A method for detecting HPV neutralizing antibodies

Abstract

The present invention provides a method for detecting HPV neutralizing antibodies, and belongs to the field of biotechnology. The method of the present invention co-transfects 293T cells with HPV capsid protein L1, L2 expression plasmids and GFP expression plasmids to obtain pseudovirus particles containing the GFP gene, incubates the pseudovirus with a serum sample to be tested and then infects the target cells, and then detects the percentage of cells expressing GFP in all cells by flow cytometry to quantify the amount of neutralizing antibodies contained in the serum sample to be tested. The method of the present invention has the characteristics of easy operation, high throughput, high sensitivity, etc., and can obtain HPV VLPs with higher titers and better infection efficiency. It is used to detect the HPV neutralizing antibody titer in serum, and its detection accuracy is higher and repeatability is good.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting HPV neutralizing antibodies. Background Art

[0002] Human papilloma virus (HPV) belongs to the genus Papillomavirus of the family Papillomaviridae. It is a spherical, non-enveloped, double-stranded circular DNA virus. The HPV genomic DNA is about 7.8 to 8.0 kb, which is divided into an early region related to viral replication and cell transformation, a late region encoding capsid protein, and a regulatory region involved in transcriptional regulation. The integration of HPV virus into the host genome usually occurs in the genomic region of transcriptional expression. The virus uses this mechanism to increase the expression of oncogenes such as E6 and E7, inactivate tumor suppressor factors p53 and retinoblastoma, and lead to uncontrolled cell proliferation and accumulation of mutations, thus causing cervical lesions.

[0003] There are more than 100 known HPV genotypes, which can be divided into "low-risk" and "high-risk" types according to their carcinogenicity. The "low-risk" types mainly cause low-grade cervical epithelial necrosis and genital warts, including HPV6, HPV11, etc.; the "high-risk" types are the main causes of cervical cancer, including HPV16, HPV18, etc.

[0004] HPV plays an important role in the pathogenesis of cervical cancer. It affects apoptosis, cell cycle, cell adhesion and DNA repair mechanisms within host cells and can also activate immune responses. Genetic susceptibility, hormonal factors, host immune responses and smoking all affect persistent HPV infection and its susceptibility to cancer. Of the more than 100 HPV types, at least 14 are considered "high risk" or carcinogenic.

[0005] HPV vaccines are a form of primary prevention of cervical cancer, helping to prevent high-risk HPV strains that cause 70% of cervical cancer cases and 90% of genital warts. The safety of Cervarix (HPV genotypes 16, 18), Gardasil (HPV genotypes 6, 11, 16, and 18), and Gardasil 9 (HPV genotypes 16, 18, 31, 33, 45, 52, and 58) has been confirmed in clinical trial data and post-marketing surveillance conducted on several continents. The key to successful HPV vaccination is the production of neutralizing antibodies against HPV, so the development of a stable and effective HPV vaccine evaluation system to detect the protective effect of the immune response is of great value for vaccine development and quality control.

[0006] Neutralization test is the "gold standard" for evaluating whether antibodies induced by HPV vaccines have protective effects. The existing methods for detecting neutralizing antibodies against HPV are mainly the following:

[0007] 1. Neutralizing antibody detection method based on human keratinocytes: This method is to pre-incubate HPV with the test serum for a certain period of time, and then infect human keratinocytes cultured in vitro. If the test serum contains HPV genotype-specific neutralizing antibodies, it can block the infection of HPV virus to keratinocytes, and the RT-PCR detection of HPV mRNA in cells is negative. The disadvantages of this method are that human keratinocytes need to be cultured, the applicable HPV genotypes are limited, the operation is complicated, time-consuming, and high throughput cannot be achieved.

