| 孙爱华,徐莹,冯燕,严杰.尖锐湿疣组织中人乳头瘤病毒6和11型的感染率及其L1基因的表达[J].Chinese journal of Epidemiology,2006,27(2):150-153 |
| 尖锐湿疣组织中人乳头瘤病毒6和11型的感染率及其L1基因的表达 |
| Study on the frequency of human papillomavirus type 6 and type 11 infection and L1 gene expression ofthe virus in biopsy samples of pointed condyloma patients |
| Received:April 11, 2005 |
| DOI： |
| KeyWord: 尖锐湿疣 人乳头瘤病毒6型/11型 L1基因型 |
| English Key Word: Pointed condyloma Human papillomavirus type 6Pt ype 11 L1 genotype |
| FundProject:浙江省医药卫生科技计划(2004A018) |
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| Abstract: |
| 目的了解尖锐湿疣组织标本中人乳头瘤病毒6型(HPV-6)和11型(HPV-11)感染率的差异,构建HPV-6型主要衣壳蛋白L1基因原核表达系统,建立ELISA方法检测标本中L1基因表达情况。方法采用基于HPV-6型和HPV-11型L1基因的双重PCR,检测尖锐湿疣组织标本中HPV-6型和HPV-11型的感染率;采用PCR扩增HPV-6型全长L1基因,T-A克隆后测序,构建原核表达系统pET32a-L1-E.coliBL21(DE3),采用SDS-PAGE检测目的重组蛋白rL1的表达。制备rL1兔抗血清,用免疫双扩散试验检测抗血清的效价;建立ELISA方法检测标本中L1基因的表达情况。结果116例患者尖锐湿疣组织标本中,92.2%(107/116)检出HPV-6型和/或HPV-11型。其中单一HPV-6型阳性者占70.1%(75/107),单一HPV-11型阳性者占23.4%(25/107),HPV-6型和HPV-11型混合感染者占6.5%(7/107)。与报道的相应序列比较,所克隆的HPV-6型L1基因核苷酸和氨基酸序列同源性分别为99.20%~99.93%和99.80%~100%。IPTG能诱导rL1表达,rL1兔抗血清免疫双扩散效价为1∶4。尖锐湿疣组织标本88.8%(103/116)检出L1蛋白。结论成功地构建了HPV-6型L1基因原核表达系统,并建立了HPV-6型和HPV-11型L1基因分型的双重PCR和L1蛋白ELISA检测方法。浙江省尖锐湿疣患者主要感染HPV-6型,病灶中HPV高频率表达病毒主要衣壳蛋白L1。 |
| English Abstract: |
| Objective To determine the different rates of human papillomavirus types 6 (HPV26)and 11 (HPV211) infection in biopsy samples from pointed condyloma patients, and to construct prokaryotic expression system of the major capsid protein L1 of the virus so as to establishe an EL ISA for detecting the expression of L1 gene in the biopsy samples. Methods Using a double PCR based on the L1 gene of HPV26 and HPV211, the infection rates of HPV26 and HPV211 in the biopsy samples were determined. The whole length of HPV26 L1 gene was amplified using PCR and the target amplification fragment was sequenced after T2A cloning. The prokaryotic expression system pET32a2L12 E. coli BL21 (DE3) was constructed and SDS2PAGE was used to measure the expression of the target recombinant protein rL1. Rabbit anti2rL1 serum was prepared and immuno2diffusion assay was applied to examine the antiserum titer. EL ISA was established to detect the expression of L1 gene in the biopsy samples. Results In the biopsy samples from 116 pointed condyloma patients, 92. 2 %(107P116) were detectable for HPV26 andPor HPV211. Of the 107 positive samples, 70. 1 %(75P107) and 23. 4 %(25P107) were positive for HPV26 or HPV211 alone and 6. 5 % (7P107) were coinfected with both HPV26 and HPV211 respectively. When compared with the reported corresponding sequences, the homology of nucleotide and sequence of the cloned HPV26 L1 gene was from 99. 20 %299. 93 % while its putative amino acid sequence homology was from 99. 80 %2100 %, suggesting IPTG could induce the expression of rL1. The immuno2diffusion titer of therabbit anti2rL1 serum was 1∶4. 88. 8 %(103P116) of the biopsy samples were the major capsid protein L1 detectable. Conclusion A prokaryotic expression system of HPV26 L1 gene,a double PCR assay for HPV26 and HPV211 genotyping,and an ELISA assay for detecting the major capsid protein L1 were successfully established in this study. The pointed condyloma patients in Zhejiang area mainly infected with HPV26. The HPV in the focus frequently expressed major capsid protein L1. |
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