The Construction Of Prokaryotic Expression System Of HPV Major Capsid Protein L1 Gene And The Frequencies Of HPV-6/11 Infection In Biopsy Samples And L1 Gene Expression Of Pointed Condyloma Patients

Human papillomavirus (HPV) is a double-stranded DNA virus with a strict epithelial-phlic characteristic. According to the difference of genomic sequence of HPV, more than 130 genotypes of the virus were identified so far. L1 and L2 genes of HPV are respectively responsible for encoding the major capsid protein L1 and the minor capsid protein L2. The proteins L1 and L2 compose the HPV capsid with a molar ratio of 25-30 to 1. The whole length of HPV L1 gene is approximate 1.5 kb and the molecular weight of its expression product is 55～60 KD. The sequence of L1 protein shows a high conservation and is a major genus-specific antigen, which inducing neutralizing antibody.HPV infects human skin and mussel tissues and cause proliferative diseases of epithelial tissues. According to the harmfulness of infection with different genotypes of HPV, it can be divided into high-, intermediate- and low-risk types. The high-risk type contains genotypes HPV-16, HPV-18 and HPV-45, while the intermediate-risk type includes genotypes HPV-33, HPV-35 and HPV-58. Cervical carcinoma, a common malignant tumor in women, was found to be closely associated with the infection of HPVs belonging to high- and intermediate-risk types. For low-risk type,HPV-6, HPV-11, HPV-42, HPV-43 and HPV-44 genotypes are involved. The infection of HPVs belonging to low-risk type causes verruca vulgaris in skin and pointed condyloma in genitals. HPV-6 and HPV-11 are the two genotypes that mostly causing agents of pointed condyloma.Since cervical carcinoma is a common tumor in women and pointed condyloma is a major sexual transmission disease, HPV vaccine becomes the key point in the corresponding research field. It was reported that virus like-particles (VLPs), consisted of the product expressed by LI gene or the products co-expressed by both LI and L2 genes, could induce experimental animals to produce protective immune response. However, the outputs of all the VLPs were reported to be low. At present, a kind of VLP vaccine to cervical carcinoma based on LI protein of HPV has been going into the phase HI of clinical check, but its practical effect for prevention remains unanswered.At the present, the laboratory diagnosis of HPV infection to detect the specific genetic fragments of the virus is dependent on molecular techniques such as PCR and Southern hybridization. However, these molecular techniques are unable to determine the immune response and state to HPV of patients. These techniques' costs are higher as well as offer a relative ratio of false positive results. It is well known that serological technique is one of the major methods to determine the infection of particular pathogens. So far, HPV have been cultivated in vitro and cannot replication in animals, resulting in the difficulty to obtain HPV antigens required in serological examinations. Therefore, by using genetic engineering techniques, the expression system of the major capsid protein LI of HPV is easily constructed, which lay a foundation to conveniently gain a great number of the target recombinant protein. The serological detection methods by using recombinant major cased protein LI (rLl) as the antigen can not only used for clinic laboratory diagnosis of pointed condyloma, but also applied as an important tool in serologically epidemiological investigation for HPV infections as well as for cervical carcinoma and its precancerous lesion. However, the data about the serological examination methods of HPV-associated diseases are absent so far.In this study, we established a multiple PCR based on LI gene to simultaneously detect both HPV-6 and HPV-11. By using this PCR, the difference of infecting frequencies in biopsy samples from pointed condyloma patients in Chain were investigated. The entire LI gene of HPV-6 was cloned from lesion tissue of the patients and a prokaryotic expression system with high efficiency of the gene was then constructed. An ELISA was established to examine the expression of HPV LI gene in the biopsy samples. All the works will offer evidences for rLl as an antigen candidate in the laboratory diagnostic methods and vaccines of HPV.MATERIALS AND METHODS1. Preparation of DNA templateThe pointed condyloma tissue from a patient was completely broken. Part of Ihe broken tissue was used for protein LI by ELISA and the rest was applied to extract genomic DNA by Genomic DNA Purification Kit (Biocolor). The concentration and purity of DNA extract were measured by ultraviolet spectrophotometry.2. Amplification of the target geneBased on the published corresponding genome sequences (Genbank No.: NC₀01668, AF067036, AF092932) and the result of endonuclease map analysis in the sequences, a pair of primer with suitable endonuclease cutting sites was designed. PCR with the designed primers was used to amplify the target gene in pointed condyloma tissues. Agarose electrophoresis was performed to detect the amplification products.3. T-A cloning, sequencing and construction of expression vectorThe target amplification DNA fragment of LI gene was cloned into pGEM-T vector by using T-A Cloning Kit (BioAsia) according to the manufacturer's instruction. The recombinant plasmid was transferred into E.coli strain DH5 a . This transferred cabterial strain was enriched and then the recombinant plasmid in the E.coli was extracted by alkaline denature method. After getting satisfactory sequencing results, the strains of E.coli DH 5 a respectively containing pGEM-T-Lland pET32a were enriched and then the plasmids were extracted. The fragments of target gene recovered from the plasmid were digested with double endonucleases to obtain the fragments of the target gene and linearlized pET32a. The two different fragments were ligased and then transferred into E.coli strain BL21DE3. The bacterial strain was enriched and the recombinant plasmid in the bacterium was extracted for sequencing again. The sequencing data was homologiously compared to the reported sequernces of HPV-6 LI gene mentioned above.4. Expression and purification of the target recombinant proteinThe prokaryotic expression system pET32a-Ll-ï¿¡.co//BL21DE3 was cultured at 37"C for 4 h for expression induced with EPTG at different disages. 