Method for assembling human papilloma virus virus-like particles

Abstract

The invention relates to the field of medicine, in particular to a method for assembling virus-like particles by human papilloma virus L1 protein. Comprising the following steps: putting various types of L1 pentamer protein solutions of HPV (human papillomavirus) into a buffer solution with the pH value of 4.5-6.0 and the salt concentration of 2-5M, and assembling the L1 pentamer protein solutions into virus-like particles under the condition that the oxygen introduction amount is 0.04-0.25 L / min. By controlling and optimizing oxygen, temperature and other conditions in the process of assembling the human papilloma virus L1 pentamer protein solution into the virus-like particles, the homogeneity and stability of the prepared virus-like particles are improved, and the antigenicity and immunogenicity of the virus-like particles are further improved.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically to a method for assembling virus-like particles from human papillomavirus L1 protein. Background Art

[0002] Human papillomavirus (HPV) is a small, non-enveloped virus. Their genetic material consists of circular double-stranded DNA, approximately 8000 base pairs. They cause lesions by infecting and replicating in the skin and mucous membranes. The double-stranded DNA virus has high stability and a low mutation rate, which allows the virus to remain consistent with the pathogen for a long time. So far, more than 200 different virus types have been identified, and 15 of them (such as HPV16, HPV18, HPV33, HPV45, and HPV59) are considered high-risk because they are the main pathogenic genes for cervical cancer and other anogenital cancers (such as anal cancer, vulvar cancer, and penile cancer). The surface of HPV is coated with two capsid proteins, L1 and L2. HPV infects epithelial cells by binding L1 protein to heparan sulfate proteoglycans (HSPGs) on the basement membrane; while the L2 gene affects the expression, localization, transport, and assembly of L1 protein in vivo. The L1 protein is the main capsid protein with a molecular weight of 53 kDa, which can spontaneously assemble into pseudovirus particles (Virus-like particles, VLPs) in an in vitro recombinant system, forming an icosahedral structure with T = 7. VLPs present a series of antigenic epitopes, similar to those found in natural virus particles, with good safety and protective efficacy, which lays the foundation for the development of HPV vaccines.

[0003] Currently, the six marketed HPV vaccines (Cervarix®, Gardasil®, Gardasil®9, Xinkening®, Wozehui®, Cervavac) all belong to VLPs vaccines. Monitoring and optimizing various characteristics of HPV VLPs (such as thermal stability, particle homogeneity, immunogenicity, etc.) are necessary for the vaccine to immunize the body to achieve the best preventive effect. Among them, the assembly process directly affects the homogeneity and stability of the particles, and thus affects the immunogenicity of the particles. Therefore, it is necessary to study and optimize the assembly method of HPV L1-VLPs. Summary of the Invention

[0004] Based on the needs of the prior art, the present invention provides a method for assembling human papillomavirus virus-like particles.

[0005] The present invention provides a method for assembling human papillomavirus virus-like particles, which comprises the following steps: Place the HPV L1 pentamer protein solution in a buffer with a pH value of 4.5 - 6.0 and a salt concentration of 2 - 5 M, and assemble it into virus-like particles under an oxygen flow rate of 0.04 - 0.25 L / min.

[0006] In a specific embodiment, the temperature of the assembly is 22°C - 28°C, preferably 24°C - 26°C, for example, 25°C.

[0007] In a preferred embodiment, the oxygen flow rate is 0.05 - 0.2 L / min.

[0008] In a specific embodiment, the assembly time is 1 - 20 h, preferably 5 - 15 h.

[0009] Specifically, the concentration of the HPV L1 pentamer protein in the buffer is 1.5 - 11 mg / ml.

[0010] Preferably, the average particle size of the assembled virus-like particles is 50 - 75 nm, and the purity is greater than 95.0%.

[0011] In a specific embodiment, the HPV L1 pentamer protein is prepared by the following method: Construct an Escherichia coli expression vector containing the coding gene of the fusion protein of HPV L1; Construct an Escherichia coli expression engineering strain containing the expression vector, induce expression in the Escherichia coli expression engineering strain, and purify the fusion protein by chromatography to obtain the HPV L1 pentamer protein; Specifically, the HPV L1 pentamer protein is the L1 pentamer protein of HPV type 6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 or HPV68.

