Recombinant protein of human papillomavirus type 52 l1 protein and application thereof

Abstract

The application discloses a recombinant protein of human papillomavirus type 52 L1 protein, and the amino acid sequence of the recombinant protein is the amino acid sequence of human papillomavirus type 52 L1 protein (1-141) with a His tag at the N terminal, wherein the amino acid sequence of human papillomavirus type 52 L1 protein (1-141) is shown as SEQ ID NO:1; the recombinant protein is applied to preparation of a reagent for detecting human papillomavirus type 52 infection, and can be used for detecting whether HPV52 antibodies exist in a vaginal washing liquid of a to-be-detected person; the method has the characteristics of high sensitivity and specificity, wide application, and is not dependent on large equipment, and has strong universality.

Description

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a human papillomavirus type 52 L1 1-141 The invention relates to a recombinant protein and its application in preparing an immunoassay reagent for human papillomavirus type 52 infection. Background Art

[0002] The occurrence and impact of human papillomavirus (HPV), particularly high-risk subtypes, are a major public health concern due to their strong association with cervical cancer. HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 73) are clearly classified as common high-risk types by the International Agency for Research on Cancer (IARC). It is estimated that at least 99.7% of cervical cancers and 50% of head and neck squamous cell carcinomas worldwide are caused by HPV. Cervical cancer is the second most common cancer among women worldwide and the leading cause of cancer in most developing countries, where 80% of cases occur. Early and accurate identification of HPV subtypes is crucial for the effective management and prevention of cervical cancer and for enabling timely therapeutic intervention.

[0003] HPV is a type of small ds DNA virus, about 8000bp in size. Each HPV virus particle consists of an icosahedral capsid of about 60nm in size, with a double-stranded DNA as the template chain. The coding chain is divided into three regions: the early region (early, E), the late region (late, L) and the non-coding region (NCR). The non-coding region is also called the long control region (LCR). The HPV late genome is about 2500bp long and encodes L1 and L2 proteins, which together constitute the viral capsid.

[0004] Existing methods for measuring specific HPV antibody levels include neutralization assays and blood tests. Neutralization assays provide meaningful assessments of humoral immune responses, but are challenging to use in large clinical trials due to their cumbersome procedures. Blood tests carry the risk of cross-infection and are cumbersome and require large equipment. The dual-antigen sandwich ELISA offers an alternative approach to overcome the limitations of HPV antibody testing. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention provides a recombinant protein based on human papillomavirus type 52 L1 protein and its use in preparing a human papillomavirus type 52 infection immunoassay reagent. The amino acid sequence of the recombinant protein of the present invention is the amino acid sequence of the human papillomavirus type 52 L1 protein at positions 1 to 141 with a His tag at the N-terminus, wherein the amino acid sequence of the human papillomavirus type 52 L1 protein at positions 1 to 141 is shown in SEQ ID NO: 1.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] 1. Preparation of recombinant protein based on human papillomavirus type 52 L1 protein

[0008] HPVL1 was obtained by PCR amplification 1-141 Gene, construction of recombinant expression vector pET-32a-HPVL1 1-141 ; The recombinant expression vector was transformed into DH5α competent cells, single clones were picked for PCR and double enzyme digestion verification, positive clones were obtained, plasmids were extracted, and sequencing was performed; the recombinant expression vector pET-32a-HPVL1 1-141 Transformed into Rosetta strain to obtain recombinant expression strain pET-32a-L1 1-141 / Rosetta; the recombinant expression strain was induced with IPTG to obtain the recombinant protein; the expression of the fusion protein was analyzed by SDS-PAGE; the recombinant protein was verified by Western blot to confirm that the expressed protein was the target protein, and the recombinant protein of human papillomavirus type 52 L1 protein was purified;

[0009] 2. Preparation of horseradish peroxidase-labeled recombinant protein HPVL1 based on human papillomavirus type 52 L1 protein 1-141 -HRP;

