Recombinant protein of 52 type human papilloma virus L1 protein and application thereof

Abstract

The invention discloses a recombinant protein of 52-type human papilloma virus L1 protein, the amino acid sequence of the recombinant protein is the 1st-141 amino acid sequence of the 52-type human papilloma virus L1 protein with a His tag at the N terminal, and the 1st-141 amino acid sequence of the 52-type human papilloma virus L1 protein is as shown in SEQ ID NO: 1; the recombinant protein is applied to preparation of a reagent for detecting 52 type human papilloma virus infection and can be used for detecting whether an HPV52 antibody exists in vaginal irrigation fluid of a person to be detected or not, and the method has the advantages of being high in sensitivity and specificity and wide in application, does not depend on large equipment and is high in 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 recombinant protein and its application in preparing immunoassay reagent for human papillomavirus type 52 infection. Background Art

[0002] The occurrence and impact of human papillomavirus (HPV), especially high-risk subtypes, are major public health challenges due to their strong association with cervical cancer. HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 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 cancer in women in most developing countries, where 80% of cases occur. Early and accurate identification of HPV subtypes is essential for the effective management and prevention of cervical cancer and to enable 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 strand. The coding strand is divided into three regions: early region (early, E), late region (late, L) and 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 tests and blood tests. Neutralization tests provide meaningful assessment of humoral immune responses, but are challenging to use in large clinical trials due to cumbersome procedures. Blood tests are a method that carries the risk of cross-infection, and the process is cumbersome and relies on large equipment. Double antigen sandwich ELISA provides an alternative method to overcome the limitations of HPV antibody testing. Summary of the invention

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

[0006] To achieve the above object, the present invention adopts the following technical scheme: 1. Preparation of recombinant protein based on human papillomavirus type 52 L1 protein PCR amplification of HPVL1 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 selected 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; use IPTG to induce the recombinant expression strain to obtain the recombinant protein; use SDS-PAGE to analyze the expression of the fusion protein; perform Western blot verification on the recombinant protein to determine that the expressed protein is the target protein, and purify the recombinant protein of human papillomavirus type 52 L1 protein; 2. Preparation of horseradish peroxidase-labeled recombinant protein HPVL1 based on human papillomavirus L1 protein type 52 1-141 -HRP; 3. Establishment of double antigen sandwich ELISA detection method 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 washing fluid, HPVL1 1-141 -HRP enzyme-labeled secondary antibody incubation and color development reaction, and then use an enzyme reader to measure the absorbance value of the reaction solution at 450nm, and calculate the critical value based on the ELISA data, which is used as the standard for subsequent sample positive and negative judgment and for establishing a double antigen ELISA detection method; 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.

[0007] The detection reagents of the present invention also include commercially available reagents conventionally used in the double antigen sandwich ELISA detection method.

[0008] The beneficial effects of the present invention are: The present invention uses vaginal washing fluid as the detection object, which not only avoids the problems existing in traditional detection methods, but also has the characteristics of high sensitivity, specificity and wide application. The double antigen sandwich ELISA detection method based on recombinant protein can be used to detect HPV antibodies, does not rely on large equipment, has strong universality, and is more helpful for the screening of HPV patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 L1 1-141 Gene sequence; Lane M: DNA Marker (50-500bp); Lanes 1 and 2: L1 1-141 Gene PCR products; Figure 2 For the recombinant expression strain pET-32a-L1 1-141 / SDS-PAGE test results of the recombinant protein expressed by Rosetta, where M is a protein marker (8kDa-180 kDa); Lane 1: uninduced bacteria; Lane 2: recombinant protein obtained after induction; Figure 3 Western blot was used to verify the expression of the recombinant protein. Lanes 1 and 2 are the supernatants of the uninduced E. coli; Lanes 3 and 4 are the supernatants of the induced expression strain after the first ultrasonic disruption and centrifugation; Lanes 5 and 6 are the supernatants of the urea-dissolved inclusion bodies after centrifugation; Figure 4 The results of identification of the expression form of the recombinant protein; Well 1 is the uninduced bacterial solution; Well 2 is the supernatant obtained by induced broken bacterial separation; Well 3 is the precipitate obtained by induced broken bacterial separation; Figure 5 The results of SDS-PAGE analysis of purified recombinant proteins, M is a protein marker (8kDa-180 kDa), lane 1 is unpurified protein; lane 2 is purified recombinant protein; Figure 6 This is a schematic diagram of the optimization results of the double antigen sandwich ELISA detection method; Figure 7 These are the results of 115 clinical samples tested using the double antigen sandwich ELISA assay. DETAILED DESCRIPTION

