【作者】 佟惠春；

【导师】 李秋香； 李冬田；

【作者基本信息】 天津医科大学 ， 病原生物学， 2007， 硕士

【摘要】 近年来，性传播疾病(STD)逐年增多，而其中尖锐湿疣(CA)的发病率占性病构成比的24.73％，居第二位。人乳头瘤病毒(HPV)型别众多，而尖锐湿疣主要是由HPV 11型引起的。HPV基因组中的晚期区基因L1编码主要衣壳蛋白，具有稳定的免疫原性。在原核表达系统中，L1基因表达后可自行组装成不含病毒DNA的病毒样颗粒(VLPs)。VLPs具有与天然病毒相似的空间构象、免疫特性和生物学活性，因此以L1为保护性抗原构建的疫苗在预防HPV感染方面发挥着重要的作用。本研究首先从天津地区尖锐湿疣患者新鲜组织标本中提取HPV DNA，并用PCR方法对其进行分型，筛选出HPV 11型病毒DNA。用PCR扩增出带有酶切识别位点的EPV11 L1基因片段。利用PCR产物两末端带有的单个A碱基，与带有单个T碱基末端的pMD18-T载体借助碱基互补连接，构建成质粒pMD18T-HPV11L1，并进行了酶切鉴定以及L1基因的PCR鉴定和序列分析。从该质粒中酶切获取L1基因片段，并将其插入带有相同粘性末端的表达载体质粒pET42a构建成表达HPV11L1蛋白的重组表达质粒pET42a-HPV11L1，并对其进行了酶切以及L1基因的PCR鉴定和序列分析。最后对该重组表达质粒pET42a-HPV11 L1在大肠杆菌BL21(DE3)中进行了初步表达。本研究将HPV11型L1基因克隆入pET42a原核表达载体，成功构建了pET42a-HPV11L1，并在大肠杆菌中得到了高效表达，为进一步展开HPV疫苗的研究创造条件。更多还原

【Abstract】 Recently, sexually transmitted diseases (STDs) have been annuallyincreased. Condyloma Acuminatum (CA), the second cause of STDs isthe pathogen of 24.73% cases of STDs. Human papillomavirus (HPV) iscomposed of various serotypes and CA is mainly caused by HPV11. Thelate gene L1 of HPV genome encodes the major capsid protein, whichcontains stable immunogenicity. In the prokaryotic expression system,L1 gene may express and automatically assemble virus-like particles(VLPs) lack of viral DNA. VLPs possess the dimensional conformation,immunological characteristics and biological properties similar to wildHPVs. Subsequently, vaccines composed of the protective antigen L1may play an important role in the prevention of HPV infections.Our study firstly got HPV DNA from fresh tissue samples of CA fromTianjin area and then typified them with PCR to screen the HPV11 DNA.The L1 gene of HPV11 with enzymatic identification sites was multipliedwith PCR. Plasmid pMD18T-HPV11L1 was constructed by basiccomplementary linkage between the single basic A at the both end of thePCR products and the single basic T at the end of vector pMD 18-T,which was then identified by enzymic digestion, PCR of L1 gene as wellas its sequence analysis. L1 gene digested from pMD18T-HPV11L1 wasinserted into the expression vector plasmid pET42a with the same cohesive ends to construct the recombinant expression plasmidpET42a-HPV11L1 expressing HPV11L1 protein, which was thensuccessfully identified by enzymic digestion PCR of L1 gene and itssequence analysis. Finally, the recombinant expression plasmidpET42a-HPV11L1 was primarily expressed in the E. coli strain BL21(DE3).Our study successfully constructed pET42a-HPV11L1 by cloning L1gene of HPV11 into the prokaryotic expression vector pET42a and thengot high expression of L1 gene in E. coli, which created conditions forfurther study of HPV vaccine.更多还原