Polynucleotide for expressing HPV 16 L1 and application thereof

Abstract

The invention relates to polynucleotide for expressing HPV (human papilloma virus) 16L1 and application of the polynucleotide, in particular to an optimized coding gene for coding main capsid protein L1 of human papilloma virus type 16, and the gene can efficiently express and recombine the main capsid protein L1 of human papilloma virus type 16 after being transferred into pichia pastoris cells.

Description

Technical Field

[0001] The present invention relates to the field of medicine and biology, and in particular to a polynucleotide expressing HPV 16 L1 and an application thereof. Background Art

[0002] Papillomavirus is a non-enveloped double-stranded DNA virus with a diameter of about 55nm, and its genome contains about 8,000 base pairs. Among the more than 300 papillomaviruses identified, more than 200 infect humans, namely human papillomavirus (HPV), of which more than 40 viruses can cause different epithelial-related diseases in humans. HPV mainly infects skin and mucous membrane tissues. In most cases, HPV infection has no obvious clinical symptoms, but some persistent HPV infections can cause skin and mucous membrane epithelial hyperplasia and even cancer, which is clinically manifested as different types of epithelial warts, cervical cancer, anal cancer and other malignant tumors. It has been confirmed that HPV infection is a necessary condition for the occurrence of cervical cancer.

[0003] L1 protein is the main structural protein of HPV, forming the HPV capsid, with a molecular weight of about 55kD. L1 proteins aggregate with each other, first forming pentamers containing 5 L1 monomers, also known as capsomeres, and 72 L1 pentamers form an icosahedral HPV capsid with T=7. Studies have found that L1 protein expressed by recombinant DNA can form a viral capsid alone, and its structure is similar to that of live virus particles, so it is also called virus-like particle (VLP).

[0004] At present, various expression systems such as insect expression system, yeast expression system, prokaryotic expression system (E. coli) and mammalian cells can obtain virus-like particles (VLP) by expressing the main capsid protein L1 alone or by jointly expressing L1+L2. Among them, the Pichia pastoris expression system has the advantages of both prokaryotic and eukaryotic expression systems. The target protein can be expressed at a high level by using this system. The HPV antigens expressed by it are closer to human cell structures than those expressed by prokaryotic expression, so they have the advantages of strong immunogenicity and high neutralization titer. Summary of the invention

[0005] Based on the existing yeast expression system, the expression amount of HPV 16 L1 protein and the stability of the protein need to be improved, and the present invention provides a gene encoding the major capsid protein L1 of human papillomavirus type 16 that can be expressed at a high level, and its expression vector, recombinant strain and application.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides an artificially designed and synthesized isolated polynucleotide, which encodes the major capsid protein L1 of human papillomavirus type 16, and the polynucleotide sequence includes one or more site mutations at positions 141, 360, 777, 1098 or 1293 based on the parent sequence, and the parent sequence is shown in SEQ ID NO.2.

[0008] Preferably, the polynucleotide sequence comprises a mutation of the base at one or more of positions 141, 360, 777, 1098 or 1293 in the parent sequence to thymine.

[0009] Preferably, the polynucleotide sequence is as shown in SEQ ID NO.1 or its complementary sequence.

[0010] In one aspect, the present invention provides a Pichia pastoris expression vector, comprising the polynucleotide described in any one of the above items.

[0011] Preferably, the expression vector is selected from one or more of pGEX-6p-1, pGEX-4T-2, pMAL, pPICZ, pPIC6, pGAPZ, pAO815 and pET28a; preferably one or more of pPICZ, pPIC6, pGAPZ, pAO815, more preferably pPICZαB.

[0012] In one aspect, the present invention provides a recombinant strain, which comprises the expression vector described above, or has any of the polynucleotides described above integrated into its genome.

[0013] Preferably, the strain is a Pichia pastoris strain.

[0014] Preferably, the method for constructing the recombinant strain comprises the following steps:

[0015] (1) designing and synthesizing the polynucleotide described in any one of the above items;

[0016] (2) constructing a Pichia pastoris expression vector containing the polynucleotide sequence obtained in step (1);

[0017] (3) Transforming the expression vector obtained in step (2) into a host cell, culturing the transformant, and obtaining the strain expressing the human papillomavirus type 16 major capsid protein L1.