[0010] The present invention is further described in detail below through examples, but the protection scope of the present invention is not limited to the contents described above. 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.

[0011] Example 1: L1 1-141 Gene preparation 1. Retrieve HPV52 type L1 from NCBI database 1-141 The protein gene coding sequence was determined by using Primer Premier 5.0 to design primers. Bm Ⅰ and SalⅠ is the restriction site, and the primers were synthesized in Beijing Qingke Biotechnology Co., Ltd. The primer sequences are as follows: F: 5'-gcgtcgacttaccttttaacctttt-3'; R: 5'- cgcggatccatggtacagatttttat-3'; 2. Use the TIANamp Genomic DNA Kit (DP304) from Tiangen Technology Co., Ltd. to extract total human papillomavirus DNA from the vaginal washing fluid of HPV patients. The extraction is carried out according to the instructions of the kit, and the extracted DNA is stored at -80℃ for future use; 3. Using the DNA extracted in step 2 as a template, perform PCR amplification using the above primers. The amplification system is as follows:

[0012] 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.

[0013] After the PCR reaction was completed, the PCR product was identified by 2% agarose gel electrophoresis, and the target gene was cut and recovered using the Zhuangmeng small agarose gel DNA recovery kit. The results are shown in 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 423bp, and the gene sequence is shown in SEQ ID NO: 2.

[0014] Example 2: Recombinant expression vector pET-32a-HPVL1 1-141 Construction and transformation 1. Double restriction enzyme digestion of pET-32a plasmid and gene Take out the frozen Escherichia coli transformed with pET-32a from the -80℃ refrigerator, dip a small amount of bacterial liquid on the LB solid culture 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 culture 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 steps in the instruction manual.

[0015] The extracted pET-32a plasmid and target gene were prepared into the double enzyme digestion reaction system as shown in the table below, and the double enzyme digestion was carried out at 37°C for 4 h;

[0016] 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.

[0017] 2. Construction and transformation of recombinant expression vector Use T4 DNA Ligase to connect the double-digested and purified vector and target gene fragment. Add reagents according to the system in the table below and perform the ligation reaction at 16°C for 8 h.

[0018] 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 culture medium was added; after shaking the bacteria at 37°C and 200 rpm for 1 hour, 900 μL of supernatant was discarded after centrifugation at 3000 rpm for 5 minutes; the remaining liquid was blown and mixed, and then spread on an LB solid culture medium plate containing 0.1 mg / mL ampicillin, and 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 was used as a template for bacterial liquid PCR identification. The PCR system was as follows, and the primers were the same as in Example 1;

[0019] The PCR reaction program was 94℃ for 5min; 94℃ for 30s, 56℃ for 30s, 72℃ for 1.5min, for a total of 30 cycles; and finally 72℃ for 5min extension. After the reaction, the PCR product of the bacterial solution was detected with 2% agarose gel, and the monoclonal colonies with positive PCR results were selected, the plasmids were extracted, and sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing. The results showed that the recombinant expression vector pET-32a-HPVL1 1-141 Build successful.

[0020] 3. Recombinant expression strain pET-32a-L1 1-141 / Rosetta Construction The plasmid obtained in step 2 was transformed into Rosetta competent cells. The method was the same as above. Positive colonies were picked, positive single clones were picked, and sequencing was performed to obtain the recombinant expression strain pET-32a-L1. 1-141 / Rosetta; Use sterilized 50% glycerol and bacterial solution in a ratio of 3:7 to preserve the bacterial solution and store it at -80℃ for later use.

