Recombinant plasmid of high-yield HPV33 subtype pseudovirus and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a recombinant plasmid of high-yield HPV33 subtype pseudovirus and application of the recombinant plasmid. The invention provides coding gene sequences of capsid proteins L1 and L2 which are subjected to codon optimization and can improve the yield of the HPV33 subtype pseudovirus, wherein the coding gene sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO.2; meanwhile, the coding gene sequences of the optimized capsid protein L1 and the optimized capsid protein L2 are subjected to secondary codon optimization to obtain coding gene sequences of the capsid protein L1 and the capsid protein L2 after secondary codon optimization, and the coding gene sequences are respectively shown as SEQ ID NO.3 and SEQ ID NO.4. The invention further discloses a preparation method of the capsid protein L1 and the capsid protein L2. Besides, the invention further provides a recombinant plasmid containing the optimized nucleic acid sequence and capable of producing the HPV33 subtype pseudovirus at high yield, the recombinant plasmid is used for expressing the HPV33 capsid protein L1 and the HPV33 capsid protein L2, the yield of the HPV33 subtype pseudovirus can be obviously increased, and a new way is provided for preparation of the HPV33 subtype pseudovirus.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology and relates to a recombinant plasmid with high yield of HPV33 subtype pseudovirus and its application. Background Art

[0002] Currently, human papillomavirus (HPV) has been recognized as a causative agent of laryngeal cancer, oral cancer, lung cancer, and anal and genital cancer. The virus is a double-stranded, non-enveloped DNA virus that infects keratinocytes of stratified squamous epithelium. The HPV genome is approximately 8,000 base pairs and encodes eight proteins required to complete the viral life cycle. The two late genes encode L1 (major) and L2 (minor) capsid proteins, which form an icosahedral lattice around the HPV genome. Among them, HPV33 is one of the common subtypes that causes genital warts and certain types of respiratory papillomas.

[0003] HPV vaccine is the most effective means to prevent HPV infection. The vaccine must be able to induce a strong and lasting immune response to produce sufficient antibodies and memory cells to resist the infection of pathogens and mediate immune protection against HPV through neutralizing antibodies. Producing HPV pseudoviruses for pseudovirus neutralizing antibody detection is one of the most effective methods to establish a reliable and effective vaccine evaluation system. Therefore, the development and optimization of HPV vaccines require a large number of pseudoviruses for evaluating antigenicity and immunogenicity. High-yield pseudoviruses can meet the antigenic materials required for large-scale vaccine research and clinical trials, promote the development of a new generation of vaccines and optimize existing vaccines. However, due to the influence of multiple factors such as inefficient expression systems, problems in the correct assembly of viral components, and unsatisfactory culture conditions, the yield of HPV pseudoviruses is usually insufficient to meet the needs of neutralization tests. John T Schiller et al. took advantage of the fact that HPV capsid proteins L1 and L2 can non-specifically package DNA, co-transfected with plasmids of HPV L1 and L2 genes with Luciferase reporter plasmids, released HPV pseudoviruses from cells by lysis after 48 hours of incubation, and purified them. The production strategy of pseudoviruses is based on maximizing the production of two capsid proteins L1 and L2 and a target reporter plasmid (pseudogenome) in mammalian cells. In order to generate a high copy number pseudogenome for packaging, the SV40 replication origin (ori) is inserted into the target plasmid and pseudoviruses are produced in cells expressing high levels of SV40 large T antigen (LT). The inventors obtained a considerable yield of HPV16, HPV18 and other subtype pseudoviruses according to the above packaging method, but the yield of HPV33 was not satisfactory.

[0004] Therefore, a method for increasing the yield of HPV33 subtype pseudovirus is urgently needed. Summary of the invention

[0005] In order to solve the above technical problems, the present application performs codon optimization on the vector backbone of the HPV33 subtype pseudovirus and the gene sequences of the L1 and L2 proteins to increase the yield of the HPV33 subtype pseudovirus.

[0006] On the one hand, the present application provides a nucleic acid sequence that highly expresses HPV33 subtype pseudovirus capsid proteins L1 and L2, wherein the nucleic acid sequence comprises any of the following: a) The nucleic acid sequences SEQ ID NO.1 and SEQ ID NO.2 respectively express the HPV33 subtype pseudovirus capsid proteins L1 and L2; b) The nucleic acid sequences SEQ ID NO.3 and SEQ ID NO.4 respectively express the HPV33 subtype pseudovirus capsid proteins L1 and L2.