[0008] 2. Enzyme-linked immunosorbent assay (ELISA) neutralizing antibody detection method: Direct ELISA is to coat the diluted virus-like particles with the ELISA plate, then add the diluted serum and enzyme-labeled secondary antibody, and determine the neutralizing antibody titer by substrate color development and ELISA reader reading. The disadvantage of direct ELISA is that it can only measure the total antibody titer instead of the neutralizing antibody titer. Competitive inhibition ELISA is to coat and seal the virus-like particles, add the serum to be tested and neutralizing monoclonal antibody labeled with horseradish peroxidase (HRP), react at room temperature, and then perform substrate color development detection. The disadvantage of competitive inhibition ELISA is that the neutralizing antibodies in the serum that do not compete with the neutralizing monoclonal antibody labeled with HRP for specific epitopes cannot be detected, resulting in false negative results. At the same time, some non-neutralizing antibodies in the serum may prevent the neutralizing antibodies from binding to the neutralizing monoclonal antibody labeled with HRP, resulting in false positive results.

[0009] 3. Pseudovirus neutralizing antibody detection method: HPV capsid protein L1, L2 expression plasmids and reporter gene expression plasmids are co-transfected into eukaryotic cells to obtain pseudovirus particles containing reporter genes. The pseudovirus is co-incubated with the serum sample to be tested and then infected into the target cells. The amount of neutralizing antibodies in the serum sample to be tested is determined by detecting whether the reporter gene is expressed in the target cells and its expression level. This method of preparing pseudoviruses using multi-plasmid co-transfection is simple to operate, short in cycle, and highly efficient, and is currently the most ideal method for neutralizing antibody detection.

[0010] However, the existing pseudovirus neutralizing antibody detection method has the problems of insufficient virus titer, low infection efficiency, and high intra-experimental and inter-experimental coefficients of variation, resulting in poor detection of HPV infection.

[0011] For example, Chinese patent document CN 103333865 B discloses an HPV pseudovirus and a kit thereof, as well as a method for detecting HPV neutralizing antibodies. This method can only detect serum samples of 6 pseudovirus types, and the detection titer of certain genotype pseudoviruses is not high enough. The intra-experimental and inter-experimental coefficient of variation (CV) of the detection method is high, and the method repeatability and precision are poor.

[0012] For another example, Chinese patent document CN 115932275 A discloses a high-throughput detection method for HPV neutralizing antibodies, which can detect 9 pseudovirus types. However, the intra-experimental variability of this method is between 7% and 28%, and the inter-experimental variability is between 12% and 23%, and there is still a problem of poor detection precision.

[0013] Therefore, how to provide a pseudovirus neutralizing antibody detection method with high virus titer, high infection efficiency, high intra-experimental coefficient of variation and inter-experimental coefficient of variation, high precision and good repeatability for detecting various types of HPV infections has become a technical problem that needs to be solved urgently. Summary of the invention

[0014] The present invention is to solve the above technical problems, thereby providing a method for packaging HPV pseudoviruses and a method for detecting HPV neutralizing antibodies. The technical purpose of the present invention is to solve the problems of low virus titer, poor detection precision and repeatability, and poor detection effect in the existing HPV neutralizing antibody detection methods.

[0015] One of the purposes of the present invention is to provide a method for packaging HPV pseudoviruses, comprising the following steps:

[0016] (1) The codon-optimized HPV capsid protein L1 and L2 expression genes were introduced into the pcDNA3.1(+) vector. The recombinant plasmids were named pXL1h and pXL2h according to the viral genotype, where X represents the genotype. The reporter plasmid was pcDNA3.1-EGFP. All plasmids were transformed into Escherichia coli DH5α strains for expansion and culture, and then the plasmids were extracted.

[0017] (2) 293T cells were cultured, and pXL1h, pXL2h, and pcDNA3.1-EGFP were mixed and added to the CaCl 2 In the solution, mix well and then add an equal volume of 2×HeBS buffer, mix well, and let stand at room temperature;

[0018] (3) After the plasmid mixture is incubated, slowly drip it into the cells in a circular motion and incubate it in an incubator;

[0019] (4) Collect cell pellets at 36, 48, 60, and 72 hours, wash, add an equal volume of 0.3 wt % Triton X-100 containing 1 wt % nuclease, resuspend the cells, and lyse at 37°C for 16 hours;

[0020] (5) After lysis, place the mixture in an ice bath for 5 minutes, add NaCl solution, place the mixture in an ice bath for 20 minutes, centrifuge at 8000 rpm for 15 minutes, and aliquot the supernatant to obtain HPV pseudovirus.