[0012] The present invention provides the human papillomavirus virus-like particles obtained by the above-mentioned assembly method.

[0013] The present invention further provides the application of the human papillomavirus virus-like particles in the preparation of HPV antigens, or HPV vaccines or diagnostic reagents.

[0014] Based on the problems discovered during the actual research process, the present invention improves the uniformity and stability of the prepared virus-like particles by controlling and optimizing the oxygen content and temperature conditions during the assembly of the human papillomavirus L1 pentamer protein solution into virus-like particles. This further enhances the antigenicity and immunogenicity of the virus-like particles and solves the problem of preparing virus-like particles in different altitude regions. Specifically, during the technology transfer of the HPV vaccine to different locations, the inventors found that ideal L1-VLPs of various HPV types could not be obtained. After repeated exploration, it was found that the relatively high altitude of the location, for example, the oxygen content in Kunming is about 80% of that in Beijing, and the lower oxygen content is not conducive to the bonding of disulfide bonds (oxidation reaction) during the assembly process. Therefore, the inventors considered controlling the oxygen conditions during the assembly of the L1 pentamer protein solution of various HPV types into virus-like particles. By increasing the oxygen concentration during the assembly process, the problem of poor assembly effect and long time consumption in high-altitude regions and other different locations was solved. At the same time, the uniformity and stability of the prepared virus-like particles were also improved, further enhancing the antigenicity and immunogenicity of the virus-like particles. Detailed implementation manners

[0015] The present invention will be described below through specific implementation manners to better understand the present invention, but it does not constitute a limitation to the present invention.

[0016] The preparation of the L1 pentamer protein solution of various HPV types can refer to the published literature of the prior art. The L1 pentamer protein solution of various HPV types can be prepared by the following methods: design of the L1 protein sequence, construction of the recombinant expression vector, expression of the recombinant host cell, purification of the recombinant protein, etc.

[0017] Taking the preparation of the HPV6 L1 pentamer protein solution as an example, the process can be referred to as follows: The artificially synthesized truncated nucleotide sequence of HPV6 L1 is (SEQ ID NO.2). First, the DNA fragment of HPV6 L1 is amplified by PCR. The L1 gene PCR fragment containing the NdeI and Xho1 restriction enzyme sites and the recombinant vector pKL1 are respectively digested with NdeI / Xho1 double restriction enzymes and the digested fragments are recovered. Then, the recovered gene fragment is ligated with the vector fragment pKL1 containing the corresponding sticky ends using T4 DNA ligase, and the ligation reaction is carried out at 16°C for 10 - 15 h.

[0018] The ligation system is as follows: 6 μl of the pKL1 vector fragment, 2 μl of the HPV6L1 gene fragment, 1 μl of T4 DNA ligase, and 1 μl of T4DNA Ligase buffer. After the ligation reaction, the ligation product is transformed into E. coliScreen the recombinants in DH5α. Expand the screened monoclonal colonies and extract the plasmids, and then perform sequencing verification to obtain the recombinant expression vector pKL1-HPV6L1.

[0019] Transform the recombinant vector with correct sequencing results into Escherichia coli BL21 host cells, and use it as an engineering bacterium for expressing recombinant proteins to express HPV6 L1 protein. The engineering bacterium medium is 2YT medium (10 g / L tryptone; 5 g / L yeast extract; 10 g / L NaCl). Pick a single colony containing the recombinant plasmid into 10 ml of 2YT medium (containing 100 μg / ml ampicillin), shake culture at 230 rpm / min at 37 °C overnight. Transfer 5 ml of the overnight culture into 500 ml (containing 100 μg / ml ampicillin) of 2YT liquid medium, shake culture at 37 °C until the recombinant engineering bacterium grows to OD600nm≈0.4 - 1, add IPTG with a final concentration of 0.2 mM for induction, and perform induction expression of recombinant proteins for more than 6 hours at 28 °C. Collect the bacteria and break them, and then collect the supernatant.

[0020] Affinity chromatography of the recombinant protein with GST tag: Load 5 ml of GST agarose affinity chromatography medium into the affinity column, equilibrate the chromatography column with buffer L (pH 8.0, 50 mM Tris, 200 mM NaCl, 5 mM DTT), then load the protein solution with GST tag, and after loading, add 3C enzyme for digestion. After digestion, wash with Buffer L until no protein flows out, collect the eluate, and the affinity chromatography is completed.