[0007] By adopting the above technical scheme, the nucleic acid sequences of HPV33 subtype pseudovirus capsid proteins L1 and L2 are subjected to primary codon optimization and secondary codon optimization, wherein the nucleic acid sequences expressing HPV33 subtype pseudovirus capsid proteins L1 and L2, respectively, are obtained after primary codon optimization as SEQ ID NO.1 and SEQ ID NO.2, and the nucleic acid sequences expressing HPV33 subtype pseudovirus capsid proteins L1 and L2, respectively, are obtained after secondary codon optimization as SEQ ID NO.3 and SEQ ID NO.4. By using the codon-optimized nucleic acid sequences for protein expression, high-yield HPV33 subtype pseudovirus capsid proteins L1 and L2 can be obtained.

[0008] On the other hand, the present application also provides a recombinant plasmid with high production of HPV33 subtype pseudovirus, wherein the recombinant plasmid uses plasmid p18sheLL as a backbone, and the nucleic acid sequences of capsid proteins L1 and L2 therein are replaced with the nucleic acid sequence described in claim 1.

[0009] Furthermore, the nucleic acid sequence of the recombinant plasmid is shown in SEQ ID NO.5.

[0010] Furthermore, the nucleic acid sequence of the recombinant plasmid is shown in SEQ ID NO.6.

[0011] Using the above technical solution, the present application compares the difference between the plasmid p18sheLL backbone of the high-yield HPV18 subtype pseudovirus and the plasmid HPV(33)p33shell_GM backbone of the HPV33 subtype pseudovirus designed in the early stage. The results show that the plasmid HPV(33)p33shell_GM backbone (SEQ ID NO.7) has an additional sequence (SEQ ID NO.9) of 56 bp in length compared to the plasmid p18sheLL (SEQ ID NO.8) backbone, which includes the T7 promoter located upstream of L1. Downstream of L1, the HPV(33)p33shell_GM plasmid backbone has a different sequence (SEQ ID NO.10) of 22 bp in length. In contrast, the p18sheLL plasmid backbone has a sequence (SEQ ID NO.11) of 42 bp in length at the corresponding position. In addition, the CMV promoter sequences of the two also differ at c.565C, with the HPV(33)p33shell_GM plasmid being a C base, while the p18sheLL plasmid backbone being an A base. Therefore, using the plasmid p18sheLL as the backbone, the capsid protein L1 and L2 nucleic acid sequences therein are replaced with the capsid protein L1 and L2 nucleic acid sequences of the HPV33 subtype pseudovirus, and the nucleic acid sequence is codon-optimized. The replaced plasmid can significantly increase the yield of the HPV33 subtype pseudovirus after virus packaging relative to the original plasmid.

[0012] On the other hand, the present application also provides a method for preparing HPV33 subtype pseudovirus, which comprises transfecting the above-mentioned recombinant plasmid into target cells, culturing the target cells into anchorage-dependent epithelial-like cells and then lysing them, collecting and purifying the expression products, and obtaining HPV33 subtype pseudovirus.

[0013] Furthermore, the target cells are obtained by infecting HEK-293T cells with lentivirus and undergoing resistance screening.

[0014] On the other hand, the present application also provides the use of a nucleic acid sequence that highly expresses HPV33 subtype pseudovirus capsid proteins L1 and L2 in the preparation of HPV33 subtype pseudovirus capsid proteins L1 and L2.

[0015] On the other hand, the present application also provides the use of a recombinant plasmid that produces a high yield of HPV33 subtype pseudovirus in the preparation of HPV33 subtype pseudovirus.

[0016] In summary, compared with the prior art, the present application uses plasmid p18sheLL as the skeleton, replaces the capsid protein L1 and L2 nucleotide sequences therein with the nucleic acid sequences encoding the capsid proteins L1 and L2 of the HPV33 subtype pseudovirus and after codon optimization, and the obtained recombinant plasmid can significantly increase the yield of the HPV33 subtype pseudovirus. The recombinant plasmid provided by the present application provides a new approach for the preparation of HPV33 subtype pseudovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings.

[0018] Figure 1 This is the plasmid map of p33sheLL OptiGM (p18sheLL OptiGM) constructed in Example 1.

[0019] Figure 2 This is the plasmid map of lenti-CMV-SV40T (Codon opt)-T2A-Puro constructed in Example 1.

[0020] Figure 3 This is a transfection flow chart of Example 1.

[0021] Figure 4 This is the plasmid map of p33sheLL OptiGM (re-Codon opt) (p18sheLL OptiGM) constructed in Example 2.

[0022] Figure 5 This is the HPV(33)p33shell_GM plasmid map constructed in Comparative Example 1.

[0023] Figure 6 This is the p18sheLL spectrum in Comparative Example 2.

[0024] Figure 7 Schematic diagram of the well plate layout for HPV33 pseudovirus TCID50 assay.

[0025] Figure 8 Schematic diagram of the gradient dilution arrangement in the HPV33 pseudovirus TCID50 assay. DETAILED DESCRIPTION

[0026] In order to make the present invention more clearly understandable, the preferred embodiments are described in detail with the accompanying drawings as follows. The present invention is further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0027] The experimental methods in the following examples where specific conditions are not specified were generally carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0028] In the examples of the present application, HPV18 plasmid p18sheLL was purchased from Biofeng Company with the product number SC06660206.