10% SDS-PAGE was used to determine the expression of the target recombinant protein rLl. The expressed rLl was collected by Ni-NTA affinity chromatography (BioAsia).5. Preparation of rLl antiserum and titer dtermiantion of the antiserumBy using routine subcutaneous immunizing method, rabbits were challenged with rLl to prepare the antiserum. Immunodiffusion assay was applied to determine the antiserum's titer.6. Detection of the LI expression in clinical sanples116 biopsy samples of pointed condyloma from patients were homogenized and then were ultrasonically broken (300V, 5 S X 3). The supernatants were collected by centriphugation at 3000 r/min for 10 min. The protein concentrations in the supernatants were measured by ultraviolet spectrophotometry. By using the ultrasonic supernatants with protein concentration of 100 ug/ml as the coated antigen, the rabbit antiserum against rLl (1:500 dilution) as the first antibody and HRP-labeling sheep anti-rabbit IgG antibody (1:3000 dilution) as the second antibody, an ELISA was established to detect LI proteins in the pointed condyloma samples mentioned above. During the detection, rLl and the ultrasonically broken supernatants of 5 preputial tissue samples with the same protein concentrations were used as the positive and negative controls, respectively. By using OPT as the substrate, the OD490 value of each the biopsy samples was measured with Enzyme LinkedImmunosorbent Detector. The value of the OD490 mean plus 3SD from the 5 cases of negative control was acted as the positive deciding standurd.7. Detection of the infraction frequencies of HPV-6/11 in pointedcondyloma samplesAccording to the deletion of amino acid residual at the 130th position and a serine (S) residual located at the 131st position in the HPV-6 LI sequences (Genbank No.: NC₀01668, AF067036, AF092932), and a glycine (G) residual and tyrosine (Y) residual respectively located at the 130th and 131st positions in the HPV-11 LI sequences (Genbank No.: U55993, M14119), and the amino acid sequence at 273-278th position for HPV-6 was EKGSG while the sequence at the same position for HPV-llwas LVKGGN, two sets of primers respectively specific to HPV-6 and HPV-11 were designed using a software Primer Designer. A multiple PCR using the two sets of specific primers was built to simultaneously detect the fragments of HPV-6 and HPV-11 LI genes. Agarose electrophoresis was performed to detect the amplification products. The expected sizes of amplification products of HPV-6 and HPV-11 LI genes were 305 bp and 444 bp, respectively.RESULTS1.PCRThe entire LI gene with 1503 bp of HPV-6 was obtained by amplification from the DNA of pointed condyloma biopsy sample of a patient (No.: H26).2. Analyzing results of the target cloned gene sequenceThe homologies of nucleotide and amino acid sequences of the cloned entire HPV-6 LI gene were 99.20-99.93% and 99.80-100%, respectively, compared to the reported sequences of HPV-6 LI genes. The sequences from T-A clone and subclone are absolutely same.3. Expression and antigenicity of target recombinant protein1.0, 0.5 and 0.1 mmol/L of IPTG were able to efficiently induce the rLl expression, but 0.1 mmol/L of IPTG displayed the inducing effect. Theimmunodifrusion titer of the rabbit anti-rLl serum was 1:4.4. The detection result of protein LI in the pointed condyloma samplesThe value of mean±SD at OD490 of the 5 cases of negative control was 0.17+ 0.06 so that the positive deciding standard was 0.35. According to the standard, 88% (103/116) of the pointed condyloma biopsy samples are positive for LI protein and its range of OD490 values were 0.46¹.15. Of these samples, 4 cases showed positive results by PCR but negative results by ELISA, and no cases with negative PCR result but with positive ELISA result could be found.5. The infection rates of HPV 11/6 in the pointed condyloma samplesIn the 116 pointed condyloma biopsy samples, the total positive detection rate of HPV-6 and HPV-11 LI genes was 92.2% (107/116). Of the 107 positive samples, 75 cases and were HPV-6 LI gene detectable, 25 cases were HPV-11 LI gene positive and 7 cases were positive for both the HPV-6 and HPV-11 LI genes.Conclusion1. In this study, we successfully constructed a prokaryotic expression system of HPV-6 LI gene with high expression efficiency.2. A multiple PCR had been established to simultaneously detect the LI genes of HPV-6 and HPV-11 in pointed condyloma biopsy samples.3. An ELISA had been established to detect the major capsid protein LI of HPV in pointed condyloma biopsy samples.4. A high positive rate of the major capsid protein LI of HPV could be found in pointed condyloma biopsy samples.5. The pointed condyloma patients in Zhejiang area were mainly infected with HPV-6. The minority of the patients showed HPV-6 and HPV-11 coinfection.