[0010] 3. Establishment of double antigen sandwich ELISA assay

[0011] The recombinant protein obtained in step 1 was diluted with PBS and added to the wells of the ELISA plate for coating overnight, followed by blocking, incubation with vaginal wash fluid, and HPVL1 1-141 -HRP enzyme-labeled secondary antibody incubation and color development reaction, and then use a microplate reader to measure the absorbance of the reaction solution at 450nm. The critical value is calculated based on the ELISA data and used as the standard for subsequent sample positive and negative judgment to establish a dual-antigen ELISA detection method;

[0012] The critical value of ELISA = average value × 2; the average value is the average value of the indirect ELISA results of negative samples. The indirect ELISA result judgment standard: the OD value of the HPV washing fluid sample is greater than or equal to the critical value and is judged as positive; the OD value of the HPV washing fluid sample is less than the critical value and is judged as negative.

[0013] The detection reagents of the present invention also include commercially available reagents conventionally used in the double antigen sandwich ELISA detection method.

[0014] The beneficial effects of the present invention are:

[0015] The application takes vaginal irrigation liquid as a detection object, avoids the problems in the traditional detection method, has high sensitivity and specificity, wide application, and the double antigen sandwich ELISA detection method based on the recombinant protein can be used for detecting HPV antibody, is not dependent on large equipment, has strong universality, and is more helpful for the screening of HPV patients. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 For the amplified L1 1-141 gene sequence; wherein lane M: DNA Marker (50-500bp); lane 1, 2: L1 1-141 gene PCR product;

[0017] Figure 2 For SDS-PAGE detection results of recombinant proteins expressed by recombinant expression strain pET-32a-L1 1-141 / Rosetta, wherein M is a protein marker (8kDa-180 kDa); lane 1: uninduced bacteria; lane 2: recombinant protein obtained after induction;

[0018] Figure 3 For Western blot verification of recombinant protein expression results, lanes 1, 2 are supernatants of uninduced E. coli broken; lanes 3, 4 are supernatants after centrifugation of the first ultrasonic broken expression strain; lanes 5, 6 are supernatants after centrifugation of urea-dissolved inclusion bodies;

[0019] Figure 4 For identification results of recombinant protein expression forms; well 1 is uninduced bacteria; well 2 is supernatant obtained by separating induced and broken bacteria; well 3 is precipitate obtained by separating induced and broken bacteria;

[0020] Figure 5 For SDS-PAGE analysis results of purified recombinant proteins, M is a protein marker (8kDa-180 kDa), lane 1 is unpurified protein; lane 2 is purified recombinant protein;

[0021] Figure 6 For schematic diagram of condition optimization results of the double antigen sandwich ELISA detection method;

[0022] Figure 7 For results of 115 clinical samples detected by the double antigen sandwich ELISA detection method. DETAILED DESCRIPTION

[0023] The application will be further described in detail through examples, but the protection scope of the application is not limited to the content described, and the methods in the examples are all conventional methods unless otherwise specified, and the reagents used are all conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified.

[0024] Example 1: L1 1-141 Gene preparation

[0025] 1. Retrieve HPV52 type L1 from the NCBI database 1-141 The protein gene coding sequence was determined by using Primer Premier 5.0 to design primers. At the same time, according to the plasmid pET-32a and gene sequence analysis, the primers were inserted into BamH Ⅰ and Sal Ⅰ is the restriction site. The primers were synthesized in Beijing Qingke Biotechnology Co., Ltd. The primer sequences are as follows: F: 5'-gcgtcgacttaccttttaacctttt-3'; R: 5'- cgcggatccatggtacagatttttat-3';

[0026] 2. Use the TIANamp Genomic DNA Kit (DP304) from Tiangen Technology Co., Ltd. to extract total human papillomavirus DNA from the vaginal wash fluid of HPV patients. The extraction was carried out according to the kit instructions. The extracted DNA was stored at -80°C until use.