[0029] In the examples of this application, the gene synthesis service is provided by Nanjing GenScript Biotechnology Co., Ltd.

[0030] The sequencing reaction service and primer synthesis service used in the examples of this application are provided by Sangon Biotechnology (Shanghai) Co., Ltd.

[0031] The plasmid extraction kit in the examples of the present application was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., with the item number DP120-01.

[0032] Example 1 Preparation of HPV33 subtype pseudovirus using p33sheLL OptiGM (p18sheLL OptiGM) plasmid 1. Plasmid preparation The plasmid map of p33sheLL OptiGM (p18sheLL OptiGM) is as follows Figure 1 As shown, the plasmid sequence is shown as SEQ ID NO.5, and its construction steps are described as follows.

[0033] The plasmid p18sheLL plasmid (SEQ ID NO.8) from Jiman Biotechnology (Shanghai) Co., Ltd. was selected, and the plasmid was double-digested with Bsp119I (BstBI) (Thermo, catalog number FD0124) and EcoRV (EcoR32I) (best-enzymes, catalog number EG15537S) and purified for use.

[0034] The nucleotide sequences of the target genes HPV33 L1 and HPV33 L2 were optimized once as shown in SEQ ID NO.1 and SEQ ID NO.2, which were provided by the plasmid provided by the gene synthesis service of Nanjing GenScript Biotechnology Co., Ltd. They were used as templates to design PCR amplification primers. The optimized target gene HPV33 L1 and HPV33 L2 DNA fragments were obtained by PCR, and then connected to the cut vector using the seamless cloning experimental method.

[0035] The amplification primers used are shown in Table 1, the PCR amplification system is shown in Table 2, and the PCR reaction conditions are pre-denaturation, 94°C, 120s, 98°C, 10s, 68°C, 30s, 29 cycles, 72°C, 30s.

[0036] Table 1 DNA fragment PCR amplification primers Table 2 PCR reaction system Hieff cloneTM Plus One Step Cloning Kit (Yeasen, Catalog No. 11205ES08) was used for seamless cloning. The ligation system used is shown in Table 3. The ligation reaction was carried out at 50°C for 20 minutes.

[0037] Table 3 Connection system Components Dosage 5×CE Buffer 4μL HPV33L1 DNA fragment 1μL HPV33L2 DNA fragment 1μL Linearized vector 1μL Seamless cloning enzyme 2μL <![CDATA[ddH2O]]> 11μL The ligation reaction product was transformed into the Escherichia coli stbl3 strain, and the plate was coated with LB medium containing 50 mg / L ampicillin, and the ampicillin-resistant transformant was isolated and the plasmid was extracted.

[0038] The prepared plasmid was selected for double enzyme digestion verification, and then sequenced to verify whether the insertion site, direction and sequence of the gene were completely correct. After sequencing verification, the p33sheLL OptiGM (p18sheLL OptiGM) plasmid prepared in this example was completely correct after sequencing. The plasmid map after sequencing verification in this example is as follows: Figure 1 Show.

[0039] 2. Cell Construction The HEK-293T SV40gp6 (HEK-293TT) Cell Line cell line used in this example was obtained by infecting HEK-293T cells with lenti-CMV-SV40T (Codon opt)-T2A-Puro (Genomeitech #58777GI-LV) lentivirus.

[0040] lenti-CMV-SV40T(Codon opt)-T2A-Puro 2.1 (Genomeitech #58777GI-LV) Plasmid construction The vector lenti-CMV-MCS-T2A-Puro (Genomeitech #19605GW-P) from Genometech (Shanghai) Co., Ltd. was selected. The vector was double-digested with XhoI (Thermo, Catalog No. FD0694) and BamHI (Thermo, Catalog No. FD0054) and purified for use.

[0041] The nucleotide sequence of the target gene SV40T (Codon opt) was derived from the plasmid lenti-EF1a-SV40T (Codon opt)-T2A-Puro (Genomeitech #GM-53061P100) of Genometech (Shanghai) Co., Ltd. It was used as a template to design PCR amplification primers. The target gene SV40T (Codon opt) DNA fragment was obtained by PCR, and then connected to the cut vector using a seamless cloning experimental method.

[0042] The specific amplification primers used are shown in Table 3, the PCR amplification system is shown in Table 4, and the PCR reaction conditions are: pre-denaturation, 94°C, 120s, 98°C, 10s, 68°C, 30s, 29 cycles, 72°C, 30s.

