痘苗病毒天坛株生物学特性研究及其基因工程减毒载体的构建

发布时间：2018-05-23 11:06

本文选题：痘苗病毒 + 天坛株　；参考：《武汉大学》2005年博士论文

【摘要】：尽管痘苗病毒天坛株曾作为天花病预防疫苗在中国大范围接种使用了几十年,但其相关的生物学特性并没有详细的研究和报导。在本文中我们对痘苗病毒天坛株的细胞宿主范围、体外复制特性和病毒体内的毒性等生物学特性进行了系统的鉴定。我们发现天坛株具有广泛的宿主范围,经研究的12种哺乳动物细胞中的11种及原代鸡胚细胞CEF均是天坛株的允许细胞,而中国仓鼠卵巢细胞CHO-K1是天坛株的非允许细胞。天坛株在几乎所有测试的细胞中均能产生严重的至细胞病变效应(Cytopathic effects,CPE),包括四种人类来源的细胞系。体内实验显示,尽管天坛株比神经毒性的WR株毒性弱很多,但天坛株仍保留相当的神经毒性,而且经鼻腔接种可引起小鼠体重严重下降。因而有必要对天坛株进行进一步的减毒,使其成为更为安全理想的天花疫苗或作为更安全的病毒载体用于构建活病毒载体疫苗。痘苗病毒的血凝素基因(Hemagglutinin,HA)与痘苗病毒的体内毒性密切相关,缺失HA可导致多种痘苗病毒株系在动物体内不同程度的减毒。在本文中,我们构建了HA基因完全失活或部分失活的重组痘苗病毒TT_(zcl)和TT_(pzcl)(HA基因的晚期启动子被插入失活)。我们发现,重组痘苗病毒在12种VTT允许细胞中的7种中有不同程度的复制水平的下降,其中TT_(zcl)在RK13和MDCK中完全失去复制能力。而TT_(pzcl)在MDCK中失去复制能力。在多数动物细胞中包括在人源细胞中重组病毒具有比野生天坛株病毒更低的细胞到细胞间的扩散水平或更小的对细胞的毒性。此外,我们还证实重组痘苗病毒在连续传代中显示具有外源基因稳定插入和稳定表达的特征。在体外实验中我们进一步检测了不同感染途径重组痘苗病毒在模型动物中的毒性。我们发现,重组病毒TT_(zcl)具有经多种接种途径接种在体内高度减毒的特征,这可能与TT_(zcl)在动物脑中枢神经系统中复制水平显著下降相关。而HA部分失活的重组痘苗病毒TT_(pzcl)在体内也显著减毒的特征,但其减毒的程度大大低于重组病毒TT_(zcl)。高度减毒的重组痘苗病毒TT_(zcl)将是更为安全的载体,可用于进一步构建病毒载体疫苗。
[Abstract]:Although vaccinia virus Temple of Heaven strain has been widely used in China for decades as a smallpox preventive vaccine, its biological characteristics have not been studied and reported in detail. In this paper, we systematically identified the biological characteristics of vaccinia virus Temple of Heaven strain, such as cell host range, in vitro replication characteristics and virulence in vivo. We found that the Temple of Heaven strain has a wide host range, 11 of the 12 mammalian cells studied and the primary chicken embryo cell CEF are all permitted cells of Temple of Heaven strain, while the Chinese hamster ovary cell CHO-K1 is the non-permissible cell of Temple of Heaven strain. Temple of Heaven strain can produce severe cytopathic effects in almost all the tested cells, including four human cell lines. In vivo experiments showed that although the Temple of Heaven strain was much less toxic than the neurotoxic WR strain, the Temple of Heaven strain still retained considerable neurotoxicity, and inoculation through nasal cavity caused severe weight loss in mice. Therefore, it is necessary to further attenuate the Temple of Heaven strain and make it a safer ideal smallpox vaccine or a safer viral vector for construction of live virus vector vaccine. Hemagglutinin gene (Hemagglutinin HA) of vaccinia virus is closely related to virulence of vaccinia virus in vivo. In this paper, we constructed recombinant vaccinia virus (TTSZ) with HA gene inactivation or partial inactivation, and the late promoter of TT_(pzcl)(HA gene was inserted into inactivation. We found that recombinant vaccinia virus has different degrees of reduction in replication level in 7 out of 12 VTT species, and TTS virus completely loses replication ability in RK13 and MDCK. TTT / Pzcls lose the ability to replicate in MDCK. Recombinant viruses in most animal cells, including human cells, have lower cell-to-cell diffusion levels or less cytotoxicity than wild Temple of Heaven strains. In addition, we also confirmed that recombinant vaccinia virus has the characteristics of stable insertion and stable expression of exogenous genes in successive passages. In vitro, we further examined the toxicity of recombinant vaccinia virus with different infection pathways in model animals. We found that the recombinant virus TTS ZL has the characteristics of highly attenuated virus in vivo by multiple inoculation routes, which may be related to the significant decrease of TTS replication in the central nervous system of animals. However, the HA partially inactivated recombinant vaccinia virus TTS / pzcll was also characterized by a marked reduction of virus in vivo, but the degree of the reduction was much lower than that of the recombinant virus. Highly attenuated recombinant vaccinia virus TTSP will be a safer vector for further construction of viral vector vaccine.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346

【引证文献】