不同病毒载体系统应用于水牛体细胞转基因的研究  

Study on the application of different viral vector systems in somatic cell transgenic of buffalo

作　　者：吴永梅 杨小玲[1] 邹灵秀[1] 潘雨 邓彦飞[1] WU Yongmei;YANG Xiaoling;ZOU Lingxiu;PAN Yu;DENG Yanfei(State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,Animal Science and Technology,Guangxi University,Nanning 530003,China)

机构地区：[1]广西大学动物科学技术学院亚热带农业生物资源保护与利用国家重点实验室,南宁530003

出　　处：《黑龙江畜牧兽医》2023年第3期7-12,131,132,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：广西创新驱动重大项目(AA17204051);广西自然科学基金面上项目(2018GXNSFAA281007);南宁市科学研究与技术开发计划项目“科技基地专项”(20192087);广西高校中青年教师科研基础能力提升项目(2019KY0048)。

摘　　要：为了筛选出能高效率感染水牛体细胞、实现基因转移的病毒表达载体系统,试验采用携带增强绿色荧光蛋白(EGFP)的3种不同病毒载体介导的转基因,即逆转录病毒载体(pMX-EGFP)、诱导型慢病毒载体(FUW-TETO-EGFP)和慢病毒载体(FUGW-EGFP)系统进行病毒包装,然后分别一次或二次感染不同的水牛体细胞[水牛胎儿成纤维细胞(BFFs)和水牛胃细胞(BSTC)],应用荧光显微镜观察EGFP是否表达,然后用流式细胞仪检测感染效率,最后用G418对感染效率低的病毒载体系统进行阳性细胞系筛选。结果表明:3种病毒载体系统均能感染BFFs和BSTC,实现了基因的转移和表达,FUGW-EGFP、FUW-TETO-EGFP系统对水牛体细胞的感染效率显著高于pMX-EGFP系统(P<0.05);除FUGW-EGFP系统感染BSTC外,同种类型病毒载体系统感染同种细胞,二次感染的感染效率高于一次感染,但均差异不显著(P>0.05);同一病毒载体系统一次感染BSTC的效率高于感染BFFs。说明FUGW-EGFP、FUW-TETO-EGFP系统对水牛体细胞的感染效率高于pMX-EGFP系统,而且BSTC比BFFs更易被病毒感染。The objective of this study is to screen out the virus expression vector system that can efficiently infect buffalo somatic cells and realize gene transfer.Three different viral vector-mediated transgene systems carrying enhanced green fluorescent protein(EGFP),namely,retrovirus vector system(pMX-EGFP),inducible lentivirus vector system(FUW-TETO-EGFP)and Lentivirus vector system(FUGW-EGFP),were used for virus packaging.Then they infected different buffalo somatic cells(Buffalo fetal fibroblasts[BFFs]and buffalo stomach cells[BSTC])once or twice.Fluorescence microscopy was used to observe the expression of EGFP,then flow cytometry was used to detect the infection efficiency,and finally G418 was used to screen the positive cell lines of the virus vector system with low infection efficiency.The results showed that BFFs and BSTC could be infected by the three viral vector systems,and gene transfer and expression were realized.The infection efficiency of FUGW-EGFP and FUW-TETO-EGFP was significantly higher than that of pMX-EGFP(P<0.05).Except for the infection of BSTC by the same type of virus carrier system,the infection efficiency of secondary infection was higher than that of primary infection,but the difference was not significant(P>0.05).The infection efficiency of BSTC with the same virus carrier system was higher than that of BFFs.These results indicated that FUGW-EGFP and FUW-TETO-EGFP infected buffalo somatic cells more efficiently than pMX-EGFP,and BSTC was more susceptible to virus infection than BFFs.

关 键 词：逆转录病毒载体系统 慢病毒载体系统 诱导型慢病毒表达载体系统 转基因 水牛体细胞 感染方式 

分 类 号：S823.83[农业科学—畜牧学] Q78[农业科学—畜牧兽医]