Sun Ai-hua, Xu Ying, Feng Yan, Yan Jie
Medical College of Zhejiang University, Hangzhou 310053, China.
Zhonghua Liu Xing Bing Xue Za Zhi. 2006 Feb;27(2):150-3.
To determine the different rates of human papillomavirus types 6 (HPV-6) and 11 (HPV-11) 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 establish an ELISA for detecting the expression of L1 gene in the biopsy samples.
Using a double PCR based on the L1 gene of HPV-6 and HPV-11, the infection rates of HPV-6 and HPV-11 in the biopsy samples were determined. The whole length of HPV-6 L1 gene was amplified using PCR and the target amplification fragment was sequenced after T-A cloning. The prokaryotic expression system pET32a-L1-E. coli BL21 (DE3) was constructed and SDS-PAGE was used to measure the expression of the target recombinant protein rL1. Rabbit anti-rL1 serum was prepared and immuno-diffusion assay was applied to examine the antiserum titer. ELISA was established to detect the expression of L1 gene in the biopsy samples.
In the biopsy samples from 116 pointed condyloma patients, 92.2% (107/116) were detectable for HPV-6 and/or HPV-11. Of the 107 positive samples, 70.1% (75/107) and 23.4% (25/107) were positive for HPV-6 or HPV-11 alone and 6.5% (7/107) were coinfected with both HPV-6 and HPV-11 respectively. When compared with the reported corresponding sequences, the homology of nucleotide and sequence of the cloned HPV-6 L1 gene was from 99.20% - 99.93% while its putative amino acid sequence homology was from 99.80% - 100%, suggesting IPTG could induce the expression of rL1. The immuno-diffusion titer of the rabbit anti-rL1 serum was 1:4. 88.8% (103/116) of the biopsy samples were the major capsid protein L1 detectable.
A prokaryotic expression system of HPV-6 L1 gene, a double PCR assay for HPV-6 and HPV-11 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 HPV-6. The HPV in the focus frequently expressed major capsid protein L1.
检测尖锐湿疣患者活检标本中6型人乳头瘤病毒（HPV-6）和11型人乳头瘤病毒（HPV-11）的感染率，构建HPV-6和HPV-11主要衣壳蛋白L1的原核表达系统，建立检测活检标本中L1基因表达的ELISA方法。
采用基于HPV-6和HPV-11 L1基因的双重PCR检测活检标本中HPV-6和HPV-11的感染率。应用PCR扩增HPV-6 L1基因全长，经T-A克隆后对目的扩增片段进行测序。构建原核表达系统pET32a-L1-大肠杆菌BL21（DE3），采用SDS-PAGE检测目的重组蛋白rL1的表达。制备兔抗rL1血清，采用免疫扩散试验检测抗血清效价。建立ELISA检测活检标本中L1基因的表达。
116例尖锐湿疣患者活检标本中，HPV-6和/或HPV-11的检出率为92.2%（107/116）。在107份阳性标本中，单纯HPV-6阳性率为70.1%（75/107），单纯HPV-11阳性率为23.4%（25/107），HPV-6和HPV-11混合感染率为6.5%（7/107）。与已报道的相应序列比较，克隆的HPV-6 L1基因核苷酸及氨基酸序列同源性分别为99.20%～99.93%和99.80%～100%，提示IPTG能诱导rL1表达。兔抗rL1血清免疫扩散效价为1:4。116例活检标本中，主要衣壳蛋白L1的检出率为88.8%（103/116）。
本研究成功构建了HPV-6 L1基因原核表达系统，建立了HPV-6和HPV-11基因分型双重PCR方法及检测主要衣壳蛋白L1的ELISA方法。浙江地区尖锐湿疣患者以HPV-6感染为主，病灶中HPV常表达主要衣壳蛋白L1。