[0021] The inventors have designed a pseudovirus packaging method, which can obtain HPVVLPs with higher titers and better infection efficiency, and use the pseudovirus packaging method to detect HPV neutralizing antibody titers in serum, with higher detection accuracy and good repeatability.

[0022] The method of the present invention co-transfects 293T cells with HPV capsid protein L1 and L2 expression plasmids and GFP expression plasmids to obtain pseudovirus particles containing the GFP gene, co-incubates the pseudovirus with the serum sample to be tested and infects the target cells, and then uses a flow cytometer to detect the percentage of cells expressing GFP in all cells to quantify the amount of neutralizing antibodies contained in the serum sample to be tested. The method of the present invention has the characteristics of easy operation, high throughput, high sensitivity, etc.

[0023] Furthermore, the HPV types described in step (1) include 9 types in total, namely HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58.

[0024] Furthermore, the mass ratio of pXL1h, pXL2h and pcDNA3.1-EGFP in step (2) is 10:1:5.

[0025] Furthermore, in step (2), when the 293T cell confluency reached 85%, the cells were cultured at 1×10 6 The cells were divided into cell culture dishes for culture.

[0026] Furthermore, in step (2), the CaCl 2 The concentration of the solution is 0.25 mol / L, and the standing time at room temperature is 20 min.

[0027] Furthermore, in step (3), the culture medium is replaced with fresh culture medium after incubation for 12 hours.

[0028] Furthermore, in step (5), 0.17 times the volume of 5M NaCl solution is added.

[0029] A second object of the present invention is to provide HPV pseudovirus obtained by the above packaging method.

[0030] A third object of the present invention is to provide a kit comprising the HPV pseudovirus obtained by the packaging method as described above.

[0031] A fourth object of the present invention is to provide a method for detecting the titer of pseudovirus using the HPV pseudovirus obtained by the packaging method as described above, comprising the following steps:

[0032] (1) Collect the cell pellet and resuspend it in a small amount of medium without antibiotics. Take 10 μL of the cell suspension for counting. Dilute the cell suspension to 7.5×103 / 100 μL, cells were seeded into 96-well plates according to 100 μL of cell suspension per well;

[0033] (2) After 6-8 hours, observe the cell adhesion. If the cell adhesion is good, perform pseudovirus titration test. Dilute the pseudovirus solution in a 10-fold gradient, set up a positive and negative control, take 100 μL and add it to the above cells, shake it back and forth to mix, and place it in a carbon dioxide incubator.

[0034] (3) After 72 h, the cells were taken out for observation and image collection, and the pseudovirus titer was calculated based on the fluorescence expression.

[0035] A fifth object of the present invention is to provide a method for detecting HPV neutralizing antibodies for non-diagnostic purposes, wherein HPV pseudoviruses are obtained by the packaging method as described above, and then neutralizing antibodies are detected according to the following method:

[0036] S1: Collect the cell pellet and resuspend it in a small amount of medium without antibiotics. Take 10 μL of the cell suspension for counting. Dilute the cell suspension to 7.5×10 3 / 100 μL, cells were seeded into 96-well plates according to 100 μL of cell suspension per well;

[0037] S2: The collected blood samples were centrifuged at 3000 rpm for 5 min, the upper serum was taken into a 5 mL EP tube, and incubated at 56°C for 30 min to inactivate;

[0038] S3: dilute the inactivated serum in a gradient manner: 1×, 8×, 40×, 200×, 1000×, 5000×, 25000×. After dilution, add 55 μL per well to a 96-well plate according to the test items;

[0039] S4: Add 55 μL of the pseudovirus solution diluted to the appropriate titer into the above serum dilution solution according to the test items, and set up negative control wells of virus + culture medium and blank control wells containing only culture medium. Tap the 96-well plate to mix it evenly, and then put it in a 4°C refrigerator for incubation for 1 hour;