[0027] 3. Using the DNA extracted in step 2 as a template, perform PCR amplification using the above primers. The amplification system is as follows:

[0028]

[0029] The PCR reaction program was 94°C for 5 min, 94°C for 30 s, 56°C for 30 s, and 72°C for 1.5 min, for a total of 35 cycles; and finally an extension at 72°C for 5 min.

[0030] After the PCR reaction was completed, the PCR products were identified by 2% agarose gel electrophoresis, and the target gene was cut and recovered using the Zhuangmeng small amount agarose gel DNA recovery kit. Figure 1 From the figure, we can see that a specific band appeared between 400-500bp, which is consistent with the target gene L1 1-141 The theoretical size is consistent with 423 bp, and the gene sequence is shown in SEQ ID NO: 2.

[0031] Example 2: Recombinant expression vector pET-32a-HPVL1 1-141 Construction and transformation

[0032] 1. Double enzyme digestion of pET-32a plasmid and gene

[0033] Take out the frozen Escherichia coli transformed with pET-32a from the -80℃ freezer, dip a small amount of bacterial liquid on LB solid medium containing 0.1 mg / mL ampicillin, and culture at 37℃ for 14 h; pick a single clone colony and culture it in LB liquid medium containing 0.1 mg / mL ampicillin until the logarithmic growth phase, take part of the bacterial liquid and use the Zhuangmeng Plasmid Mini Extraction Kit (ZP101) to extract the plasmid according to the operating procedures in the instruction manual.

[0034] Prepare the extracted pET-32a plasmid and target gene into the double enzyme digestion reaction system shown in the table below, and perform double enzyme digestion at 37°C for 4 h.

[0035]

[0036] The product obtained after double enzyme digestion was identified by 2% agarose gel, and the band of the target gene size was cut and recovered using Zhuangmeng Small Volume Agarose Gel DNA Recovery Kit (ZP202). The concentration of the recovered DNA was measured using a micro-spectrophotometer.

[0037] 2. Construction and transformation of recombinant expression vector

[0038] Use T4 DNA Ligase to ligate the double-digested and purified vector and target gene fragment. Add reagents according to the system in the table below and conduct the ligation reaction at 16°C for 8 hours.

[0039]

[0040] The ligation product was transformed into DH5α competent cells. Specifically, the competent cells were taken out of the -80°C refrigerator and placed on ice for 20 minutes; the ligation product was slowly added to the competent cells with a pipette and mixed, and the reaction was carried out on ice for 30 minutes; it was placed in a 42°C water bath for 60 seconds, handled with care, and then placed on ice for 2 minutes, and 1 mL of LB medium was added; after shaking the bacteria at 37°C and 200 rpm for 1 hour, 900 μL of supernatant was discarded using a centrifuge at 3000 rpm for 5 minutes; the remaining liquid was pipetted and mixed, and then spread on an LB solid culture medium plate containing 0.1 mg / mL ampicillin, and incubated inverted in a 37°C constant temperature incubator for 14 hours; when the plaque on the culture medium grew to an appropriate size, 5 single colonies were randomly picked from the LB plate and placed in LB liquid culture medium containing 0.1 mg / ml ampicillin, and cultured at 200 rpm and 37°C for 5 hours until the bacterial liquid became turbid. In a sterile operating table, bacterial liquid PCR identification was performed using the bacterial liquid as a template. The PCR system was as follows, and the primers were the same as in Example 1;

[0041]

[0042] The PCR reaction program was 94°C for 5 minutes; 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1.5 minutes, for a total of 30 cycles; and a final extension at 72°C for 5 minutes. After the reaction, the PCR product of the bacterial solution was detected on a 2% agarose gel. Monoclonal colonies with positive PCR results were selected, plasmids were extracted, and DNA sequencing was performed at Beijing Qingke Biotechnology Co., Ltd. The results showed that the recombinant expression vector pET-32a-HPVL1 1-141 Build successful.