[0018] Preferably, in step (3), the culturing is carried out in a YPDS medium plate containing antibiotics.

[0019] Preferably, the specific steps of culturing are: spreading the transformant on a YPDS medium plate containing 80-120 μg / ml antibiotics for culture, picking positive clones and spreading them on a YPDS medium plate containing 400-600 μg / ml antibiotics for culture, picking positive clones and spreading them on a YPDS medium plate containing 800-1200 μg / ml antibiotics for culture, picking positive clones and spreading them on a YPDS medium plate containing 1500-2500 μg / ml antibiotics for culture, and isolating the strain expressing the human papillomavirus type 16 major capsid protein L1.

[0020] Preferably, the expression vector is selected from one or more of pGEX-6p-1, pGEX-4T-2, pMAL, pPICZ, pPIC6, pGAPZ, pAO815 and pET28a, preferably one or more of pPICZ, pPIC6, pGAPZ, pAO815, and more preferably pPICZαB.

[0021] Preferably, the antibiotic is bleomycin.

[0022] In one aspect, the present invention provides a method for preparing human papillomavirus type 16 major capsid protein L1, the method comprising culturing the strain as described above, and optionally isolating human papillomavirus type 16 major capsid protein L1 from the culture medium.

[0023] Preferably, the method further comprises the step of purifying human papillomavirus type 16 major capsid protein L1.

[0024] Preferably, the step of purifying the human papillomavirus type 16 major capsid protein L1 is to purify using a POROS50HS chromatography column and a CHT II chromatography column in sequence.

[0025] On the one hand, the present invention provides use of any of the polynucleotides described above, or the expression vectors described above, or the recombinant strains described above in the preparation of HPV vaccines.

[0026] Compared with the prior art, the main beneficial technical effects of the present invention are:

[0027] The present invention modifies the natural HPV 16 L1 gene, performs a Pichia codon preference analysis on all its amino acids, and uses the most frequently used codons as much as possible, while avoiding too high a GC ratio of the transcribed mRNA, and avoiding commonly used restriction enzyme cleavage sites and other information to design a new HPV 16 L1 sequence, the yield of the encoded HPV 16 L1 protein is much higher than that of the parent sequence. Moreover, the encoded HPV 16 L1 protein has better thermal stability, stronger affinity for neutralizing antibodies, and better uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the electrophoresis diagram of PCR amplification in Example 2, where M is DL5000 DNA Marker, and Lanes 1 to 2 are the amplified products of the target gene;

[0029] Figure 2 This is the electrophoresis diagram of colony PCR amplification in Example 2, where M is DL5000 DNA Marker and Lane 1-8 are amplified single colonies;

[0030] Figure 3 This is a diagram of the high pressure screening of Pichia pastoris YPDS in Example 3;

[0031] Figure 4 This is the thermal stability spectrum of the protein stock solution measured by turbidity method in Example 7;

[0032] Figure 5 The thermal stability spectrum of the protein stock solution measured by the DSF method in Example 7;

[0033] Figure 6 This is a schematic diagram of the results of SEC-MALS determination of HPV 16 L1 protein molecular weight in Example 7;

[0034] Figure 7 This is a TEM transmission electron microscope of the protein stock solution of the present application in Example 7;

[0035] Figure 8 This is a TEM transmission electron microscope image of the control protein stock solution in Example 7;

[0036] Fig. 9 Schematic diagram of the binding ability of V5 neutralizing mouse monoclonal antibody to 16 L1 protein in Example 7. DETAILED DESCRIPTION

[0037] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0038] The advantage of the Pichia pastoris expression system is that it has the advantages of both prokaryotic and eukaryotic expression systems. This system can be used to express proteins at high levels. The HPV antigens expressed are closer to human cell structures and therefore have stronger immunogenicity. The present invention uses the Pichia pastoris expression system to design a new HPV DNA sequence based on the sequence shown in SEQ ID NO.2, using the codons with the highest usage frequency for all amino acids, and obtains a 16L1 protein stock solution with high expression and high stability, which can be used for the production of vaccine products. Based on this, the present invention is completed.

