences are AGGAGATATA, AGGAAACAGTA, AGGAGGAATAA, and AGGAGGAATTA.

2. The plasmid expression vector according to claim 1, characterized in that the frame of the plasmid expression vector of Tac promoter is a pGEX vector, preferably in which the GST gene is knocked out.

3. 3. Use of the plasmid expression vector of claim 1 or 2 in the expression of a foreign gene.

4. The use according to claim 3, characterized in that the foreign gene is a gene encoding an antigenic protein or polypeptide of a microorganism including a virus or a bacterium, and a gene encoding a tumor antigen, for example, the foreign gene is a DNA sequence of human papillomavirus L1 protein (HPVL1), a gene encoding human rhinovirus 3C protease (HPV3C, abbreviated as 3C enzyme), a gene encoding human enterovirus type 71 (EV71) capsid protein, or a gene encoding norovirus (NoV) capsid protein.

5. The use according to claim 4, characterized in that the host cell used for expression is Escherichia coli, including but not limited to DH5α, GI698, ER2566, BL21(DE3), XA90, B834(DE3) or BLR(DE3).

6. A method for constructing an expression vector, which is a method for gene recombination, comprising: S1: Designing a mutagenic primer based on the SD sequence, wherein the SD sequence is AGGAGATATA, AGGAAACAGTA, AGGAGGAATAA, AGGAGGAATTA; S2: A step of performing mutagenic PCR using the pGEX vector as a template to introduce an NdeI cleavage site into the original vector; S3: Removing the GST gene sequence by NdeI / BamHI double enzyme digestion; S4: Complementing sticky ends with DNA polymerase I, Klenow fragment; S5: Ligating with T4 DNA ligase to obtain a closed circular plasmid; S6: Designing primers and performing PCR to newly introduce the original cleavage site into the plasmid; Including, Optionally, S7: A construction method further comprising the steps of expanding and propagating a clone with the correct sequence, preserving the strain, extracting the plasmid, and preserving the plasmid.

7. A method for expressing a human papillomavirus vaccine antigen using the plasmid expression vector of claim 1 or 2, comprising: The antigen-encoding gene is cloned into the plasmid expression vector and expressed by the host cell, preferably the L1 protein, more specifically the L1 protein of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68; Preferably, the method comprises modifying the L1 protein, for example by truncating one or more amino acids from the N-terminus, one or more amino acids from the C-terminus, or one or more amino acids from both the N-terminus and the C-terminus.

8. The method of claim 7, characterized in that the amino acid sequence of the L1 protein is set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO:

15.

9. 9. The method of claim 7 or 8, wherein the host cell is Escherichia coli, including but not limited to DH5α, GI698, ER2566, BL21(DE3), XA90, B834(DE3) or BLR(DE3).

10. 10. The method of claim 9, further comprising the step of collecting and purifying the antigen.

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