[0021] Purification by gel filtration chromatography: Further purify the HPV6 L1 pentamer protein purified by affinity chromatography collected in the previous step. It can first pass through ion exchange chromatography to collect the HPV6 L1 pentamer protein, or directly use the gel filtration medium of Superdex200 (produced by GE) for further gel filtration chromatography without passing through the ion exchange chromatography step. The mobile phase is pH 8.0, 10 mM Tris, 100 mM NaCl, and collect the HPV6 L1 pentamer protein solution.

[0022] The nucleotide sequence of the truncated HPV6L1 mentioned above refers to the amino acid sequence used in Example 1 of Patent CN202410202916.9, and the SEQ ID NO.1 in its sequence listing (the nucleotide sequence is SEQ ID NO.2). The acquisition of the L1 pentamer protein solutions of HPV33, HPV45, HPV16, and HPV31 types involved in the following examples all refer to Example 1 of Patent CN202410202916.9, and the amino acid sequences used are SEQ ID NO.11 (nucleotide sequence is SEQ ID NO.12), SEQ ID NO.13 (nucleotide sequence is SEQ ID NO.13), SEQ ID NO.5 (nucleotide sequence is SEQ ID NO.6), and SEQ ID NO.9 (nucleotide sequence is SEQ ID NO.10) in its sequence listing respectively.

[0023] Example 1. Study on the assembly of HPV33 L1 pentamer protein solution into virus-like particles The assembly of the HPV33 L1 pentamer protein solution was carried out in Beijing and Kunming respectively.

[0024] In Beijing and Kunming, the method for assembling the HPV33 type L1 pentamer protein solution into virus-like particles includes the following steps: placing the HPV33 type L1 pentamer protein solution in a constant temperature water bath at 25°C for assembly respectively; the assembly solution in the constant temperature water bath is a buffer solution with a pH value of 5.25 and a salt concentration of 3M; two groups are set respectively (30 ml of each group of samples), one group of samples is placed normally (control group); the other group of samples is aerated with oxygen (oxygen aeration group). Specifically, a household handheld oxygen cylinder is connected to a clean silica gel tube, and the end of the silica gel tube is inserted into the bottom of the sample. Under the oxygen aeration amount of 0.05 L / min, oxygen is aerated for 1 minute, and then sealed and left to stand. After standing, HPV33 type virus-like particles are obtained; samples are taken at 1 hour, 3 hours, 6 hours, 9 hours, 15 hours, and 19 hours of standing respectively. After each sampling, oxygen is aerated for 1 minute and then sealed and left to stand. Sampling is carried out according to this cycle.

[0025] After 1 hour of assembly, the particle size and protein content of the two groups of samples were detected respectively as the initial data. After 3, 6, 9, 15, and 19 hours, the particle size, relative antigenic potency, HPLC purity, and disulfide bond content of the two groups of samples were detected respectively to investigate the influence of oxygen on the physical and chemical properties of the protein samples at different time points of 3 hours, 6 hours, 9 hours, 15 hours, and 19 hours. The detection results of HPV33 type virus-like particles are shown in Table 1.

[0026] The detection methods of the protein samples at the above different time points are as follows: Particle size: The particle size of the sample was determined by dynamic light scattering method. 1 mL of the test solution sampled at each time point was added to a colorimetric cell dedicated for particle size detection. The particle size and particle size distribution of the sample at each assembly time point were measured using a dynamic light scattering nanoparticle size analyzer, Malvern Zetasizer Nano ZS (the particle size distribution coefficient PdI value was used as an index of particle size dispersion, a sample with a value less than 0.05 was a highly homogeneous sample; a sample with a value of 0.05 - 0.1 was a quasi-homogeneous sample, a sample with a value of 0.1 - 0.3 was a sample with poor homogeneity, and a sample with a value greater than 0.3 was a heterogeneous sample). By comparing the changes in the particle size and uniformity of VLP at the same time point, the effect of oxygen on the after-ripening process of the protein sample was explored.