[0039] On the one hand, the present invention provides an artificially designed synthetic polynucleotide, which encodes the major capsid protein L1 of human papillomavirus type 16, characterized in that the polynucleotide sequence includes one or more site mutations at positions 141, 360, 777, 1098 or 1293 based on the parent sequence, and the parent sequence is shown in SEQ ID NO.2.

[0040] In certain embodiments, the polynucleotide sequence comprises a mutation of the base at one or more of positions 141, 360, 777, 1098 or 1293 in the parent sequence to thymine.

[0041] In certain embodiments, the polynucleotide sequence is as shown in SEQ ID NO.1 or its complementary sequence.

[0042] The words “containing,” “having,” or “including” used in this article include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0043] In one aspect, the present invention provides a Pichia pastoris expression vector, comprising the polynucleotide described in any one of the above items.

[0044] In certain embodiments, the expression vector is selected from one or more of pGEX-6p-1, pGEX-4T-2, pMAL, pPICZ, pPIC6, pGAPZ, pAO815 and pET28a.

[0045] In certain embodiments, the expression vector is selected from one or more of pPICZ, pPIC6, pGAPZ, and pAO815.

[0046] In certain embodiments, the expression vector is pPICZαB.

[0047] In one aspect, the present invention provides a recombinant strain, which comprises the expression vector described above, or has any of the polynucleotides described above integrated into its genome.

[0048] In certain embodiments, the strain is a Pichia strain.

[0049] In certain embodiments, the Pichia strain is selected from the group consisting of Pichia X-33, GS115, KM71, and SMD1168 strains.

[0050] In certain embodiments, the method for constructing the recombinant strain comprises the following steps:

[0051] (1) designing and synthesizing the polynucleotide described in any one of the above items;

[0052] (2) constructing a Pichia pastoris expression vector containing the polynucleotide sequence obtained in step (1);

[0053] (3) Transforming the expression vector obtained in step (2) into a host cell, culturing the transformant, and obtaining the strain expressing the human papillomavirus type 16 major capsid protein L1.

[0054] In certain embodiments, in step (3), the culturing is performed in a YPDS medium plate containing antibiotics.

[0055] In some embodiments, the culturing step comprises: spreading the transformant on a YPDS medium plate containing 80 to 120 μg / ml (e.g., 85 μg / ml, 90 μg / ml, 95 μg / ml, 100 μg / ml, 105 μg / ml, 110 μg / ml, 115 μg / ml, 120 μg / ml, etc.) of antibiotics, picking positive clones and spreading them on a YPDS medium plate containing 400 to 600 μg / ml (e.g., 410 μg / ml, 420 μg / ml, 430 μg / ml, 440 μg / ml, The cells were cultured on YPDS medium plates containing 450 μg / ml, 460 μg / ml, 470 μg / ml, 480 μg / ml, 490 μg / ml, 500 μg / ml, 510 μg / ml, 520 μg / ml, 530 μg / ml, 540 μg / ml, 550 μg / ml, 560 μg / ml, 570 μg / ml, 580 μg / ml, 590 μg / ml, 600 μg / ml, etc.) of antibiotics, and positive clones were selected and spread on YPDS medium plates containing 800-1200 μg / ml (e.g. 820 μg / ml). / ml, 840μg / ml, 860μg / ml, 880μg / ml, 900μg / ml, 920μg / ml, 940μg / ml, 960μg / ml, 980μg / ml, 1000μg / ml, 1050μg / ml, 1100μg / ml, 1150μg / ml, 1200μg / ml, etc.) of antibiotics were cultured on YPDS medium plates, and positive clones were picked and spread on plates containing 1500-2500μg / ml (for example, 1550μg / ml, 1600μg / ml, 16 The strain expressing the human papillomavirus type 16 major capsid protein L1 is isolated by culturing on YPDS medium plates containing antibiotics (50μg / ml, 1700μg / ml, 1750μg / ml, 1800μg / ml, 1850μg / ml, 1900μg / ml, 1950μg / ml, 2000μg / ml, 2050μg / ml, 2100μg / ml, 2150μg / ml, 2200μg / ml, 2300μg / ml, 2400μg / ml, 2500μg / ml, etc.).

[0056] The present invention adopts Plate PTVA (a step-by-step increase method of antibiotic plates) to achieve the effect of vector amplification of the number of copies of exogenous genes, so as to obtain strains with higher numbers of copies of exogenous genes.