[0043] Table 3 PCR amplification primers for target gene SV40T (Codon opt) DNA fragment Table 4 PCR system Components Dosage 10×PCR Buffer for KOD-Plus-Neo 5μL KOD-Plus-Neo (1.0U / μL) 1μL dNTPs(2mM) 5μL <![CDATA[MgSO4(25mM)]]> 3μL Plasmid template 1μL Amplification primers (10 μM each) 1.5μL <![CDATA[ddH2O]]> 29μL Hieff cloneTM Plus One Step Cloning Kit (Yeasen, Catalog No. 11205ES08) was used for seamless cloning. The ligation system used is shown in Table 5. The ligation reaction was carried out at 50°C for 20 minutes.

[0044] Table 5 Connection system Components Dosage 5×CE Buffer 4μL SV40T (Codon opt) DNA fragment 1μL Linearized vector 1μL Seamless cloning enzyme 2μL <![CDATA[ddH2O]]> 12μL The ligation reaction product was transformed into the Escherichia coli stbl3 strain, and the plate was coated with LB medium containing 50 mg / L ampicillin, and the ampicillin-resistant transformant was isolated and the plasmid was extracted.

[0045] The prepared plasmid was selected for double enzyme digestion verification, and then sequenced to verify whether the insertion site, direction and sequence of the gene were completely correct. After sequencing verification, the prepared lenti-CMV-SV40T (Codon opt)-T2A-Puro plasmid was completely correct after sequencing. The plasmid map after sequencing verification in this example is as follows Figure 2 Show.

[0046] 2.2 Lentivirus packaging process 1) HEK-293T cell splitting: One day before transfection, subculture cells with a density of more than 80% into 10 cm culture dishes at an appropriate ratio. When the cell density reaches 70%-80%, prepare for transfection.

[0047] 2) Transfection: Take a sterile 15 mL centrifuge tube and pack 4 plates with a transfection system / plate. The transfection system is shown in Table 6.

[0048] Table 6 Transfection system Components Volume / Mass DMEM 850μL Plasmids 10 μg <![CDATA[GM easy TM Lentiviral Mix]]> 10μL(10μg) <![CDATA[HG Transgene TM Reagent]]> 60μg 3) After mixing, place at room temperature for 15-20 minutes, evenly add to the culture dish to be transfected, and then place in a CO2 incubator for culture.

[0049] 4) Add Enhancing buffer: 10-12 hours after transfection, evenly add 100× Enhancing buffer to promote transfection, 120 μL / dish.

[0050] 5) Change medium: 18 to 20 hours after transfection, carefully aspirate the cell culture medium and discard it in a waste liquid cup containing disinfectant (Note: the culture medium now contains a small amount of virus, which must be treated before being discarded, and the pipette tip used must be soaked in disinfectant for at least 15 minutes before being discarded), then add 12 mL of replacement medium and continue culturing.

[0051] 6) Virus collection: 48 hours after the fluid change, aspirate the cell supernatant into a 50 mL centrifuge tube, centrifuge at 4°C, 3500 g for 5 min, filter the supernatant with a 0.45 μm filter and transfer to a new centrifuge tube. Finally, transfer the filtrate in batches to a 100KD ultrafiltration tube, centrifuge at 4°C, 3000 g for 10 min, discard the lower layer of liquid into a waste liquid cup filled with disinfectant, adjust the centrifugation time again according to the volume required for the virus, and centrifuge the ultrafiltration tube until the centrifugation is completed.

[0052] 7) Virus packaging and storage: The virus was packaged and stored at -80°C.

[0053] 2.3 Lentiviral titer determination 2.3.1 RNA solution preparation 1) 293T cells were cultured to the logarithmic growth phase, and the culture medium used for virus dilution was DMEM (without serum and PS) cell culture medium.

[0054] 2) On the first day, after cell digestion and counting, 2×10 5 cells were inoculated into each well of a 12-well plate and cultured at 37° C. overnight. During infection, the cells grew to a confluence density of 20-30%.

[0055] 3) On the second day, when the 12-well plate was transfected on the same day, the virus solution stored in the -80°C refrigerator was thawed in an ice bath, and gradient dilutions were performed with DMEM (without serum and PS) culture medium. The standard dilution was 4 gradients, and the target sample was diluted 2 gradients: Dilution No. 1: 12 μL virus solution + 108 μL virus dilution medium Dilution No. 2: 12 μL dilution No. 1 + 108 μL virus dilution medium Dilution No. 3: 12 μL dilution No. 2 + 108 μL virus dilution medium Dilution No. 4: 12 μL dilution No. 3 + 108 μL virus dilution medium Select the desired cell wells, gently mix the lentiviral dilutions in each tube, take 100 μL and add it to each well of cells, and culture it in a cell culture incubator at 37°C overnight.

[0056] 4) On the fifth day, the cells were observed to be in good condition and free of contamination, and the cell density was higher than 80%. RNA was extracted for RT-qPCR.

[0057] 5) Total RNA extraction: 1×10 6 Add 350 μL of lysis buffer LB to each cell and mix by repeatedly pipetting (until no cell clusters are visible).