[0040] S5: After the incubation is completed, 100 μL of serum + pseudovirus mixture is added to the cells along the well wall and placed in a carbon dioxide incubator;

[0041] S6: After 72 hours, the samples were taken out to observe the GFP expression and the number of fluorescent spots in each reaction well was read using an enzyme-linked immunosorbent assay (ELISA) spot analyzer. The data were analyzed using Origin. The serum dilution gradient corresponding to the time when the GFP fluorescence expression reached half of the negative control was the neutralizing antibody titer of the sample, and a value ≥40 was considered positive.

[0042] The beneficial effects of the present invention are as follows:

[0043] (1) The method of the present invention is used for the detection of HPV neutralizing antibodies and has the advantages of high precision and good repeatability;

[0044] (2) The detection cost of the method of the present invention is low;

[0045] (3) The method of the present invention requires a small amount of sample, and the serum requirement for a single sample is as low as 500 μL, which is suitable for the analysis of large sample volumes. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The titer test results are repeated 3 times in the same sample experiment;

[0047] Figure 2 The titer test results were repeated 3 times between experiments for the same sample;

[0048] Figure 3 This is the plasmid map of pcDNA3.1-EGFP. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0050] The invention provides a method for packaging a HPV pseudovirus, a method for detecting a pseudovirus titer and a method for detecting an HPV neutralizing antibody.

[0051] (I) Packaging of fake viruses

[0052] 1. The codon-optimized capsid protein L1 and L2 expression genes of 9 HPV types, including HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58, were introduced into the pcDNA3.1(+) vector. The recombinant plasmids were named pXL1h and pXL2h according to the viral genotype, where X is the genotype; the reporter plasmid was pcDNA3.1-EGFP. All plasmids were transformed into E. coli DH5α strains for expansion and culture, and then the plasmids were extracted.

[0053] 2. When the 293T cell confluence reaches 85%, the cells are plated at 1×10 6 The density was divided into 6 cm cell culture dishes. After the cells attached to the wall, pXL1h, pXL2h and pcDNA3.1-EGFP were added to 1 mL 0.25 mol / L CaCl in a certain ratio (2:2:1, 2:1:1, 10:1:5). 2After mixing evenly, add an equal volume of 2×HeBS buffer and let stand at room temperature for 20 min.

[0054] 3. After the plasmid mixture is incubated, slowly add it to the cells in a circular motion and incubate it in the incubator for 12 hours, then replace it with fresh culture medium.

[0055] 4. Collect cell pellets at 36, 48, 60, and 72 hours, wash once with PBS, add an equal volume of 0.3% TritonX-100 (containing 1% nuclease) to resuspend the cells, and lyse at 37°C for 16 hours.

[0056] 5. After lysis, place the mixture in an ice bath for 5 minutes, add 0.17 times the volume of 5M NaCl solution, place the mixture in an ice bath for 20 minutes, centrifuge at 8000 rpm for 15 minutes, take the supernatant and store it in aliquots, and determine the titer.

[0057] (II) Pseudovirus titer detection

[0058] 1. Collect the cell pellet and resuspend it in a small amount of culture medium without antibiotics. Take 10 μL of cell suspension for counting and dilute the cell suspension to 7.5×10 3 / 100 μL, and cells were seeded into 96-well plates with 100 μL of cell suspension per well.

[0059] 2. After 6-8 hours, observe the cell adhesion. If the cell adhesion is good, perform pseudovirus titration test. Dilute the pseudovirus solution in 10-fold gradients (10 -1 -10 -6 ), and set up positive and negative controls, take 100 μL and add it to the above cells, shake it back and forth to mix, and put it into a carbon dioxide incubator.

[0060] 3. After 72 hours, the cells were taken out for observation and image collection, and the pseudovirus titer was calculated based on the fluorescence expression. (Pseudovirus titer (TU / mL = GFP expression percentage × dilution factor × 10 3 ))

[0061] (III) HPV neutralizing antibody titer detection

[0062] 1. Collect the cell pellet and resuspend it in a small amount of culture medium without antibiotics. Take 10 μL of cell suspension for counting and dilute the cell suspension to 7.5×10 3 / 100 μL, and cells were seeded into 96-well plates with 100 μL of cell suspension per well.

