稳定表达鸡氨肽酶N的MDCK细胞系建立及其初步鉴定

发布时间：2018-02-11 06:41

本文关键词： 鸡传染性支气管炎病毒氨基肽酶N 稳定表达细胞系感染　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：鸡传染性支气管炎病毒(IBV)是冠状病毒科(Coronaviridae)的单股正链RNA病毒。临床上传染性支气管炎(IB)可以导致患病鸡产蛋量下降,肉鸡的生长速度减慢,给养殖业造成重大的经济损失。IBV不同血清型的毒株众多,在同一地区可能同时出现多个血清型的毒株,且他们之间交叉保护效果较低,给IB的综合防治带来巨大的困难。IB致病机制的阐明,尤其是对IBV感染宿主细胞过程的研究是该病防治的基础,有研究表明鸡氨肽酶N(c APN)在IBV感染宿主细胞的过程中发挥重要作用。为了进一步研究c APN的生物学功能及其对IBV感染宿主细胞的影响,我们开展了稳定表达c APN的MDCK细胞系建立及其初步特性的研究。本文构建了p ET30a-c APN原核重组质粒,鉴定正确的质粒转化Rosetta宿主菌中进行诱导表达,SDS-PAGE表明重组表达蛋白分子量为113Ku,且以包涵体的形式表达。重组蛋白纯化和复性后作为免疫原免疫新西兰大白兔制备其多克隆抗体。为了进行c APN稳定表达细胞系的筛选,将pc DNA3.1-c APN转染MDCK细胞,通过G418筛选和多次克隆筛选出稳定表达cAPN基因的细胞株,RT-PCR和间接免疫荧光检测表明c APN在获得的MDCK细胞中可以稳定表达,并命名为MDCK-c APN。病毒感染实验表明IBV可以感染MDCK-c APN细胞,并且在接种后24 h后出现了典型的细胞病变,而对照MDCK细胞不能感染IBV,说明cAPN在IBV感染细胞的过程中可能作为细胞受体发挥着重要的生物学功能。总之,本实验筛选成功稳定表达cAPN的MDCK-c APN细胞系,为进一步研究IB致病机理及在IBV毒株的分离和生物学特性鉴定方面奠定了基础。
[Abstract]:Infectious bronchitis virus (IBV) is a coronavirus (Coronaviridae) positive strand RNA virus. The clinical infectious bronchitis (IB) can cause the ill chicken egg production decreased, broiler growth slowed, resulting in significant economic losses to the aquaculture industry.IBV different serotype strains are numerous, and there were several serum the strain could be in the same area, and their cross protection is low, to clarify prevention and cure of IB difficult.IB pathogenic mechanism of huge, especially on IBV infection of host cells is the basis for the disease prevention and treatment, research has shown that chicken aminopeptidase N (C APN) play an important in the process of IBV infection of host cells. In order to further study the biological function of C APN and IBV infection of the host cells, and the characteristics of the MDCK cell line was established we carried out the stable expression of C APN The study of P ET30a-c APN. We construct the Prokaryotic Recombinant Plasmid, the plasmid and transformed into E.coli Rosetta for expression, SDS-PAGE showed that the recombinant protein molecular weight is 113Ku, and the expression in the form of inclusion bodies. After purification and renaturation of recombinant proteins as by immunization of New Zealand white rabbits to prepare polyclonal in order to carry out the screening of antibody. Cells stably expressing C APN, PC DNA3.1-c APN was transfected into MDCK cells by G418 screening and repeatedly screened clones with stable expression of cAPN gene in cell lines, RT-PCR and indirect immunofluorescence assay showed that C APN can be stably expressed in the MDCK cells, and named IBV that can be infected with MDCK-c APN cells MDCK-c APN. virus infection, and at 24 h after inoculation appeared typical cytopathic effect, while the control MDCK cells can not be infected with IBV, cAPN in IBV infected cells. In the process, it may play an important biological function as a cell receptor. In short, we successfully screened MDCK-c APN cell lines stably expressing cAPN, which laid the foundation for further study of IB pathogenesis and isolation and biological characteristics identification of IBV strains.

【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【共引文献】