[0058] 6) Add the treated homogenate to a DNA removal / RNA adsorption universal column (the column is placed in a 2 mL collection tube), centrifuge at 13,000 rpm for 1 min, and collect the filtrate containing RNA.

[0059] 7) Accurately estimate the volume of the filtrate and add an equal volume of binding solution BD (please confirm that anhydrous ethanol has been added first), and immediately mix gently by pipetting.

[0060] 8) Add all the above mixed solutions to a new DNA removal / RNA adsorption universal column, centrifuge at 13,000 rpm for 30 seconds, and discard the filtrate.

[0061] 9) Add 700 μL of deproteinization solution, centrifuge at room temperature for 30 seconds, and centrifuge at 13,000 rpm for 30 seconds. Discard the filtrate and put the DNA removal / RNA adsorption universal column back into the 2 mL collection tube.

[0062] 10) Add 500 μL of rinse solution W (please make sure that anhydrous ethanol has been added first), centrifuge at 13,000 rpm for 30 seconds, and discard the filtrate.

[0063] 11) Repeat step 6).

[0064] 12) Place the DNA removal / RNA adsorption universal column back into the 2 mL collection tube, centrifuge the empty column at 13,000 rpm for 2 min, and remove the residual rinse solution.

[0065] 13) Place the DNA cleanup / RNA adsorption universal column in a new 1.5 mL RNase-free centrifuge tube, add 30-50 μL RNase-free H2O to the center of the membrane, leave at room temperature for 1 min, then centrifuge at 13,000 rpm for 1 min, and collect the filtrate, which is the RNA solution.

[0066] 14) The samples can be stored at -80℃ for a long time.

[0067] 2.3.2 Reverse transcription of cDNA 1) Take a sterilized RNase-free eppendorf tube and add the Mix in Table 7 to each sample; Table 7 Mix system 2) After gentle mixing, incubate at 37°C for 2 min; 3) Incubate at 55°C for 15 min; 4) Incubate at 85°C for 5 min to terminate the reaction; 5) Add 40 μL ultrapure water to dilute to 50 μL; 6) The obtained cDNA was stored at -20°C for future use.

[0068] 2.3.3 Real-time PCR detection The cDNA samples were respectively configured with real-time fluorescence quantitative PCR reaction system, the solution was mixed by flicking the bottom of the tube, and the tube was placed on a Realtime PCR instrument for PCR amplification reaction after a brief centrifugation. The Realtime PCR instrument used the Q9604 system of BIO-GENER Company, the detection primer sequences of the target gene and the internal reference gene were shown in Table 8, the PCR system was shown in Table 9, and the PCR reaction program was as follows: pre-denaturation: 95℃, 30s, PCR cycle: 95℃, 30s, 60℃, 60s, 40cycles, melting curve, 60℃→95℃.

[0069] Table 8 Primer sequences for detection of target genes and internal reference genes Table 9 PCR system 2.3.4 Titer determination results The standard products are: Well 1: Dilution No. 1, containing 1×10 -2mL lentiviral solution; Well No. 2: Dilution No. 2, containing 1×10 -3 mL lentiviral solution; Well 3: No. 3 dilution solution, containing 1×10 - 4 mL lentiviral solution; Well 4: dilution No. 4, containing 1×10 -5 mL of lentiviral solution, and the specific parameters are shown in Table 10.

[0070] Table 10 Standard product parameters Cell samples with different amounts of virus were added, and total RNA was extracted and reverse transcribed into cDNA, and then quantitative PCR was performed. The titer value was determined by comparing the Ct value differences between the control group, standard curve and test group.

[0071] The results are shown in Table 11. The titer of lentivirus lenti-CMV-SV40T (Codon opt)-T2A-Puro was 3.47×10 8 TU / mL.

[0072] Table 11 Lentivirus assay results 2.4 Preparation of HEK-293T SV40gp6 (HEK-293TT) Cell Line Stable Strain HEK-293T cells were infected with lenti-CMV-SV40T (Codon opt)-T2A-Puro (Genomeitech #58777GI-LV) lentivirus to obtain the HEK-293T SV40gp6 (HEK-293TT) Cell Line stable strain.

[0073] On day 1, HEK-293T cells were seeded at 2E5 cells in a 12-well plate.

[0074] 2d, before infection, the virus stock solution was taken out from the -80℃ refrigerator and thawed in an ice bath, and the virus stock solution was diluted with complete culture medium (DMEM+10% FBS+1% PS) at MOI=10. The original culture medium of the treatment group was removed, and 500 μL of the culture medium containing the diluted lentivirus solution was added to the cells of the treatment group.

[0075] On day 3, the culture medium was replaced. 16 h after infection, the culture medium containing the lentivirus was replaced with 1 mL of complete culture medium.