[0027] Protein content: The protein content of the sample was determined by the BCA method. The protein content determination standard product HSA was taken and serially diluted to prepare a standard curve of gradient concentrations; the test samples were diluted to different gradients with a diluent so that the diluted concentration gradients were within the concentration range of the standard curve. The diluted standard solution and test solution were added to a 96-well plate at 20 μL / well. Finally, the alkaline copper test solution was added to the 96-well plate at 200 μL / well, the sealing film was covered, and the plate was placed in a 37 °C electrothermal constant temperature incubator for 30 minutes. The absorbance was measured at a wavelength of 570 nm. The linear regression equation was calculated using the series of standard concentrations and their corresponding absorbances, and the absorbance of the test sample was substituted into the equation to obtain the protein content of the test sample.

[0028] Relative antigenic potency: The relative antigenic potency was determined by ELISA method. Sample preparation: After diluting the reference substance and the test samples sampled at each time point to the same concentration, they were each serially diluted by a factor of two to 7 concentration gradients; the corresponding type of ELISA microtiter plate that had been coated and blocked was taken, and the reference substance and test samples were each added to the plate at 100 μL / well and incubated at 37 °C for 1 hour; the enzyme-labeled antibody was diluted to an appropriate concentration and added to the plate at 100 μL / well and incubated at 37 °C for 1 hour; freshly prepared chromogenic solution was added at 100 μL / well and incubated at room temperature in the dark for 5 minutes; the stop solution was added at 50 μL / well to terminate the reaction; the plate was placed in an ELISA reader and read at a wavelength of 450 nm; the biological assay statistical program BS2000 of the Chinese Pharmacopoeia was used, and the quantal response parallel line 3.3 method was selected for analysis to obtain the relative potency PT value of the test sample relative to the activity of the reference substance.

[0029] Purity (molecular exclusion chromatography): The purity was determined by high performance liquid chromatography. The test solution sampled at each time point was diluted to 1 mg / mL with the mobile phase. After centrifuging at 10,000 rpm for 10 minutes using a centrifuge, 50 μL of the supernatant was taken and loaded onto a sample vial for injection. High performance liquid chromatography conditions: detection wavelength 280 nm, column temperature 25 °C, isocratic elution flow rate 0.5 mL / minute, elution time 35 minutes. Result analysis: The purity of L1-VLP was analyzed by the area normalization method.

[0030] Content of disulfide bond: The detection principle is that 5,5'-dithiobis(2-nitrobenzoic acid) (abbreviated as DTNB or Ellman's Reagent) can undergo a thiol-disulfide exchange reaction with L-cysteine to generate 2-nitro-5-thiobenzoic acid (abbreviated as TNB). TNB is yellow in an alkaline environment and has a strong absorption peak at 412 nm. Therefore, the thiol (-SH) can be specifically detected through this reaction, and thus the content of L-cysteine can be quantitatively detected. Using the above method, the total thiol content and free thiol content of the samples at each assembly time point were measured respectively, and the ratio of the difference between the two to the total thiol content is the content of disulfide bond in the sample: Content of disulfide bond (%) = (Total thiol content - Free thiol content) / Total thiol content * 100%.

[0031] The above same detection method was used for the detection of each sample involved in the following examples.

[0032] Table 1. Detection results of various samples of HPV33 virus-like particles

[0033] Note: " / " indicates that this item was not detected at this sampling time point.

[0034] The detection results of various samples of HPV33 virus-like particles in Beijing and Kunming shown in Table 1 indicate that at each time point (3h, 6h, 9h, 15h, 19h), the particle size of the oxygenated group and the control group of HPV33 (Beijing) L1-VLP protein solution changed little. Judging from the PdI data, after 3h of assembly, the PdI data of the oxygenated group was the smallest. From the results of the relative antigenic potency, at each time point (3h, 6h, 9h, 15h, 19h), the relative antigenic potency of both the oxygenated group and the control group increased, and at 19h, the relative antigenic potency of the oxygenated group was 3.5 times that of the control group. The HPLC detection results showed that at each time point (3h, 6h, 9h, 15h), the purity of both the oxygenated group and the control group was greater than 97%, and the purity of the oxygenated group was the highest at 15h, reaching 99.1%. From the results of the content and proportion data of disulfide bonds, as the time increased to 19h, both the content and proportion of disulfide bonds in the oxygenated group showed an increasing trend. At 19h, the content of disulfide bonds in the oxygenated group increased by 4.4% compared with the control group, and the proportion of disulfide bonds in the oxygenated group increased by 13.5% compared with the control group.