[0057] In some embodiments, the specific steps of culturing are: spreading the transformant on a YPDS culture medium plate containing 90-110 μg / ml antibiotics, picking positive clones and spreading them on a YPDS culture medium plate containing 450-550 μg / ml antibiotics, picking positive clones and spreading them on a YPDS culture medium plate containing 900-1100 μg / ml antibiotics, picking positive clones and spreading them on a YPDS culture medium plate containing 1800-2200 μg / ml antibiotics to isolate and obtain the strain expressing the major capsid protein L1 of human papillomavirus type 16.

[0058] In some embodiments, the specific steps of culturing are: spreading the transformant on a YPDS medium plate containing 100 μg / ml antibiotics for culturing, picking positive clones and spreading them on a YPDS medium plate containing 500 μg / ml antibiotics for culturing, picking positive clones and spreading them on a YPDS medium plate containing 1000 μg / ml antibiotics for culturing, picking positive clones and spreading them on a YPDS medium plate containing 2000 μg / ml antibiotics for culturing to isolate the strain expressing the major capsid protein L1 of human papillomavirus type 16.

[0059] In certain embodiments, the expression vector is selected from one or more of pGEX-6p-1, pGEX-4T-2, pMAL, pPICZ, pPIC6, pGAPZ, pAO815 and pET28a.

[0060] In certain embodiments, the expression vector is selected from one or more of pPICZ, pPIC6, pGAPZ, and pAO815.

[0061] In certain embodiments, the expression vector is pPICZαB.

[0062] In certain embodiments, the antibiotic is bleomycin.

[0063] In one aspect, the present invention provides a method for preparing human papillomavirus type 16 major capsid protein L1, the method comprising culturing the strain as described above, and optionally isolating human papillomavirus type 16 major capsid protein L1 from the culture medium.

[0064] In certain embodiments, the method further comprises the step of purifying the human papillomavirus type 16 major capsid protein L1.

[0065] In certain embodiments, the step of purifying the human papillomavirus type 16 major capsid protein L1 is performed by sequentially using a POROS 50HS chromatography column and a CHT II chromatography column.

[0066] On the one hand, the present invention provides the use of any of the polynucleotides described above, or the expression vectors described above, or the recombinant strains described above in the preparation of HPV vaccines. The HPV vaccine prepared by using the major capsid protein L1 of human papillomavirus type 16 expressed by the polynucleotides provided in the present application has good immunogenicity and can be used as a potential candidate HPV vaccine.

[0067] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0068] Example 1 Gene Design and Synthesis of HPV Type 16 L1 Protein

[0069] On the basis of the parent sequence (SEQ ID NO: 2), the preferred codons of Pichia pastoris were optimized to avoid too high a GC ratio of the transcribed mRNA, the influence of the secondary structure of the mRNA on the translation efficiency, and the commonly used restriction sites (BstBI, KpnI, SacI, DraI, etc.), and a new HPV 16L1 protein DNA sequence was designed. A BstBI restriction site (TTCGAA) was added to the 3' end of the sequence, a KpnI restriction site (GGTACC) was added to the 5' end, and a Kozak sequence (GAAACG) was added, wherein the 1st to 6th bases are BstBI restriction sites, the 4th to 9th bases are Kozak sequences, and the 1531st to 1536th bases are KpnI restriction sites, see SEQ ID NO: 1.

[0070] SEQ ID NO: 1

[0071]

[0072] SEQ ID NO:2

[0073]

[0074] Example 2 Construction of pPICZαB recombinant expression vector

[0075] Anhui General Biotechnology was commissioned to synthesize the above gene sequence. The target sequence was amplified by PCR (G0241170-1_1 and G0241170-1_28) to obtain the target gene product; then the product was detected by gel electrophoresis, and the position of the product electrophoresis band was compared with the marker. It was found that the size of the amplified product was consistent with the theory ( Figure 1 ) to confirm that the target gene was amplified correctly.