[0043] 3. Recombinant expression strain pET-32a-L1 1-141 / Rosetta Construction

[0044] The plasmid obtained in step 2 was transformed into Rosetta competent cells. The method was the same as above. Positive colonies were picked and positive single clones were selected for sequencing verification to obtain the recombinant expression strain pET-32a-L1. 1-141 / Rosetta; preserve the bacterial solution with sterilized 50% glycerol and bacterial solution in a ratio of 3:7 and store at -80℃ until use.

[0045] Example 3: Inducible expression of recombinant protein

[0046] 1. Take frozen pET-32a-HPVL1 1-141 Streak the / Rosetta bacterial suspension onto a LB (ampicillin) solid plate and invert the plate in a 37°C constant temperature incubator. When the plaques grow to an appropriate size, randomly pick several single colonies and culture them in 1 mL LB (ampicillin) liquid medium at 37°C and 200 rpm for 5 h. Then transfer them to 300 mL LB liquid medium containing 20 μg / mL ampicillin resistance and culture them at 37°C and 200 rpm. When the OD600 of the bacterial suspension is between 0.4 and 0.6, add IPTG to the liquid medium to a final concentration of 0.5 mmoL / L. Induce expression at 26°C and 120 rpm / min for 12 h. Collect the bacterial suspension and use the pre-induction bacterial cell as a control. Verify by SDS-PAGE that the recombinant protein of approximately 34 kD is obtained after induction, which is in line with the expected size. The results are shown in Figure 2. Figure 2 shown.

[0047] 2. Western blot verification

[0048] In order to further verify the expression of recombinant protein, the recombinant protein was identified by Western blot targeting the expression tag His.

[0049] After induction of expression, the cells were collected, ultrasonically disrupted, and centrifuged at 4°C and 12,000 g for 10 min; the supernatant was discarded, the pellet was resuspended in PBS, centrifuged and the supernatant was discarded, and the process was repeated twice until the pellet was clean and milky white; the supernatant was discarded, 8 mM urea was added, and the pellet was ice-bathed for 1 hour to dissolve; the supernatant was collected after being boiled at 95°C for 10 minutes and then subjected to SDS-PAGE. At the same time, the supernatant of uninduced E. coli disruption was set as a control. After 2 hours of transfer, the antibody was incubated: the NC membrane was removed and blocked with 5% skim milk at 4°C for 2 hours. The NC membrane was removed and incubated with mouse His tag monoclonal antibody (1:2000 dilution) diluted in blocking buffer at 37°C for 2 hours. The membrane was washed 5 times with PBST for 5 minutes each; the HRP-labeled goat anti-mouse polyclonal antibody (1:10,000 dilution in blocking buffer) was incubated at 37°C for 1 hour. The membrane was washed 3 times with PBST for 5 minutes each; the membrane was developed and exposed; the results are shown in Figure 2. Figure 3 As shown, it shows that the obtained protein is a His-tagged recombinant protein.

[0050] Example 4: Identification of recombinant protein expression and protein purification

[0051] After induction of expression, the collected cells were resuspended in PBS, lysed by ultrasonication, and the supernatant and precipitate were separated by centrifugation. The expression form was analyzed by SDS-PAGE. It was verified that the expression product was inclusion body. The results are as follows Figure 4 As shown, inclusion body purification was performed. After protein expression was induced, ultrasonically disrupted and centrifuged at 4°C and 12000g for 10 minutes; the supernatant was discarded, the precipitate was resuspended in PBS, centrifuged and the supernatant was discarded, and the process was repeated twice until the inclusion body precipitate was clean milky white; the supernatant was discarded, 8mM urea was added, and the inclusion body was dissolved in an ice bath for 1 hour; the supernatant was taken after centrifugation at 4°C and 12000g for 20 minutes, the supernatant was taken, filtered through a 0.22μm microporous membrane, and filled with Ni agarose gel column, and the equilibrium solution was used; the protein liquid was loaded onto the column and flowed through naturally; the column was rinsed with 2 times the column volume of washing solution; the eluate was collected with 1 times the column volume of elution solution. Take an appropriate amount of eluate, add 5× protein electrophoresis SDS Buffer to cook the sample, and analyze by SDS-PAGE. The results are shown as follows Figure 5 As shown, lane 2 has a distinct and single band at 34 kD, indicating that the recombinant protein was successfully obtained with high purity.