[0035] Table 1 also shows that the particle size results of the oxygenated group and the control group of the HPV33 type (Kunming) L1-VLP protein solution are not very different. From the PdI data, the PdI of the oxygenated group at 19 h is lower than that of the control group. From the results of the relative antigenic potency, at 9 h, 15 h, and 19 h, the relative antigenic potency of the oxygenated group and the control group shows a gradually increasing trend, and at 9 h, the relative antigenic potency of the oxygenated group is higher than that of the control group. The relative antigenic potency is positively correlated with its immunogenicity. The relative potency experiment directly detects the antigen-antibody binding ability by ELISA method. The increase in relative antigenic potency means that the conformational correctness of the recombinant protein is improved (such as being closer to the structure of the natural pathogen), making the antigenic epitopes more fully exposed and having a stronger ability to bind antibodies in ELISA. At the same time, it also indicates that the functional antigenicity of the vaccine is good, providing a basis for immunogenicity.

[0036] This application uses the truncated L1 protein of HPV33 type, mainly to improve the soluble expression of the L1 pentamer protein by Escherichia coli, and does not affect the immunogenicity of the L1-VLPs protein. Only part of the C-terminal and N-terminal of the HPV33 type L1 protein are truncated, and the oxygen content in the above assembly method is sufficient, so it is considered that the full-length sequence (not truncated) of the HPV33 type L1 protein is also applicable to the above assembly method.

[0037] Based on the above analysis, increasing the oxygen concentration during the assembly process helps to accelerate the assembly process and improve the potency of the HPV33 type L1 pentamer protein solution.

[0038] Example 2. Study on the assembly of HPV45 L1 pentamer protein solution into virus-like particles The assembly of the HPV45 L1 pentamer protein solution was carried out in Beijing and Kunming respectively.

[0039] In Beijing and Kunming, the method for assembling the HPV45 L1 pentamer protein solution into virus-like particles includes the following steps: placing the HPV45 L1 pentamer protein solution in a 28°C constant temperature water bath for assembly; the assembly solution in the constant temperature water bath is a buffer solution with a pH value of 5.25 and a salt concentration of 4.5 M; two groups are set up respectively (30 ml of each group of samples), one group with the samples placed normally (control group); the other group with the samples oxygenated (oxygenated group), specifically using a household handheld oxygen cylinder connected to a clean silica gel tube, inserting the end of the silica gel tube into the bottom of the sample, oxygenating for 1 minute at an oxygen flow rate of 0.1 L / min, then sealing and starting to place, and obtaining HPV45 virus-like particles after placement; sampling at 1 hour, 9 hours, 15 hours, and 19 hours after placement respectively, and oxygenating for 1 minute again after each sampling and then sealing and placing, and sampling according to this cycle.

[0040] One hour after assembly, the particle size and protein content of the two groups of samples were detected respectively as the initial data. After 9, 15, and 19 hours, the particle size, relative antigenic potency, HPLC purity, and disulfide bond content of the two groups of samples were detected respectively. The detection methods were the same as those in Example 1 above. The influence of oxygen on the physicochemical properties of the protein samples at different time points of 9 hours, 15 hours, and 19 hours was investigated. The detection results of HPV45 virus-like particles are shown in Table 2.

[0041] Table 2. Detection Results of Each Sample of HPV45 Virus-Like Particles

[0042] Note: " / " indicates that this item was not detected at this sampling time point.

[0043] Table 2 shows the detection results of each sample of HPV45 virus-like particles in Beijing and Kunming. From the results in Table 2, it can be seen that as the time increased to 19 h, the particle sizes of the oxygen-added group and the control group of the HPV45 (Beijing) L1-VLP protein solution were both smaller than those at 1 h. Judging from the PdI data, at each time point (1 h, 9 h, 15 h, 19 h), the PdI values changed little. From the results of relative antigenic potency, at 9 h, 15 h, and 19 h, the relative antigenic potencies of the oxygen-added group and the control group both increased, and at 19 h, the relative antigenic potency of the oxygen-added group was 8.5 times that of the control group. The HPLC detection results showed that at each time point (1 h, 9 h, 15 h, 19 h), the purities of the oxygen-added group and the control group were both greater than 99%. From the results of the disulfide bond content data, as the time increased to 19 h, the disulfide bond content of the oxygen-added group showed an increasing trend. Compared with that at 1 h, it increased by 8.2%, and compared with the control group, the disulfide bond content of the oxygen-added group at 19 h was 5.7% higher than that of the control group. From the results of the disulfide bond ratio data, as the time increased to 19 h, the disulfide bond ratios of the oxygen-added group and the control group changed little.