[0076] The pPICZαB vector was digested with BstBⅠ-KpnⅠ endonucleases, and the PCR amplification product was then connected to the pPICZαB linearized vector by recombination. Finally, the ligation product was transferred into E. coli Top10 host cells and spread on bleomycin-resistant plate culture medium, and placed in a 37°C incubator for overnight culture. After the plate colonies grew, 8 monoclonal colonies were picked, and cultured and revived after adding culture medium. G0241170-1_1 and G0241170-1_14 were used as primers for colony PCR amplification to screen positive monoclones of the target gene. The amplified product was taken for gel electrophoresis detection, and the band size was consistent with the theoretical (801bp) ( Figure 2 ), the first clone numbered C30262 was selected for sequencing and restriction digestion, and the sequence was confirmed to be correct. At the same time, the parent sequence (shown in SEQ ID NO: 2) was constructed on the pPICZαB vector and transformed into Escherichia coli Top10 host cells as a control group using the same method.

[0077] Table 1

[0078]

[0079] Example 3 Construction of a recombinant strain expressing HPV type 16 L1 protein

[0080] The positive clone strains (C30262 Top 10 strains) and the control strains obtained according to the method of Example 2 were respectively amplified and cultured in LB liquid medium containing Zeocin antibiotics, and the plasmid containing the target gene (pPICZ-16L1) was extracted using the Kangwei Century Plasmid Extraction Kit (CAT: CW21055), and linearized by single enzyme digestion with Sac I restriction endonuclease. After the enzyme digestion, the purity of the plasmid was identified by 1% agarose gel electrophoresis, and the linearized fragment was recovered (concentration of 578ng / μl) and electroporated to Pichia pastoris X-33 strain (Invitrogen), and the electroporation conditions were DNA 5μg, voltage 1.5Kv, and electric shock time 4.1ms.

[0081] The electroporation product was spread on a YPDS plate containing 100 μg / ml Zeocin. Since the pPICZαB vector contains a Zeocin resistance gene, the transformation product can grow on a YPDS agar plate containing Zeocin. The positive strain on the plate was spotted on a YPDS plate containing 500 μg / ml Zeocin and inverted at 30°C. After 24 hours of incubation, the positive clone was transferred to a YPDS plate containing 1000 μg / ml Zeocin. Repeat the operation and transfer to a YPDS plate containing 2000 μg / ml Zeocin. Single colonies were isolated to obtain a Pichia pastoris expression strain of HPV 16 L1 with a high copy of the plasmid (see Figure 3 ).

[0082] Example 4 Induced expression and purification of HPV 16 L1 in Pichia pastoris

[0083] The Pichia pastoris expression strain obtained according to the method of Example 2 was inoculated into BMGY liquid culture medium and cultured at 30°C and 250rpm for 28h until OD 600 >1.0, take an appropriate amount of bacterial solution and centrifuge it to replace the medium with BMMY medium, so that the OD 600 The final value was controlled at about 1.0, and 0.5% methanol was added every 24 hours to induce expression. After 72 hours of expression, the bacterial liquid was harvested by centrifugation and broken. The antigen expression level of the supernatant was detected by double antibody sandwich ELISA method.

[0084] The supernatant of the broken bacterial cells is purified by chromatography, and the virus-like particles formed by self-assembly of the L1 protein are obtained by disassembly and reassembly steps. The specific steps are as follows:

[0085] The crushed supernatant was initially purified by a POROS 50HS chromatography column, and the elution method was to wash with 35% B solution and 65% A solution, and gradient elution was performed with 35% to 70% B solution (containing 65% to 30% A solution), and the eluted components were collected, wherein the A solution was 0.5 mol / L sodium chloride, 0.05 mol / L MOPS, 0.05% (w / v) polysorbate 80, pH 7.00±0.10, and the B solution was 1.5 mol / L sodium chloride, 0.05 mol / L MOPS, 0.05% (w / v) polysorbate 80, pH 7.00±0.10.

[0086] After the components containing HPV 16L1 protein were mixed, they were purified using a CHT II chromatography column, and the elution method was a linear gradient elution from 100% A solution to 100% B solution, and the eluate was harvested. The A solution was 0.6 mol / L sodium chloride, 0.05 mol / L MOPS, 0.036 mol / L sodium dihydrogen phosphate, 0.05% (w / v) polysorbate 80, pH 6.50±0.10; the B solution was 0.6 mol / L sodium chloride, 0.182 mol / L sodium dihydrogen phosphate, 0.05% (w / v) polysorbate 80, pH 6.50±0.10.

