电转染法制备奶牛转溶葡球菌酶(Lysostaphin)和内溶素(Endolysin)基因胚胎  

Construction of Bovine(Bos taurus) Transgenic Cloned Embryos with Lysostaphin and Endolysin Genes by Electronic Transfection

作　　者：杨林[1,2] 杜卫华[2] 郝海生[2] 刘岩[2] 秦彤[2] 赵学明[2] 王栋[2] 朱化彬[2] 王宗礼[1] 

机构地区：[1]中国农业科学院草原研究所,呼和浩特010010 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《农业生物技术学报》2013年第2期206-215,共10页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育科技重大专项(No.2009ZX08010-015B)和(No.2009ZX08011-031B)

摘　　要：溶葡球菌酶(Lysostaphin)是一种含锌的单链蛋白酶,能有效地杀灭金黄色葡萄球菌。内溶素(Endolysin)是双链DNA噬菌体所特有,是一类胞壁质水解酶,具有广泛的抗菌效果。内溶素与抗生素之间有高效的协同作用。本研究通过BTX电转染和AMAXA核转染的方法将含有溶葡球菌酶(Lysostaphin)和内溶素(Endolysin)两个目的基因(Seq2和Seq3)及绿色荧光蛋白(enhenced green fluorescent protein,EGFP)和新霉素(neomycin,neo)两个标记基因的载体pBC1-seq2+seq3-EGFP-neo,导入牛(Bos taurus)胎儿成纤维细胞,经荧光观察、G418筛选和PCR鉴定后建立稳定转染的单克隆阳性细胞系。以其作为核供体,体细胞核移植制备转基因胚胎。通过4组不同转染程序(A-023,V-013,V-023和T-016)转染牛胎儿成纤维细胞,结果表明,转染参数T-016最适合本实验,其AMAXA转染效率(20.11%)为BTX电转染转染效率的5倍。转基因囊胚率为(20.08±4.19)%,并发育正常。本研究建立了牛胎儿成纤维细胞系,筛选出高效的转染参数,并获得了含溶葡球菌酶、内溶素的转基因细胞系和转基因囊胚,为制备抗乳房炎转基因牛及寻找防治奶牛乳房炎新途径提供了技术支持。Lysostaphin is a single chain protease containing zinc which can kill staphylococcus aureus effectively. Endolysin which is the peculiar of the double-stranded DNA bacteriophages is a murein hydrolytic enzyme, it has a wide range of antibacterial effect. Lysostaphin and Endolysin have the high synergistic effect. In this study, the vectors pBCl-seq2＋seq3-EGFP-neo containing Endolysin and Lysostaphin genes and two other marker genes of enhenced green fluorescent protein （EGFP） and neomycin （neo） were transfected into bovine （Bos taurus） fetal fibroblast by electroporation and nucleofector of AMAXA. Stable transfected monoclonal cells which were identified to be the positive-cells in the way of PCR technique were obtained through fluorescence and G418 selection. Using transfected cells as the donor, transfected embryos were produced with somatic cell nuclear transfer, we used different conditions of AMAXA nuleofecor（A-023,V-013, V-023 and T-016） to transfect bovine fetal fibroblast, the results showed the suitable program was T-016. There were 5 times transfection efficient of AMAXA nuleofecor （20.11%） than it of electroporation. The blastocyst developed normally and the rate was of it 20.08%. In our study, we built up bovine fetal fibroblast cell line, sought out transfection parameter of high transfection efficiency, and acquired transgenic cell lines and transgenic blastocyst containing Lysostaphin and Endolysin genes. In conclusion, the results can provide technology supporting for producing anti-mastitis transgenic bovine and searching the new therapy way of mastitis.

关 键 词：溶葡萄球菌酶 内溶素 电转染 转基因 乳房炎 

分 类 号：S858.237.2[农业科学—临床兽医学]