【作者】 陈邦党；

【导师】 张富春；

【作者基本信息】 新疆大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 1.人乳头瘤病毒16型L1基因重组腺病毒载体的构建及表达人乳头瘤病毒(Human papillomavirus, HPV )是一种双链的小DNA病毒。高危型HPV可引起宫颈癌等恶性肿瘤,其中最常见的是HPV 16型和HPV 18型。大量研究资料证明,50%的宫颈癌与HPV16感染相关。因HPV有潜在的致癌性,故HPV疫苗的制备不能采用传统的减毒活疫苗或灭活疫苗。目前,HPV16疫苗研究集中于晚期蛋白L1及L2组成的基因工程亚单位疫苗。HPV16晚期基因L1编码病毒的主要衣壳蛋白,在真核细胞中产生的HPV16L1或L1+L2可高效自行装配成病毒样颗粒(virus like particles, VLPs)。由于VLPs不含DNA成分,且结构和天然病毒颗粒相似,能刺激机体产生抗体,保护机体不受同型别HPV感染而成为引人关注的免疫原。在前期的研究中,我们已经从新疆南部地区维吾尔族妇女宫颈癌组织中克隆获得HPV16 L1基因。该基因发生多位点变异,并形成突变的主流模式,这些突变引起HPV16 L1蛋白疏水性和抗原性的改变。L1基因的突变可导致HPV16逃避机体免疫识别,造成HPV16的持续感染。以新疆地区宫颈癌患者组织中的HPV16 L1基因开展疫苗研究,对预防HPV 16新疆流行株的感染将更为有效。本研究将本室克隆的新疆株HPV16 L1基因亚克隆到腺病毒穿梭质粒pShuttle-CMV上,采用细菌内同源重组“两步转化法”构建携带L1基因的重组腺病毒质粒pAd-L1,以获得的重组质粒线性化后转染HEK293细胞,获得重组腺病毒;PCR法对重组腺病毒基因鉴定表明L1基因已经整合到腺病毒基因组中,空斑法测定重组腺病毒RAd-L1的滴度为1.5×109 pfu /L,RT-PCR检测表明该重组腺病毒感染后的宿主细胞具有HPV16L1对应的mRNA表达;Western blot和间接免疫荧光检测表明L1基因在HEK293细胞能够有效的表达。制备了表达新疆人乳头瘤病毒16型L1基因的非复制型重组腺病毒,为后续进行重组腺病毒在动物体内诱导抗L1免疫应答能力的研究奠定了基础。2.小鼠、草原兔尾鼠卵透明带3基因重组腺病毒载体的构建及表达哺乳动物卵透明带(zonapellucida,ZP)是由卵泡细胞和卵细胞合成和分泌的,由ZP1、ZP2、ZP3三种硫酸化的糖蛋白组成,是受精过程中精子必须穿过的障碍。其中ZP3在受精过程中有两个重要作用:一是与精子专一性地结合;二是与精子结合后诱导精子的顶体反应。大量实验证明,阻断精子与ZP3的结合就可以阻断小鼠、猪等哺乳动物的生殖过程,因此,ZP3是利用免疫不育技术控制有害动物种群数量的理想靶抗原。重组腺病毒载体可模仿病毒天然的感染方式,有效地将外源DNA导入宿主细胞内并表达天然的、保持完整生物活性的抗原蛋白,能够诱导有效的特异性体液免疫应答和强烈的细胞免疫应答。本研究所用病毒载体为缺失E1和E3基因的人血清型5型复制缺陷性腺病毒,插入外源基因可达7.5kb;宿主范围广,可转染分化及未分化细胞;可获得高滴度的病毒,而且腺病毒并不整合到宿主基因组上,相对安全,基因转染后不会激活或者抑制宿主细胞基因的表达,因此作为免疫不育疫苗载体具有明显的优势。本研究将本室克隆的草原兔尾鼠和小鼠的zp3基因亚克隆到腺病毒穿梭质粒pShuttle-CMV上,采用细菌内同源重组“两步转化法”构建携带zp3基因的重组腺病毒质粒pAd-mzp3和pAd-lzp3,以获得的重组质粒线性化后转染HEK293细胞,包装成重组腺病毒;PCR法对重组腺病毒基因鉴定表明mzp3和lzp3基因已经整合到腺病毒基因组中;间接免疫荧光检测表明zp3基因在HEK293细胞中能够有效表达;病毒的滴度可达1.2×109pfu /L和1.3×109pfu/L;继而用鉴定正确的重组腺病毒感染HeLa细胞,RT-PCR检测表明构建的重组腺病毒感染后的宿主细胞具有mzp3和lzp3对应的mRNA表达,Western blot检测表明感染的HeLa细胞中zp3基因能够有效表达。本研究探索制备免疫不育疫苗的新途径,获得草原兔尾鼠卵透明带3(lzp3)和小鼠卵透明带3(mzp3)重组腺病毒载体疫苗,为进一步进行重组腺病毒活疫苗免疫不育技术控制鼠害的研究奠定基础。更多还原