[0087] The components containing 16L1 protein were replaced with disassembly buffer by hollow fiber ultrafiltration, and the disassembly was completed by stirring in an ice water bath for 45 to 60 minutes. The hollow fiber ultrafiltration system was used to replace the liquid with reassembly buffer to obtain virus-like particles formed by self-assembly of L1 protein, wherein the disassembly buffer was 0.63 mol / L sodium chloride, 0.1 mol / L Tris, 0.035 mol / L sodium dihydrogen phosphate, 0.002 mol / L disodium edetate, pH 8.20±0.10; the reassembly buffer was 1.0 mol / L sodium chloride, 0.05 mol / L citric acid, 0.002 mol / L calcium chloride, pH 6.20±0.10.

[0088] Example 5 Detection of exogenous gene copy number

[0089] The yeast genome was extracted from the fermentation culture obtained in Example 4 using a genome extraction kit (Gene JET Genomic DNA Purification Kit, Thermo), and accurately quantified using a nucleic acid quantifier. The genome was diluted 50 times, 250 times, 1250 times and 6250 times with purified water, and the amplification Ct values ​​of the GAPDH reference gene and the 16 L1 target gene were detected using qPCR technology, and the target gene copy number was calculated according to the following formula:

[0090] Target gene copy number = 2n, n = (n1 + n2 + n3 + n4) / 4, where

[0091] n 1: 50-fold dilution (GADPH Ct value - target gene amplification Ct value);

[0092] n 2: dilute 250 times (GADPH Ct value - target gene amplification Ct value);

[0093] n 3: dilution 1250 times (GADPH Ct value - target gene amplification Ct value);

[0094] n 4: dilute 6250 times (GADPH Ct value - target gene amplification Ct value).

[0095] The test results are shown in the following Table 2, and it can be seen that the copy number of the optimized strain provided by the present invention is significantly higher than that of the control strain.

[0096] Table 2

[0097] Sample name Exogenous gene copy number Control strain - fermentation broth 21 New strains - fermentation broth 48

[0098] Example 6 Antigen expression rate detection

[0099] Using purified HPV 16L1 protein stock solution as the standard, the double antibody sandwich ELISA method was used to detect the antigen content in the fermentation culture supernatant, and the BCA method was used to detect the total protein content in the supernatant, and the antigen expression rate was calculated.

[0100] The steps for antigen content detection are as follows:

[0101] After diluting the coating antibody (rabbit polyclonal antibody) 500 times with the coating solution, add it to the ELISA plate at 100 μl / well. After overnight at 4°C, remove the coating solution, wash the wells 6 times with PBST solution, pat dry, add 200 μl / well blocking solution, block at 37°C for 2h, wash 6 times with PBST and pat dry for later use.

[0102] The standard was diluted to 120 μg / ml with diluent, and then diluted 3 times to 0.002 μg / ml, for a total of 11 points. The supernatant of the test sample was diluted 1000 times and added to the blocked ELISA plate. After incubation at 37°C for 1 hour, the antigen solution was discarded, and the plate was washed 6 times with PBST and patted dry. Then, 100 μl / well of V5 neutralizing mouse monoclonal antibody diluted 2500 times was added to the ELISA plate, and the plate was incubated at 37°C for 1 hour. The antibody solution was discarded, and the plate was washed 6 times with PBST, and the plate was patted dry. 100 μl / well of HRP-labeled goat anti-mouse IgG antibody diluted 10000 times was added, and the plate was incubated at 37°C for 1 hour. The antibody solution was discarded, and the plate was washed 6 times with PBST, and the plate was patted dry. 100 μl / well of TMB colorimetric solution was added, and the plate was incubated at 37°C for 15 minutes. 50 μl / well of 2M H2SO4 stop solution was added to terminate the reaction, and the OD was read with an ELISA reader. 450nm Numeric value.

[0103] The concentration of the standard at different dilution multiples and its corresponding OD 450nm The antigen content in the test sample was calculated by fitting a four-parameter curve and substituting the test sample measured value into the standard curve and multiplying it by the dilution factor (1000).

[0104] The total protein content in the supernatant of the test sample was detected using a BCA detection kit (Thermo), and the antigen expression rate in the fermentation culture medium was calculated according to the calculation formula (antigen expression rate (%) = antigen content / total protein × 100%).