[0076] 5d, select appropriate eukaryotic resistance screening cells (Puromycin full lethal concentration 1.5μg / mL, maintenance concentration 0.75μg / mL), generally two rounds of drug screening (1 round 3 days, determined by the antibiotic preliminary experiment), the cells can be stable, after stabilization, use DMEM+10% FBS+1% P.S+0.75μg / mL Puromycin complete medium to maintain the cells.

[0077] 3. Virus packaging HPV pseudovirus packaging cells used H_SV40T AAV-293T Cell Line anchorage-dependent epithelial cells, and the culture medium used DMEM (containing 10% FBS, 1% PS).

[0078] 3.1 Transfection: One day before, subculture the grown cells in a 10 cm culture dish at an appropriate ratio. When the cells grow to 70% to 80%, prepare for transfection. Use the plasmid prepared in step 1 for transfection. The specific transfection process is as follows: Figure 3 shown.

[0079] 3.2 Collect cells: 48 hours after changing the medium, collect the cell supernatant and adherent cells together, centrifuge, discard the supernatant, and collect the cell pellet. Record the volume V of the cell pellet. Add 1.4V DPBS to the EP tube containing the cell pellet, then add 9.5mM MgCl2 (Shanghai test #10012818) and antibiotic-antimycotic (100X) (Gibco #15240096), and suspend the cells by briefly vortexing the test tube.

[0080] 3.3 Lyse cells: Add 1 / 20V of 10% Triton X-100 (Yeasen#20107ES76) (i.e., final Triton concentration 0.5%), 0.1% universal nuclease (Yeasen#20156ES25), 1 / 40V 1M ammonium sulfate (pH=9) (Shanghai test); the lysate can be buffered by adding 40mM sodium phosphate (sodium dihydrogen phosphate: Beyotime#ST1660-250g, sodium dihydrogen phosphate: Beyotime#ST330). The cell lysate was cultured overnight at 37°C.

[0081] 3.4 Virus purification: Place the mature lysate in an ice bath for 5 minutes; add 5M NaCl to make the salt concentration reach 850mM. Centrifuge at 5000xg for 5 minutes to clarify the salt lysate and collect the supernatant. Add the clarified supernatant to an ultracentrifuge tube, then add 1.8mL of 27%, 33%, and 39% OptiprepTM in sequence, and fill the ultracentrifuge tube with DPBS to a total volume of about 11mL. Use an ultracentrifuge (Hitachi #CP70ME) OptiprepTM (Axis-shield #AS1114542) for gradient centrifugation at 16°C, 234,000xg, for 3.5h.

[0082] Gradient centrifugation layering method: 46%(wt / vol)Optiprep TM :(38.3mL60% Optiprep TM +11.5mL DPBS) 33%(wt / vol)Optiprep TM :(16.1mL 46% Optiprep TM +6.4mL DPBS) 39%(wt / vol)Optiprep TM :(19mL 46% Optiprep TM +3.4mL DPBS) 27%(wt / vol)Optiprep TM :(13.2mL 46% Optiprep TM + 9.3 mL DPBS).

[0083] Collect the virus: There is a membrane layer slightly above one-third of the gradient. Discard about 6 ml of the supernatant and collect the remaining virus liquid.

[0084] Example 2 Preparation of HPV33 subtype pseudovirus using p33sheLL OptiGM (re-Codon opt) (p18sheLL OptiGM) plasmid 1. Plasmid preparation The plasmid map of p33sheLL OptiGM (re-Codon opt) (p18sheLL OptiGM) is as follows Figure 4 As shown, the plasmid sequence is shown as SEQ ID NO.6, and its construction steps are described as follows.

[0085] The plasmid p18sheLL plasmid (SEQ ID NO.8) from Jiman Biotechnology (Shanghai) Co., Ltd. was selected, and the plasmid was double-digested with Bsp119I (BstBI) (Thermo, catalog number FD0124) and EcoRV (EcoR32I) (best-enzymes, catalog number EG15537S) and purified for use.

[0086] The target gene HPV33 L1 and HPV33 L2 nucleotide sequences were optimized for the second time as shown in SEQ ID NO.3 (re-Codon opt) and SEQ ID NO.4 (re-Codon opt), which were provided by the plasmid of the gene synthesis service of Nanjing GenScript Biotechnology Co., Ltd. and used as templates to design PCR amplification primers. The target gene HPV33L1 and HPV33 L2 nucleotide sequences were optimized for the second time by PCR, and then connected to the cut vector using the seamless cloning experimental method.

[0087] The amplification primers used are shown in Table 12, the PCR amplification system is shown in Table 13, and the PCR reaction conditions are pre-denaturation, 94°C, 120s, 98°C, 10s, 68°C, 30s, 29 cycles, 72°C, 30s.