【Abstract】 1. Construction and Expression of the Recombinant Adenovirus with L1 Gene of Human Papillomavirus Type 16 Human papillomavirus (HPV)is a double strand DNA virus. HPV with high risk, such as the familiar the most type16 and type 18, can lead to the malignant tumor, e.g., cervical carcinoma. There are large amounts of data identified that 50 percent of cervical carcinoma is due to the infection of HPV16. It is not an appropriate method to develop an attenuated vaccine or inactivated vaccine of HPV because it contains DNA which has potential carcinogenesis. It is now focused on the subunit vaccine of later protein L1 and L2 to develop HPV16 vaccine.The later gene L1 of HPV16 codes for major capsid protein in virus. HPV16 L1 or HPV16 L1+L2 produced in eukaryotes can automatically and efficiently assemble to virus like particles. The VLPs become a concerning immunogenic material because it doesn’t contain DNA and shares the similarity with the crude virus in structure , therefore , it can stimulate organism to secrete antibody to prevent human from infection by same type of HPV , so L1 protein becomes an important target antigen to produce HPV vaccine. HPV16 L1 gene which was previously acquired from cervical carcinoma tissue of Xinjiang Uygur women in our lab occurs variation multiple sites hence forming the main mode of mutations. The mutation can cause the hydrophobic and antigenic changes of HPV16L1. The mutation in L1 gene will give rise to HPV16 escaping from immunity recognition which can cause continuous infection of HPV16. Research in DNA vaccine aimed at HPV16 L1 in xingjiang will greatly protect people from HPV16 infection.The HPV16 L1 gene cloned previously by our lab from cervical carcinoma tissue of Xinjiang Uygur women, was subcloned into shuttle vector pShuttle, a two-step transformation protocol was employed for the construction of recombinant adenoviral plasmid pAd-L1, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. PCR test indicated that L1 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.5×10~9 pfu/L, meanwhile, RT-PCR demonstrated the transcription of L1 in infected cells, Western blot and IFA revealed that L1 protein can express in HEK293 cells, which will be potentially used as a candidate vaccine for the prophylaxis and treatment of HPV infection.2. Construction and Expression of the Recombinant Adenovirus with Zona Pellucida 3 Gene of Murine and Lagurus lagurus The mammalian oocyte is surrounded by an extracellular translucent envelope termed the zona pellucida (ZP). The ZP mediates the initial recognition and binding events of sperm to the oocyte in a relatively species specific manner, thus playing a vital role in fertilization. In most species, ZP is composed of three biochemically and immunologically distinct glycoproteins, ZP1, ZP2, and ZP3, which can recognize and combine with sperm and induce acrosome reaction. It has been widely concentrated that fertilization could be successfully prevented when the combination of ZP3 and sperm was blocked by monoclonal antibodies both in vivo and in vitro, and so ZP3 glycoprotein has been considered a good candidate for developing an immunocontraceptive vaccine.Recombinant adenoviral vectors can imitate virus inartificial infection manners, delivery the foreign genes effectively to host cells, then express natural antigens with full bioactivity , which can induce not only effective and specific humoral but also intense cell mediated immune responses. The most commonly used adenoviral vector is human adenovirus serotype 5, which is rendered replication defective by the deletion of the E1 and E3 genes. Not only do these deletions render the virus incapable of replicating itself, but they also create space for up to 7.5 kb of foreign DNA. Recombinant adenovirus can infect both dividing and non-dividing cells, and produce high titers as well, the recombinant adenovirus remains epichromosomal in the human host cell, there is no possibility of activation or inactivation of host cell genes resulting from interruption by the transfected gene(s),so replication defective adenovirus vectors have distinct advantages as an immunocontraceptive vaccine vectors.The lzp3 and mzp3 genes cloned by our lab from Lagurus lagurus and mouse , were subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with lzp3 and mzp3, which were digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. PCR test indicated that zp3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2×109 pfu/L and1.3×109 pfu/L, IFA revealed that ZP3 protein can express in HEK293 cells. Then HeLa cells were infected by the recombinant adenovirus. RT-PCR demonstrated the transcription of zp3 in infected cells; Western blot revealed that the recombinant adenovirus RAd-lzp3 and RAd-mzp3 can be successfully expressed in the infected HeLa cells, which lays the foundation for developing a new immunocontraceptive vaccine against Lagurus lagurus and murine zona pellucida 3.更多还原