[0044] Table 2 also shows that the particle size of the oxygen-added group and the control group of the HPV45 (Kunming) L1-VLP protein solution increased by about 7 nm at 19 h compared with that at 1 h, and the PdI values of the two groups changed little. From the results of relative antigenic potency, at the three time points of 9 h, 15 h, and 19 h, the relative antigenic potency of the oxygen-added group was higher than that of the control group, and at 19 h, the relative antigenic potency of the oxygen-added group was the highest. Similar to the description of the full-length sequence of the HPV33 L1 protein in Example 1, the full-length sequence of the HPV45 L1 protein is also applicable to the above-mentioned assembly method.

[0045] Based on the above analysis, increasing the oxygen concentration during the assembly process helps to accelerate the assembly process of the HPV45 L1 pentamer protein solution.

[0046] Study on the Assembly of HPV6 L1 Pentamer Protein Solution into Virus-like Particles The method for assembling the HPV6 L1 pentamer protein solution into virus-like particles comprises the following steps: placing the HPV6 L1 pentamer protein solution in a constant temperature water bath at 24°C for assembly; the assembly solution in the constant temperature water bath is a buffer solution with a pH value of 5.25 and a salt concentration of 2.5 M; two groups are set respectively (with a sample volume of 30 ml in each group), in one group, the sample is placed normally (control group); in the other group, the sample is aerated with oxygen (oxygen-aerated group), specifically, a household handheld oxygen cylinder is connected to a clean silica gel tube, the end of the silica gel tube is inserted into the bottom of the sample, and oxygen is aerated for 1 minute at an oxygen flow rate of 0.2 L / min, then sealed and left to stand, and HPV6 virus-like particles are obtained after standing; samples are taken at 1 hour, 3 hours, 6 hours, 9 hours, 15 hours, and 19 hours respectively, and after each sampling, oxygen is aerated for 1 minute again and then sealed and left to stand, and sampling is carried out in this cycle.

[0047] After 1 hour of assembly, the particle size and protein content of the two groups of samples are detected respectively as the initial data. After 3 hours, 6 hours, 9 hours, 15 hours, and 19 hours, the particle size, relative antigenic potency, HPLC purity, and disulfide bond content of the two groups of samples are detected respectively, and the detection methods are the same as those in Example 1 above; the influence of oxygen on the physicochemical properties of the protein samples at different time points of 3 hours, 6 hours, 9 hours, 15 hours, and 19 hours is investigated. The detection results of the HPV6 virus-like particles are shown in Table 3.

[0048] Table 3. Detection Results of Each Sample of HPV6 Virus-like Particles

[0049] Note: " / " indicates that this item was not detected at this sampling time point.

[0050] Table 3 shows the detection results of various samples of HPV6 virus-like particles. From the results in Table 3, it can be seen that at each time point (1h, 3h, 6h, 9h, 15h, and 19h), the particle size of the HPV6 L1-VLP protein solution in the oxygen addition group and the control group changed little. From the PdI data, after 3h of assembly, the PdI data of the oxygen addition group and the control group were the smallest. From the relative antigen potency data, it can be seen that at 3h, the relative antigen potency of the oxygen addition group was much higher than that of the control group. At 19h, this gap decreased, but the relative antigen potency of the oxygen addition group was still twice that of the control group. The HPLC detection results showed that at each time point (3h, 6h, 9h, 15h, 19h), the purity was greater than 95%. From the data on the content of disulfide bonds, the content of disulfide bonds in the oxygen addition group and the control group changed little at each time point (3h, 6h, 9h, 15h, 19h). From the perspective of the disulfide bond ratio, the disulfide bond ratio of the oxygen addition group showed an increasing trend with the increase of time, and at 6h, 9h, 15h, and 19h, the disulfide bond ratio of the oxygen addition group was higher than that of the control group. By 19h, the disulfide bond ratio of the oxygen addition group increased by 9% compared with the control group. Similar to the description of the full-length sequence of the HPV33 type L1 protein in Example 1, the full-length sequence of the HPV6 type L1 protein is also applicable to the above assembly method.