[0021] Example 3: Inducible expression of recombinant protein 1. Take the frozen pET-32a-HPVL1 1-141 / Rosetta bacterial liquid was streaked onto LB (ampicillin) solid plates and inverted in a 37℃ constant temperature incubator. When the plaques grew to an appropriate size, several single clones were randomly picked and cultured in 1mL LB (ampicillin) liquid culture medium at 37℃ and 200rpm for 5h, and then transferred to 300mL LB liquid culture medium containing 20μg / mL ampicillin resistance at 37˚C and 200rpm. When the OD600 of the bacterial liquid was between 0.4-0.6, IPTG was added to the liquid culture medium to a final concentration of 0.5mmoL / L, and the expression was induced at 26℃ and 120rpm / min for 12h. The bacterial liquid was collected, and the bacteria before induction were set as the control. SDS-PAGE was used for verification. After induction, a recombinant protein of about 34kD was obtained, which was in line with expectations. The results are shown in Figure 2 shown.

[0022] 2. Western blot verification In order to further verify the expression of the recombinant protein, the recombinant protein was identified by Western blot targeting the expression tag His. After induction of expression, the cells were collected, ultrasonically disrupted, and centrifuged at 4°C and 12000g for 10 min; the supernatant was discarded, the precipitate was resuspended in PBS, centrifuged and the supernatant was discarded, and the process was repeated twice until the precipitate was clean and milky white; the supernatant was discarded, 8mM urea was added, and the precipitate was dissolved in an ice bath for 1h; the supernatant was taken after centrifugation at 4°C and 12000g for 20min; the sample was boiled at 95°C for 10min and then SDS-PAGE was performed. At the same time, the supernatant of the uninduced Escherichia coli disruption was set as a control. After 2h of transfer, antibody incubation was performed: the NC membrane was removed, and 5% skim milk was used for blocking at 4°C for 2h. The NC membrane was removed and the mouse His tag monoclonal antibody (1:2000 dilution) diluted in the blocking solution was used for incubation at 37°C for 2h, and the membrane was washed 5 times with PBST for 5min each time; the HRP-labeled goat anti-mouse polyclonal antibody (1:10000 dilution in the blocking solution) was used for incubation at 37°C for 1h, and the membrane was washed 3 times with PBST for 5min each time; the development and exposure were performed; the results are shown in Figure 2. Figure 3 As shown, it indicates that the obtained protein is a His-tagged recombinant protein.

[0023] Example 4: Identification of recombinant protein expression and protein purification After induction of expression, the collected bacteria 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, ultrasonic disruption was performed and centrifuged at 4°C and 12000g for 10 min; 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 1h; centrifuged at 4°C and 12000g for 20min, the supernatant was taken, filtered through a 0.22μm microporous membrane, Ni agarose gel column was filled, and a balance solution was used; the protein liquid was loaded onto the column and allowed to flow naturally; the column was rinsed with 2 times the column volume of washing solution; the eluent was collected with 1 times the column volume of eluent. Take an appropriate amount of eluent, add 5× protein electrophoresis SDS Buffer to cook the sample, and analyze it by SDS-PAGE. The results are as shown Figure 5 As shown, lane 2 has an obvious and single band at 34KD, and a high purity recombinant protein was successfully obtained.

[0024] Example 5: Horseradish peroxidase labeled recombinant protein HPVL1 1-141 -HRP preparation (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 well by pipetting, and react at room temperature in the dark for 2 h. (2) After 2 h of reaction, add 0.75 mL of 0.1 mol / L sodium carbonate solution and mix by pipetting; (3) Add 10 mg of purified HPVL1 1-141 Mix the recombinant protein by pipetting; (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. (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 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 NaBH4 (5 mg / mL) is added again, and the reaction is carried out at room temperature for 1 hour; (6) After the reaction is completed, the liquid flows out through the bottom valve and is collected in the EP tube to obtain HPVL1. 1-141 -HRP; 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.