[0052] Example 5: Horseradish peroxidase-labeled recombinant protein HPVL1 1-141 -HRP preparation

[0053] (1) Dissolve 5 mg of HRP in 0.5 mL of 0.1 mol / L NaHCO3 solution, add 0.5 mL of 10 mM NaIO4 solution, mix thoroughly by pipetting, and react at room temperature in the dark for 2 h.

[0054] (2) After 2 h of reaction, add 0.75 mL of 0.1 mol / L sodium carbonate solution and mix thoroughly by pipetting;

[0055] (3) Add 10 mg of purified HPVL1 1-141 Mix the recombinant protein by pipetting;

[0056] (4) Place 0.3 g of Sephadex G25 dry powder into a syringe with a filter membrane at the bottom, draw the mixture obtained in the previous step into the syringe, and react at room temperature in the dark for 3 h.

[0057] (5) After the reaction is completed, the liquid in the sleeve is discharged through the bottom valve and washed with about 0.5 mL of PBS. The above effluent is collected and 1 / 20 of the volume of the mixed solution of NaBH4 (5 mg / mL) is added to the collected effluent. After reacting at room temperature for 30 minutes, 3 / 20 of the volume of the mixed solution of NaBH4 (5 mg / mL) is added and reacted at room temperature for 1 hour.

[0058] (6) After the reaction is completed, the liquid is discharged through the bottom valve and collected in the EP tube to obtain HPVL1. 1-141 -HRP;

[0059] The prepared HPVL1 1-141 -HRP was aliquoted and stored at -80°C for long-term use. For short-term use, an equal volume of glycerol was added and stored at -20°C.

[0060] Example 6: Condition Optimization of Double Antigen Sandwich ELISA Method

[0061] HPV-positive and HPV-negative vaginal lavage fluid was collected and purified recombinant protein was used as the antigen to establish a double-antigen sandwich ELISA method to detect antibodies in the vaginal lavage fluid. During this process, the antigen coating concentration, serum dilution, type of blocking solution, secondary antibody dilution, and color development time were optimized. When determining the optimal reaction conditions, the positive OD value was usually greater than 2 times the negative average value, and the difference between the positive OD value and the negative OD value was obvious, that is, the positive OD value / negative OD value (P / N value) was relatively large, which was the optimal reaction condition. The specific steps are as follows:

[0062] 1. Coating: Dilute the recombinant protein with carbonate buffer to a concentration of 5 μg / mL, 10 μg / mL, or 20 μg / mL per well, add it to a 96-well ELISA plate (Corning), and coat overnight at 4°C.

[0063] 2. Blocking: After coating, discard the liquid in the wells, wash 5 times with 300μL PBST, block with 5% skim milk, incubate at room temperature for 2 hours, wash 5 times with PBST, and pat dry the liquid in the wells on paper;

[0064] 3. Incubate with vaginal douches: Add 200 μL of vaginal douches to each well and incubate at 4°C overnight.

[0065] 4. Incubate enzyme-labeled secondary antibody HPVL1 1-141 -HRP: After the vaginal wash incubation was completed, the cells were washed with PBST for 5 times and 100 μL of enzyme-labeled secondary antibody L1 at concentrations of 1:100, 1:200, and 1:400 was added. 1-141 -HRP was added to the wells and incubated at 37°C for 1 h.

[0066] 5. Color reaction: L1 1-141 After incubation with the HRP enzyme-labeled secondary antibody, the cells were washed five times with PBST, 100 μL of TMB color development solution (Tiangen Company) was added to each well, and the cells were incubated at room temperature for 20 min. 2 M H2SO4 stop solution was then added to each well, and the absorbance of each reaction well at 450 nm was measured using a microplate reader.