[0105] The antigen expression rate in the fermentation culture medium is shown in Table 3:

[0106] Table 3

[0107] Sample name Antigen content (μg / ml) Total protein content (μg / ml) Antigen expression rate (%) Control strain - fermentation broth 98.32 2424.99 4.05 New strains - fermentation broth 125.44 2670.73 4.70

[0108] It can be seen from Table 3 that the optimized sequence of the HPV 16L1 protein gene of the present invention can not only express the HPV 16L1 protein in Pichia pastoris, but also increase the expression rate by 16% compared with the control strain.

[0109] Example 7 Antigen Characterization Item Detection

[0110] The purified samples were subjected to stock solution characterization analysis: thermal stability analysis, SEC-MALS molecular weight detection, TEM electron microscopy, Octet affinity detection, etc.

[0111] (1) Using turbidity and DSF to test the thermal stability of purified antigens

[0112] 1) Turbidimetric thermal stability analysis:

[0113] The 16L1 protein sample was placed in a 50°C constant temperature metal bath, and the absorbance was measured at 350 nm using an ELISA reader every 10 min. The data were collected after 60 min, with the observation time as the horizontal axis and OD as the horizontal axis. 350nm The value is the ordinate, and curve fitting is performed, OD 350nm The faster the increase, the more unstable the sample. Figure 4 ,Depend on Figure 4 It can be seen that the stability of the 16L1 protein in the stock solution of the present application (new stock solution) is significantly better than that in the control stock solution.

[0114] 2)DSF thermal stability analysis

[0115] Take 20μl 16L1 protein sample and add it to the PCR plate, add 5μl SYPRO Orange dye (diluted 400 times), shake it thoroughly, seal the film, and perform DSF thermal stability test on the Bio-Rad CFX96 TOUCH PCR instrument. The program is "Sample Volume 25μl, Melt Curve 25℃to 95℃, increment 0.5℃", select FRET as the channel, wait for the reaction to end, stop the machine, open the Bio-Rad CFX Manager software for data analysis, and the results are as follows: Figure 5As shown by Figure 5 It can be seen that the new stock solution is more stable than the control stock solution.

[0116] (2) SEC-MALS molecular weight detection of 16 L1 protein stock solution

[0117] Dilute 16 L1 protein stock solution with mobile phase to between 0.3 and 0.7 mg / ml, centrifuge at 12000 rpm for 10 min, take the supernatant and place it in a sample bottle for testing.

[0118] Chromatographic column: Bio Core SEC-1000A (5 μm, 7.8 × 300 mm), manufacturer: NanoPu Biotechnology;

[0119] Mobile phase: 0.15 M ammonium formate + 0.25 M sodium chloride, pH 6.5, flow rate 0.8 ml / min, detection wavelength 280 nm;

[0120] The molecular weight of VLPs was analyzed using an Agilent 1260 Infinity II HPLC instrument and a multi-angle laser light scattering instrument (MALS, WYATT).

[0121] Depend on Figure 6 It can be seen that the molecular weight of the main peak protein stock solution is 17326 kDa, which is slightly higher than the control stock solution (10713 kDa) and closer to the theoretical size.

[0122] (3) TEM detection of 16 L1 protein stock solution

[0123] The 16L1 protein sample was subjected to negative staining by TEM. Figure 7 (TEM transmission electron microscopy of protein stock solution in this application) and Figure 8 (TEM transmission electron microscopy of control protein stock solution). Both the protein stock solution of the present application and the control stock solution can form VLP structure. At the same time, the particle size of the stock solution of the present application is slightly larger than that of the control stock solution, the structure is more compact, the assembly efficiency is higher, and the uniformity is better.

[0124] (4) Perform affinity testing of antigen-antibody interaction (Octet)

[0125] The V5 neutralizing mouse monoclonal antibody was diluted to 10 μg / ml with PBST solution and coupled to the AMC Biosensors (Sartorius) probe. The purified 16 L1 protein was diluted to a uniform concentration of 60 μg / ml with PBST solution. The Octet (Sartorius) analysis software was used. The Octet instrument program settings are shown in Table 4 to investigate the binding ability of the V5 neutralizing mouse monoclonal antibody to the 16 L1 protein.