[0088] Table 12 DNA fragment PCR amplification primers Hieff cloneTM Plus One Step Cloning Kit (Yeasen, Catalog No. 11205ES08) was used for seamless cloning. The ligation system used is shown in Table 13. The ligation reaction was carried out at 50°C for 20 minutes.

[0089] Table 13 Connection system Components Dosage 5×CE Buffer 4μL HPV33L1 (Codon opt) DNA fragment 1μL HPV33L2 (Codon opt) DNA fragment 1μL Linearized vector 1μL Seamless cloning enzyme 2μL <![CDATA[ddH2O]]> 11μL The ligation reaction product was transformed into the Escherichia coli stbl3 strain, and the plate was coated with LB medium containing 50 mg / L ampicillin, and the ampicillin-resistant transformant was isolated and the plasmid was extracted.

[0090] The prepared plasmid was selected and double enzyme digestion was performed for verification, and then sequencing was performed to verify whether the insertion site, direction and sequence of the gene were completely correct. After sequencing verification, the p33sheLL OptiGM (re-Codon opt) (p18sheLL OptiGM) plasmid prepared in this example was completely correct after sequencing. The plasmid map after sequencing verification in this example is as follows: Figure 4 Show.

[0091] Steps 2-3 of this embodiment are the same as steps 2-3 of embodiment 1.

[0092] Comparative Example 1 Preparation of HPV33 subtype pseudovirus from HPV(33) p33shell_GM plasmid HPV(33)p33shell_GM plasmid was independently developed and preserved by Jiman Biotechnology (Shanghai) Co., Ltd. Its plasmid map is as follows Figure 5 The plasmid sequence is shown in SEQ ID NO.7. The plasmid preparation includes transformation, plaque picking, shaking, extraction and QC.

[0093] 1. Plasmid Preparation 1.1 Plasmid transformation Stb13 competent cells (prepared by Jiman Biotechnology (Shanghai) Co., Ltd.) were placed on ice and allowed to thaw naturally. All the ligation products were added to the competent cells, placed on ice for 20 minutes, and then heat-shocked in a 42°C water bath for 60 seconds. Then quickly placed on ice for 2-3 minutes. Add 1000 μL of SOC culture medium without antibiotics and culture at 37°C, 150 rpm and shake for 45 minutes. Centrifuge at 3000 rpm for 2 minutes, discard about 850 μL of supernatant, blow and disperse the bacterial liquid at the bottom of the tube, add it to the culture dish containing the corresponding resistance, spread it evenly with a sterilized applicator, and invert it in a 37°C constant temperature incubator for overnight culture.

[0094] 1.2 Plasmid picking Take out the solid culture medium that was cultured at constant temperature last night from the constant temperature incubator, and observe the growth of spots on the surface of the culture medium. Take out the SOC culture medium from the refrigerator; determine the amount of antibiotics to be added according to the amount of SOC in the bottle; add 100μL of Amp antibiotics for every 100mL SOC; use tweezers sterilized by the flame of an alcohol lamp to clamp the white tip of the gun, open the solid culture medium, pick up a full single colony, and place it in the corresponding well. Seal the 96-well plate with sealing film and place it in a shaker for constant temperature shaking at 37℃.

[0095] 1.3 Plasmid shaking Add 12.5 mL of LB medium containing ampicillin to a centrifuge tube with a bottom. Then take out the 96-well plate from the 4°C refrigerator, aspirate 200 μL of bacterial solution from the corresponding wells, place in a shaker, and culture overnight at 250 rpm and 37°C.

[0096] 1.4 Plasmid extraction The extraction was performed using a high-purity plasmid mini-preparation kit (TIANGEN / DP107).

[0097] 1.5 Plasmid QC The extracted plasmid was selected, double enzyme digestion was performed, and then sequencing was performed to verify whether the insertion site, direction and sequence of the gene were completely correct. After sequencing verification, the HPV (33) p33shell_GM plasmid prepared in this example was completely correct after sequencing identification. The plasmid map after sequencing identification in this example is as follows Figure 5 Show.

[0098] Steps 2-3 in this comparative example are the same as steps 2-3 in Example 1.

[0099] Comparative Example 2 Preparation of HPV18 Subtype Pseudovirus Using p18sheLL Plasmid The p18sheLL plasmid was purchased from Biofeng Company with the catalog number SC06660206. The plasmid map is shown in Figure 6 As shown, the plasmid sequence is shown as SEQ ID NO.8.

[0100] Steps 2-3 in this comparative example are the same as steps 2-3 in Example 1.

[0101] HPV33 pseudovirus TCID50 assay 20-24 hours before the experiment, the HEK-293T SV40gp6 (HEK-293TT) Cell Line cells in Example 1-2 and Comparative Example 1-2 were digested from the culture dishes, and the cells were resuspended in fresh culture medium to detect cell viability and count. The cells were collected by centrifugation at 130-200×g, and the cell concentration was adjusted to 1×10 5 Use a spray gun to add 100 μL of cells / well to the middle 60 wells, add 100 μL of PBS to the surrounding wells, cover the plate, and incubate overnight in an incubator. Figure 7 shown.