[0051] In summary, increasing the oxygen concentration during the assembly process helps to accelerate the assembly process of the HPV6 type L1 pentamer protein solution.

[0052] Example 4. Study on the assembly of HPV16 L1 pentamer protein solution into virus-like particles The method for assembling the HPV16 L1 pentamer protein solution into virus-like particles includes the following steps: placing the HPV16 L1 pentamer protein solution in a constant temperature water bath at 28°C for assembly; the assembly solution in the constant temperature water bath is a buffer solution with a pH value of 6.0 and a salt concentration of 3M; two groups are set respectively (30 ml of each group of samples), one group of samples is placed normally (control group); in the other group, the samples are aerated with oxygen (oxygen aeration group). Specifically, a household handheld oxygen cylinder is connected to a clean silica gel tube, and the end of the silica gel tube is inserted into the bottom of the sample. Under an oxygen flow rate of 0.15 L / min, oxygen is aerated for 1 minute, and then sealed and placed. After placement, HPV16 virus-like particles are obtained; samples are taken at 1 hour, 3 hours, 6 hours, 9 hours, 15 hours, and 19 hours after placement. After each sampling, oxygen is aerated for 1 minute and then sealed and placed, and sampling is carried out in this cycle.

[0053] After 1 hour of assembly, the particle size and protein content of the two groups of samples were detected respectively as the initial data. After 3, 6, 9, 15, and 19 hours, the particle size, relative antigenic potency, HPLC purity, and disulfide bond content of the two groups of samples were detected respectively, and the detection methods were the same as those in Example 1 above; the influence of oxygen on the physicochemical properties of the protein samples at different time points of 3 hours, 6 hours, 9 hours, 15 hours, and 19 hours was investigated. The detection results of HPV16 virus-like particles are shown in Table 4.

[0054] Table 4. Detection results of various samples of HPV16 virus-like particles

[0055] Note: " / " indicates that this item was not detected at this sampling time point.

[0056] Table 4 shows the detection results of various samples of HPV16 virus-like particles. From the results in Table 4, it can be seen that at each time point (1h, 3h, 6h, 9h, 15h, 19h), the particle size of the HPV16 L1-VLP protein solutions in the oxygen-added group and the control group did not change significantly. From the PdI data, at 15h, the PdI values of the oxygen-added group and the control group were the smallest. From the relative antigenic potency data, it can be seen that at each time point (3h, 6h, 9h, 15h, 19h), the relative antigenic potency of the oxygen-added group and the control group showed an increasing trend, reaching the highest at 15h and continuing to 19h. At 19h, the relative antigenic potency of the oxygen-added group was 5.5 times that of the control group. The HPLC detection results showed that at each time point (3h, 6h, 9h, 15h, 19h), the purity was greater than 98%; and the HPLC purity of the oxygen-added group and the control group reached 100% at 6h and 9h. From the disulfide bond content data, the disulfide bond content of the oxygen-added group and the control group reached the highest at 15h; although the disulfide bond content decreased at 19h, the disulfide bond content and ratio of the oxygen-added group were significantly higher than those of the control group. From the analysis of the disulfide bond ratio data, at 9h, the disulfide bond ratio of the oxygen-added group increased by 12.2% compared with the control group, and at 19h, the disulfide bond ratio of the oxygen-added group increased by 8.2% compared with the control group. Similar to the description of the full-length sequence of HPV33 L1 protein in Example 1, the full-length sequence of HPV16 L1 protein is also applicable to the above assembly method.

[0057] In summary, increasing the oxygen concentration during the assembly process helps to accelerate the assembly process of the HPV16 L1 pentamer protein solution.