[0025] Example 6: Condition optimization of double antigen sandwich ELISA method HPV-positive and HPV-negative vaginal washing fluids were taken, and the purified recombinant protein was used as the antigen to establish a double antigen sandwich ELISA method to detect antibodies in the vaginal washing fluid. In 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 is usually selected to be greater than 2 times the negative average value, and the positive OD value and the negative OD value are significantly different, that is, the positive OD value / negative OD value (P / N value) is relatively large. The specific steps are as follows: 1. Coating: dilute the recombinant protein with carbonate buffer at a concentration of 5 μg / mL, 10 μg / mL, and 20 μg / mL per well, add to a 96-well ELISA plate (Corning), and coat overnight at 4°C; 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 2h, wash 5 times with PBST, and pat dry the liquid in the wells on paper; 3. Incubate vaginal rinsing solution: add 200 μL vaginal rinsing solution to each well and incubate at 4°C overnight; 4. Incubate enzyme-labeled secondary antibody HPVL1 1-141 -HRP: After the vaginal wash incubation, wash with PBST for 5 times and add 100 μL of enzyme-labeled secondary antibody L1 at a concentration of 1:100, 1:200, and 1:400 1-141 -HRP was added to the wells and incubated at 37°C for 1 h.

[0026] 5. Color reaction: L1 1-141 After the incubation with -HRP enzyme-labeled secondary antibody was completed, the wells were washed 5 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 min, 2 M H2SO4 stop solution was added to each well, and the absorbance of each reaction well at 450 nm was measured with an enzyme-labeled instrument.

[0027] 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.

[0028] Example 8: Detection and compliance analysis of the double antigen sandwich ELISA method Among the 115 vaginal washing fluid samples collected from the First People's Hospital of Yunnan Province, 85 samples were HPV positive and 31 samples were HPV negative. The double antigen sandwich ELISA test was performed using the method established by the present invention, and the steps are as follows: 1. Coating: dilute the recombinant protein with carbonate buffer at a concentration of 20 μg / mL per well, add it to a 96-well ELISA plate (Corning), and coat it overnight at 4°C; 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 2h, wash 5 times with PBST, and pat dry the liquid in the wells on paper; 3. Incubate vaginal rinsing solution: add 200 μL vaginal rinsing solution to each well and incubate at 37°C for 2 h; 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 of diluted enzyme-labeled secondary antibody HPVL1 1-141 -HRP was added to the wells and incubated at room temperature for 1 h; 5. Enzyme-labeled secondary antibody HPVL1 1-141 After the HRP incubation was completed, the wells were washed 5 times with PBST, 100 μL of TMB colorimetric 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 with an enzyme-labeled instrument; The results are shown in Figure 7 , 79 positive samples and 36 negative samples were detected among 115 samples by the method of the present invention, and the compliance rate of this experimental method was 94%.

[0029] Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) and the main cause of cervical cancer development. More than 90% of deaths are caused by lack of access to screening and treatment, and almost all cervical cancer cases are caused by human papillomavirus (HPV) infection. Among more than 200 types of HPV, at least 12 are considered high-risk HPV52, which is the fourth most common HPV type in cervical cancer cases in China. The double antigen sandwich ELISA of the present invention uses vaginal lavage fluid (the lavage fluid is PBS) for direct detection, which not only avoids the problems existing in traditional detection methods, but also has the characteristics of high sensitivity and specificity, wide application, does not rely on large equipment, has strong universality, and is more helpful for the screening of HPV patients. In addition, to date, the development and use of the double antigen sandwich ELISA has not been used for the detection of HPV 52 antibodies.

Claims

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

1.

2. Use of the recombinant protein based on 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: The double antigen sandwich ELISA method was used to detect whether there was HPV52 antibody 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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