[0063] 2. Centrifuge the collected blood sample at 3000rpm for 5 minutes, take the upper serum into a 5mL EP tube, and incubate at 56℃ for 30 minutes to inactivate.

[0064] 3. Dilute the inactivated serum in a gradient manner: 1×, 8×, 40×, 200×, 1000×, 5000×, 25000×. After dilution, add 55 μL per well to a 96-well plate according to the items to be tested.

[0065] 4. Add 55 μL of the pseudovirus solution diluted to the appropriate titer to the above serum dilution solution according to the test items per well, and set up negative control wells of virus + culture medium and blank control wells containing only culture medium. Tap the 96-well plate to mix it evenly, and then place it in a 4°C refrigerator for incubation for 1 hour.

[0066] 5. After incubation, add 100 μL of serum + pseudovirus mixture into the cells along the well wall and place in a carbon dioxide incubator.

[0067] 6. After 72 hours, the samples were taken out to observe GFP expression and the number of fluorescent spots in each reaction well was read using an enzyme-linked immunosorbent assay (ELISA) spot analyzer. The data were analyzed using Origin. The serum dilution gradient corresponding to the time when GFP fluorescence expression reached half of that of the negative control was the neutralizing antibody titer of the sample, and a value ≥40 was considered positive.

[0068] Example 1

[0069] Fake virus packaging

[0070] 1. Co-transfection of structural gene expression plasmid and reporter plasmid into 293T cells

[0071] When the 293T cell confluence reached 85%, the total number of cells was 1×10 6 The cells were divided into 6 cm cell culture dishes at different densities. After the cells attached to the wall, pXL1h, pXL2h and pcDNA3.1-EGFP were added into 1 mL 0.25 mol / L CaCl2 solution in a certain ratio (10:1:5). At the same time, pcDNA3.1-EGFP plasmid was transfected alone as a negative control. After mixing evenly, an equal volume of 2×HeBS buffer was added to mix well and the mixture was allowed to stand at room temperature for 20 min.

[0072] c. After the plasmid mixture is incubated, slowly drip it into the cells in a circle and incubate it in the incubator for 12 hours, then replace it with fresh culture medium.

[0073] d. Collect cell pellets at 36, 48, 60, and 72 hours, wash once with PBS, add an equal volume of 0.3% Triton X-100 (containing 1% nuclease) to resuspend the cells, and lyse at 37°C for 16 hours.

[0074] e. After lysis, place the mixture in an ice bath for 5 minutes, add 0.17 times the volume of 5M NaCl solution, place the mixture in an ice bath for 20 minutes, centrifuge at 8000 rpm for 15 minutes, aliquot the supernatant and store it, and determine the titer.

[0075] f. Collect the cell pellet and resuspend it in a small amount of medium without antibiotics. Take 10 μL of the cell suspension for counting and dilute the cell suspension to 7.5×10 3 / 100 μL, and cells were seeded into 96-well plates with 100 μL of cell suspension per well.

[0076] g. After 6-8 hours, observe the cell adhesion. If the cell adhesion is good, perform pseudovirus titration test. Dilute the pseudovirus solution in a 10-fold gradient (10 -1 -10 -6 ), and set up positive and negative controls, take 100 μL and add it to the above cells, shake it back and forth to mix, and put it into a carbon dioxide incubator.

[0077] h. After 72h, the cells were taken out for observation and image collection, and the pseudovirus titer was calculated based on the fluorescence expression. The titer detection results are shown in Table 1.

[0078] Pseudovirus titer (TU / mL = GFP expression percentage × dilution factor × 10 3 )

[0079] Table 1

[0080]

[0081] Example 2

[0082] Detection of HPV neutralizing antibody titer in serum

[0083] a. Among the samples in this embodiment, 2 were from Chongqing People's Hospital, 2 were from the Second Xiangya Hospital of Central South University, and 1 was from Guizhou People's Hospital. The following samples were tested for nine-valent recombinant human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 45, 52, and 58).