[0067] By comparing the absorbance at 450 nm (OD 450 ) and the positive / negative (P / N) ratio, select the condition that produces the maximum P / N value, and the result is as follows Figure 6 As shown, the optimal antigen coating concentration was determined to be 20 μg / mL, and the optimal secondary antibody dilution concentration was 1:200.

[0068] Example 8: Detection and Coincidence Analysis of Double Antigen Sandwich ELISA Method

[0069] 115 vaginal douches collected from the First People's Hospital of Yunnan Province were tested for HPV, including 85 HPV-positive samples and 31 HPV-negative samples. The double-antigen sandwich ELISA test was performed using the method established in the present invention. The steps are as follows:

[0070] 1. Coating: Dilute the recombinant protein with carbonate buffer to a concentration of 20 μg / mL per well, add it to a 96-well ELISA plate (Corning), and coat it overnight at 4°C.

[0071] 2. Blocking: After coating, discard the liquid in the wells, wash 5 times with 300μL PBST, block with 5% skim milk, incubate at room temperature for 2 hours, wash 5 times with PBST, and pat dry the liquid in the wells on paper;

[0072] 3. Incubate vaginal douches: Add 200 μL of vaginal douches to each well and incubate at 37°C for 2 h.

[0073] 4. Incubate enzyme-labeled secondary antibody HPVL1 1-141 -HRP: After the vaginal wash incubation is complete, wash with PBST 5 times and add 100 μL diluted enzyme-labeled secondary antibody HPVL1 1-141 -HRP was added to the wells and incubated at room temperature for 1 h;

[0074] 5. Enzyme-labeled secondary antibody against HPVL1 1-141 After the HRP incubation was completed, the wells were washed five times with PBST, 100 μL of TMB color development solution (Tiangen Company) was added to each well, and after incubation at room temperature for 20 minutes, 2 M H2SO4 stop solution was added to each well, and the absorbance of each reaction well at 450 nm was measured using a microplate reader;

[0075] The results are as follows Figure 7 , using the method of the present invention, 79 of the 115 samples were positive and 36 were negative, and the compliance rate of this experimental method was 94%.

[0076] Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) and the leading cause of cervical cancer. Over 90% of cervical cancer deaths are caused by a lack of access to screening and treatment, and nearly all cervical cancer cases are caused by HPV infection. Of over 200 HPV types, at least 12 are considered high-risk. HPV52 is the fourth most common HPV type in cervical cancer cases in China. The dual-antigen sandwich ELISA developed in this paper utilizes vaginal douches (PBS) for direct detection, avoiding the challenges of traditional detection methods while offering high sensitivity, specificity, and widespread applicability. It does not rely on large equipment and is highly universal, making it a valuable tool for HPV screening. Furthermore, to date, the development and use of the dual-antigen sandwich ELISA has not yet been applied to the detection of HPV 52 antibodies.

Claims

1. A recombinant protein of human papillomavirus type 52 L1 protein, the amino acid sequence of which is the amino acid sequence of amino acids 1-141 of human papillomavirus type 52 L1 protein with a His tag at the N-terminus, wherein the amino acid sequence of amino acids 1-141 of human papillomavirus type 52 L1 protein is shown in SEQ ID NO:

1.

2. Use of the recombinant protein of human papillomavirus type 52 L1 protein according to claim 1 in the preparation of a reagent for detecting human papillomavirus type 52 infection.

3. The use according to claim 2, characterized in that: Double antigen sandwich ELISA was used to detect the presence of HPV52 antibodies in the vaginal washing fluid of the subjects.

4. The use according to claim 3, characterized in that: The detection reagent used in the double antigen sandwich ELISA detection method also includes a recombinant protein based on human papillomavirus type 52 L1 protein labeled with horseradish peroxidase as a detection antigen.

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