[0058] Example 5. Study on the assembly of HPV31 L1 pentamer protein solution into virus-like particles The method for assembling the HPV31 L1 pentamer protein solution into virus-like particles includes the following steps: placing the HPV31 L1 pentamer protein solution in a constant temperature water bath at 22°C for assembly; the assembly solution in the constant temperature water bath is a buffer solution with a pH value of 4.5 and a salt concentration of 3.5 M; two groups are set respectively (with a sample volume of 30 ml in each group), in one group, the sample is placed normally (control group); in the other group, the sample is aerated with oxygen (oxygen aeration group), specifically, a household handheld oxygen cylinder is connected to a clean silica gel tube, the end of the silica gel tube is inserted into the bottom of the sample, and oxygen is aerated at a rate of 0.12 L / min for 1 minute, then sealed and left to stand, and HPV31 virus-like particles are obtained after standing; samples are taken at 1 hour, 3 hours, 7 hours, 9 hours, 12 hours, 15 hours, and 19 hours after standing, and after each sampling, oxygen is aerated for 1 minute and then sealed and left to stand, and sampling is carried out in this cycle.

[0059] After 1 hour of assembly, the particle size and protein content of the two groups of samples are detected respectively as the initial data. After 3, 7, 9, 12, 15, and 19 hours, the particle size and relative antigenic potency of the two groups of samples are detected respectively, and the detection method is the same as that in Example 1 above; the influence of oxygen on the physicochemical properties of the protein samples at different time points of 3 hours, 7 hours, 12 hours, and 19 hours is investigated. The detection results of the HPV31 virus-like particles are shown in Table 5.

[0060] Table 5. Detection results of various samples of HPV31 virus-like particles

[0061] Table 5 shows the detection results of various samples of HPV31 virus-like particles. It can be seen from the results in Table 5 that as time increases to 19 h, the particle sizes of the samples in the oxygen addition group and the control group both increase. Judging from the PdI data, the PdI values of the oxygen addition group and the control group change little. It can be seen from the relative antigenic potency data that at the three time points of 3 h, 7 h, and 12 h, the relative antigenic potency of the oxygen addition group is higher than that of the control group, and reaches the highest at 12 h, about 1.2 times that of the control group; at 19 h, the relative antigenic potencies of the two groups tend to be the same. Similar to the statement of the full-length sequence of the HPV33 type L1 protein in Example 1, the full-length sequence of the HPV31 type L1 protein is also applicable to the above assembly method.

[0062] In summary, increasing the oxygen concentration during the assembly process helps to accelerate the assembly process of the HPV31 L1 pentamer protein solution.

Claims

1. A method for assembling human papillomavirus virus-like particles, characterized in that: The following steps are involved: The HPV L1 pentamer protein solution was placed in a buffer solution with a pH value of 4.5-6.0 and a salt concentration of 2-5 M, and assembled into virus-like particles at an oxygen flow rate of 0.04-0.25 L / min.

2. The assembly method according to claim 1, characterized in that: The assembly temperature is 22°C to 28°C, preferably 24°C to 26°C, for example 25°C.

3. The assembly method according to claim 1, characterized in that: The oxygen flow rate is 0.05~0.2 L / min.

4. The assembly method according to claim 1, characterized in that: The assembly time is 1-20 hours, preferably 5-15 hours.

5. The assembly method according to claim 1, characterized in that: The concentration of the HPV L1 pentamer protein in the buffer is 1.5-11 mg / ml.

6. The assembly method according to claim 1, characterized in that: The average particle size of the assembled virus-like particles is 50-75 nm, and the purity is greater than 95.0%.

7. The assembly method according to claim 1, characterized in that: The HPV L1 pentamer protein was prepared by the following method: Constructing an Escherichia coli expression vector containing a gene encoding a fusion protein of HPV L1; An Escherichia coli expression engineering strain containing the expression vector is constructed, expression is induced in the Escherichia coli expression engineering strain, and the fusion protein is purified by a chromatography method to obtain the HPV L1 pentamer protein.

8. The assembly method according to any one of claims 1 to 7, characterized in that: The L1 pentamer protein of HPV is L1 pentamer protein of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 or HPV68 type.

9. The human papillomavirus virus-like particle obtained by the assembly method according to any one of claims 1 to 8.

10. Use of the human papillomavirus virus-like particle according to claim 9 in the preparation of HPV antigens, or HPV vaccines or diagnostic reagents.

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