[0084] Sample number: 23PJ000224, gender: female, age: 26, sample type: peripheral blood;

[0085] Sample number: 23PJ000379, gender: female, age: 22, sample type: peripheral blood;

[0086] Sample number: 23PJ000812, gender: female, age: 25, sample type: peripheral blood;

[0087] Sample number: 23PJ001071, gender: female, age: 26, sample type: peripheral blood;

[0088] Sample number: 23PJ002028, gender: female, age: 23, sample type: peripheral blood;

[0089] b. The sample was centrifuged at 3000 rpm for 5 minutes and the serum was collected in a sterile screw-capped plastic tube for the above neutralization experiment.

[0090] After collecting the cell pellet, resuspend it in a small amount of medium without antibiotics, take 10 μL of the cell suspension for counting, and dilute the cell suspension to 7.5×10 3 / 100 μL, and cells were seeded into 96-well plates with 100 μL of cell suspension per well.

[0091] c. Centrifuge the collected blood sample at 3000 rpm for 5 min, take the upper serum into a 5 mL EP tube, and incubate at 56°C for 30 min to inactivate.

[0092] d. Dilute the inactivated serum in a gradient manner: 1×, 8×, 40×, 200×, 1000×, 5000×, 25000×. After dilution, add 55 μL per well to a 96-well plate according to the items to be tested.

[0093] e. Add 55 μL of the pseudovirus solution diluted to the appropriate titer (see Table 2 below) to the above serum dilution solution according to the item to be tested, and set up negative control wells of virus + culture medium and blank control wells containing only culture medium. Tap the 96-well plate to mix it, and then place it in a 4°C refrigerator for incubation for 1 hour.

[0094] Table 2

[0095] Virus type 6 11 16 18 31 33 45 52 58 Dilution multiple 500 1000 500 1000 1000 1000 500 1000 1000

[0096] f. After incubation, add 100 μL of serum + pseudovirus mixture into the cells along the well wall and place in a carbon dioxide incubator.

[0097] g. After 72 hours, the samples were taken out to observe the GFP expression and the number of fluorescent spots in each reaction well was read using an enzyme-linked immunosorbent assay (ELISA) spot analyzer. The data obtained were analyzed using Origin. The serum dilution gradient corresponding to the time when the GFP fluorescence expression reached half of the negative control was the neutralizing antibody titer of the sample, and a value ≥40 was considered positive.

[0098] Table 3 Neutralizing antibody titer test results in sample serum

[0099]

[0100]

[0101] Example 3

[0102] Repeatability of the test

[0103] The same serum sample was used for three times in the same experiment and the same serum sample was used for three times in different experiments. The results of the neutralizing antibody titer test were as follows: Figure 1 and Figure 2 As shown, the intra-experimental coefficient of variation (CV) was 5.8%, less than 10%, and the inter-experimental coefficient of variation (CV) was 13.7%, less than 15%. The above test results show that the method for detecting HPV neutralizing antibody titer of the present invention has good repeatability.

Claims

1. A method for packaging HPV pseudovirus, characterized in that: The following steps are involved: (1) The codon-optimized HPV capsid protein L1 and L2 expression genes were introduced into the pcDNA3.1(+) vector. The recombinant plasmids were named pXL1h and pXL2h according to the viral genotype, where X represents the genotype. The reporter plasmid was pcDNA3.1-EGFP. All plasmids were transformed into Escherichia coli DH5α strains for expansion and culture, and then the plasmids were extracted. (2) 293T cells were cultured, and then pXL1h, pXL2h, and pcDNA3.1-EGFP were mixed and added to a CaCl2 solution. After mixing evenly, an equal volume of 2×HeBS buffer was added and mixed, and the mixture was allowed to stand at room temperature; (3) After the plasmid mixture is incubated, slowly drip it into the cells in a circular motion and incubate it in an incubator; (4) Collect cell pellets at 36, 48, 60, and 72 hours, wash, add an equal volume of 0.3 wt % Triton X-100 containing 1 wt % nuclease, resuspend the cells, and lyse at 37°C for 16 hours; (5) After lysis, place the mixture in an ice bath for 5 minutes, add NaCl solution, place the mixture in an ice bath for 20 minutes, centrifuge at 8000 rpm for 15 minutes, and aliquot the supernatant to obtain HPV pseudovirus.