[0126] Table 4

[0127] Procedure Phase Time(s) Remark Baseline 60 PBST solution Loading 180 V5 Antibody Baseline 2 60 PBST solution Association 90 16 L1 antigen Dissociation 120 PBST solution Regeneration 30 Glycine solution

[0128] The binding ability of V5 to 16 L1 protein is as follows Fig. 9 As shown by Fig. 9 It can be seen that under the same conditions, the binding height (binding amount) of the protein solution of the present application to the V5 neutralizing mouse monoclonal antibody is significantly higher than that of the protein solution of the control group, and the affinity Kd (M) values ​​are both between 10 -11 There is no significant difference in magnitude.

[0129] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. An artificially designed polynucleotide encoding the major capsid protein L1 of human papillomavirus type 16, characterized in that: The polynucleotide sequence includes one or more site mutations at positions 141, 360, 777, 1098 or 1293 based on the parent sequence, and the parent sequence is shown in SEQ ID NO.

2.

2. The polynucleotide according to claim 1, characterized in that The polynucleotide sequence comprises a mutation of the base at one or more of positions 141, 360, 777, 1098 or 1293 to thymine in the parent sequence.

3. The polynucleotide according to claim 2, characterized in that The polynucleotide sequence is shown in SEQ ID NO.1 or its complementary sequence.

4. A Pichia pastoris expression vector, characterized in that: The vector comprises the polynucleotide according to any one of claims 1 to 3.

5. The expression vector according to claim 4, characterized in that The expression vector is selected from one or more of pGEX-6p-1, pGEX-4T-2, pMAL, pPICZ, pPIC6, pGAPZ, pAO815 and pET28a; preferably one or more of pPICZ, pPIC6, pGAPZ, pAO815, and more preferably pPICZαB.

6. A recombinant strain, characterized in that: The strain comprises the expression vector of claim 4 or 5, or the polynucleotide of any one of claims 1 to 3 is integrated into its genome; Preferably, the strain is a Pichia pastoris strain.

7. The recombinant strain according to claim 6, characterized in that The method for constructing the recombinant strain comprises the following steps: (1) Designing and synthesizing the polynucleotide according to any one of claims 1 to 3; (2) constructing a Pichia pastoris expression vector containing the polynucleotide sequence obtained in step (1); (3) Transforming the expression vector obtained in step (2) into a host cell, culturing the transformant, and obtaining the strain expressing the human papillomavirus type 16 major capsid protein L1.

8. The recombinant strain according to claim 7, characterized in that In step (3), the culturing is carried out in a YPDS medium plate containing antibiotics; Preferably, the specific steps of culturing are: spreading the transformant on a YPDS medium plate containing 80-120 μg / ml antibiotics for culturing, picking positive clones and spreading them on a YPDS medium plate containing 400-600 μg / ml antibiotics for culturing, picking positive clones and spreading them on a YPDS medium plate containing 800-1200 μg / ml antibiotics for culturing, picking positive clones and spreading them on a YPDS medium plate containing 1500-2500 μg / ml antibiotics for culturing to isolate the strain expressing the major capsid protein L1 of human papillomavirus type 16; Preferably, the expression vector is selected from one or more of pGEX-6p-1, pGEX-4T-2, pMAL, pPICZ, pPIC6, pGAPZ, pAO815 and pET28a, preferably one or more of pPICZ, pPIC6, pGAPZ, pAO815, more preferably pPICZαB; Preferably, the antibiotic is bleomycin.

9. A method for preparing human papillomavirus type 16 major capsid protein L1, characterized in that: The method comprises culturing the strain as claimed in claim 6, and optionally isolating the major capsid protein L1 of human papillomavirus type 16 from the culture fluid; Preferably, the method further comprises the step of purifying human papillomavirus type 16 major capsid protein L1; Preferably, the step of purifying the human papillomavirus type 16 major capsid protein L1 is to purify using a POROS 50HS chromatography column and a CHT II chromatography column in sequence.

10. Use of the polynucleotide according to any one of claims 1 to 3, or the expression vector according to claim 4 or 5, or the recombinant strain according to any one of claims 6 to 8 in the preparation of an HPV vaccine.

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