【作者】 张宇；

【导师】 孔维；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 获得人乳头瘤病毒16亚型主要衣壳蛋白(L1)在Pichia Pastoris酵母分泌型表达系统中的高效表达。首先将人乳头瘤病毒16亚型主要衣壳蛋白L1基因重组到分泌型酵母表达载体(pPICZαB)形成整合质粒,电转酵母细胞GS115,经甲醇诱导表达,SDS-PAGE电泳、Western-Blot检测和PCR鉴定,筛选多拷贝阳性整合克隆,经过184个克隆筛选,获得了表达量较高的表达菌株。该菌株甲醇诱导后,发酵上清经SDS-PAGE电泳检测显示,上清中有特异蛋白条带,且在第四天表达量最高,表达产物单体分子量为55,000Da左右。发酵上清液经纯化,可获得纯度为90%以上的HPV16L1蛋白,电镜观察,所得HPV16L1蛋白可自组装成病毒样颗粒(VLPs),直径约为55nm。结果表明,人乳头瘤病毒主要衣壳蛋白L1基因在毕赤酵母表达系统中获得了分泌表达。更多还原

【Abstract】 Human papillomavirus (HPV) infection is a common sexually transmitteddisease.Although most infections are benign,persistent infection is associatedwith the development of cervical and other anogenital cancers.Until now more than 100 types of HPV have been identified.The typesassociated with diseases of the anogenital tract can be classified on the basis ofphylogenetic Relationship and of association frequencies with benign ormalignant cervical lesions as low-risk types (HPV-6, -11, -34, -40, -42, -43,-44) and high-risk types (HPV-16,-18, -31, -33, -35, -39, -45, -51, -52, -54, -56,-58, -59, and -66). The most common HR types are HPV-16, -18, -31, -33, and-45.The most commonly found LR types are HPV-6 and -11. HPV are small(55-60nm),non-enveloped icosahedral DNA viruses whichinfect cutaneous and mucosal epithelia。It has ten open reading frame(ORF),earlyregion、late region、long control region.(1)Earlyregion include six open reading frames:E1、2、4-7。Humanpapillomavirus (HPV) is etiologically involved in virtually all cervical cancers,and the early .HPV oncoproteins E6 and E7 are responsible for themalignant .phenotype, mainly through inactivation of tumor suppressorproteins such as p53 and pRB. Previous work evaluating different markers ofexposure and viral activity in tumors indicates that HPV may also play a rolein some cancers of the oral cavity and oropharynx. Several studies haveinvestigated prevalence of HPV in these cancers, but the prevalence of HPVdetection varies broadly, depending on the population, combination of subsites,type of specimen, and detection method.(2)Late region(LR) include L1、L2. Expressed L1 or L1/L2 protein couldbe assembled into virus-like particles (VLPs) in vitro retaining their naturalconformation and biological activity as capsid proteins. Nevertheless, for allthe existing protocols of HPV16 VLPs purification, CsCl ultra-centrifugationwas obligatory. This procedure is tedious and costly, thus limiting its widerapplication, especially in developing countries.(3) Long control region(LCR) call noncoding region(NCR),too.Worldwide, cancer of the cervix is the second leading cause of cancerdeath in women (behind breast cancer) and is the most common form ofcancer among women indeveloping countries, with an estimated 500,000 casesdiagnosed each year, resulting in over 200,000 deaths annually.To clone and express in vitro Human papillomavirus type 16 l1(HPV16L1) gene ,and provide a good basis for further research DNA vaccineagainst HPV16 infection and human cervical cancer.To construct eukaryoticexpression plasmid of Human Papillomavirus type 16 (HPV16) gene L1 fromwhich we may select recombinant that can express HPV16L1 in a high level.Human papillomavirus type 16 (HPV16) recombined into Picha Pastorisexpression vector(pPICZ3.6KaB).The recombinant plasmid was transformedto GS115 for expression and the expressed protein was identified byWestern—Blot and SDS. We achieved the high-level recombinant in 134recombinant, and the constructed recombinant plasmid aB-16 was highlyexpressed in P.pastoris. Western-Blot showed the specific bands of theexpressed product.The high-level recombinant was successfully expressed in P. pastoris.Therecombinant plasmid pPICZaB3. 5kb-HPV16L1 could be induced to expressthe HPV16 late protein L1 by methanol in methanol-trophic yeast expressionsystem. The L1 protein expressed could assemble automatously to form theVLPs.更多还原