2. The packaging method according to claim 1, characterized in that: The HPV types described in step (1) include 9 types, including HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58.

3. The packaging method according to claim 1, characterized in that: The mass ratio of pXL1h, pXL2h and pcDNA3.1-EGFP in step (2) is 10:1:

5.

4. The packaging method according to claim 1, characterized in that: In step (2), when the 293T cell confluency reached 85%, the cells were cultured at 1×10 6 The cells were divided into cell culture dishes for culture.

5. The packaging method according to claim 1, characterized in that: The concentration of the CaCl2 solution in step (2) is 0.25 mol / L, and the standing time at room temperature is 20 min.

6. The packaging method according to claim 1, characterized in that: In step (3), the culture medium was replaced with fresh medium after incubation for 12 hours.

7. The packaging method according to claim 1, characterized in that: In step (5), 0.17 times the volume of 5M NaCl solution was added.

8. A kit comprising the HPV pseudovirus obtained by the packaging method according to any one of claims 1 to 7.

9. A method for detecting the titer of pseudoviruses using the HPV pseudovirus obtained by the packaging method of claim 1, comprising the following steps: (1) Collect the cell pellet and resuspend it in a small amount of medium without antibiotics. Take 10 μL of the cell suspension for counting. Dilute the cell suspension to 7.5×10 3 / 100 μL, cells were seeded into 96-well plates according to 100 μL of cell suspension per well; (2) After 6-8 hours, observe the cell adhesion. If the cell adhesion is good, perform pseudovirus titration test. Dilute the pseudovirus solution in a 10-fold gradient, set up a positive and negative control, take 100 μL and add it to the above cells, shake it back and forth to mix, and place it in a carbon dioxide incubator. (3) After 72 h, the cells were taken out for observation and image collection, and the pseudovirus titer was calculated based on the fluorescence expression.

10. A method for detecting HPV neutralizing antibodies for non-diagnostic purposes, characterized in that: HPV pseudovirus is obtained by the packaging method of claim 1, and then neutralizing antibody detection is performed according to the following method: S1: Collect the cell pellet and resuspend it in a small amount of medium without antibiotics. Take 10 μL of the cell suspension for counting. Dilute the cell suspension to 7.5×10 3 / 100 μL, cells were seeded into 96-well plates according to 100 μL of cell suspension per well; S2: The collected blood samples were centrifuged at 3000 rpm for 5 min, the upper serum was taken into a 5 mL EP tube, and incubated at 56°C for 30 min to inactivate; S3: dilute the inactivated serum in a gradient manner: 1×, 8×, 40×, 200×, 1000×, 5000×, 25000×. After dilution, add 55 μL per well to a 96-well plate according to the test items; S4: Add 55 μL of the pseudovirus solution diluted to the appropriate titer into the above serum dilution solution according to the test items, and set up negative control wells of virus + culture medium and blank control wells containing only culture medium. Tap the 96-well plate to mix it evenly, and then put it in a 4°C refrigerator for incubation for 1 hour; S5: After the incubation is completed, 100 μL of serum + pseudovirus mixture is added to the cells along the well wall and placed in a carbon dioxide incubator; S6: After 72 hours, the samples were taken out to observe the GFP expression and the number of fluorescent spots in each reaction well was read using an enzyme-linked immunosorbent assay (ELISA) spot analyzer. The data were analyzed using Origin. The serum dilution gradient corresponding to the time when the GFP fluorescence expression reached half of the negative control was the neutralizing antibody titer of the sample, and a value ≥40 was considered positive.

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