[0102] The virus solution was diluted with complete DMEM medium (10% FBS, 1% PS), with 10 μL virus in the first well, 10-fold gradient dilution, 9 gradients, and the last well as medium, and repeated 6 times. The specific gradient dilution arrangement is as follows: Figure 8 shown.

[0103] The cell culture plate that was plated the day before was discarded of the supernatant culture medium, and 100 μL of the pseudovirus dilution solution was added to the corresponding cell wells, and placed in a cell culture incubator at 37°C. After 6 hours, the cell culture medium containing 10% FBS and 1% PS was replaced; after 72 hours, the Luc value was detected on a microplate reader (Moleculardevices / SpectraMax L) using a luciferase detection kit (GMOne-Step 2.0 Luciferase Reporter Gene Assay Kit / GM-040513A).

[0104] TCID50 (Tissue Culture Infective Dose 50) is a standard method for measuring viral titers, which represents the dose of virus required to infect 50% of a cell culture. The calculation of TCID50 is usually based on the results of cell culture infection after inoculation of a series of dilutions of a virus suspension.

[0105] Commonly used calculation methods include the Reed-Muench method: 1. Calculate distance ratio Distance ratio = (the first percentage with a lesion rate higher than 50% - 50%) / (the first percentage with a lesion rate higher than 50% - the first percentage with a lesion rate lower than 50%) 2. Calculate 1g TCID50 = distance ratio × difference between the logarithms of dilution + the logarithm of the dilution with a lesion rate higher than 50%.

[0106] The above method was used to detect the TCID50 of Example 1-2 and Comparative Example 1-2. The test results are shown in Table 14.

[0107] Table 14 TCID50 measurement values It can be clearly seen from Table 14 that in Example 1, the HPV33 original packaging plasmid backbone was replaced with the HPV18 plasmid backbone, and the coding genes of the HPV33 pseudovirus capsid proteins L1 and L2 were codon optimized to obtain the recombinant plasmid p33sheLLOptiGM (p18sheLL OptiGM). The TCID50 / mL of the HPV33 pseudovirus prepared using the plasmid was 10 6.5 Compared with comparative example 1, the yield of HPV33 pseudovirus was significantly improved; further, in Example 2, the coding gene sequence codons of HPV33 major capsid proteins L1 and L2 were further optimized to obtain the recombinant plasmid p33sheLL OptiGM (re-Codon opt) (p18sheLL OptiGM), and the HPV33 pseudovirus prepared by the plasmid had a TCID50 / mL of 10 7.5 , which can further increase the yield of HPV33 pseudovirus, which is equivalent to the yield of HPV18 pseudovirus in Comparative Example 2. Therefore, the recombinant plasmid of the present application can effectively increase the yield of plasmid HPV33 pseudovirus, and the recombinant plasmid provided by the present application has a high application value.

[0108] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. Highly expressed nucleic acid sequences of HPV33 subtype pseudovirus capsid proteins L1 and L2, It is characterized in that The nucleic acid sequence comprises any of the following, a) nucleic acid sequences SEQ ID NO.1 and SEQ ID NO.2 expressing HPV33 subtype pseudovirus capsid proteins L1 and L2, respectively; b) The nucleic acid sequences SEQ ID NO.3 and SEQ ID NO.4 respectively express the HPV33 subtype pseudovirus capsid proteins L1 and L2.

2. A recombinant plasmid with high yield of HPV33 subtype pseudovirus, characterized in that: The recombinant plasmid uses plasmid p18sheLL as a skeleton, and the capsid protein L1 and L2 nucleic acid sequences therein are replaced with the nucleic acid sequence described in claim 1.

3. The recombinant plasmid according to claim 2, characterized in that The nucleic acid sequence of the recombinant plasmid is shown as SEQ ID NO.

5.

4. The recombinant plasmid according to claim 2, characterized in that The nucleic acid sequence of the recombinant plasmid is shown as SEQ ID NO.

6.

5. A method for preparing a HPV33 subtype pseudovirus, characterized in that: The method comprises transfecting the recombinant plasmid described in any one of claims 2 to 4 into target cells, lysing the target cells after culturing them with anchorage-dependent epithelial cells, and collecting and purifying the expression products to obtain HPV33 subtype pseudoviruses.

6. The preparation method according to claim 5, characterized in that: The target cells are obtained by infecting HEK-293T cells with lentivirus and undergoing resistance screening.

7. Use of the nucleic acid sequence according to claim 1 in the preparation of HPV33 subtype pseudovirus capsid proteins L1 and L2.

8. Use of the recombinant plasmid according to any one of claims 2 to 4 in preparing HPV33 subtype pseudovirus.

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