重组人SOD1/3转基因山羊的制备及表达产物的检测  

Preparation of Recombinant Human SOD1/3 Transgenic Goat and Detection of Expressed Products

作　　者：周敏雅 陆睿 张婷[1] 袁婷婷 卢瑶瑶 严坤宁 袁玉国[1] 成勇[1] ZHOU Min-ya;LU Rui;ZHANG Ting;YUAN Ting-ting;LU Yao-yao;YAN Kun-ning;YUAN Yu-guo;CHENG Yong(College of Veterinary Medicine Yangzhou University,Yangzhou 225009;College of Medicine Yangzhou University,Yangzhou 225009)

机构地区：[1]扬州大学兽医学院,扬州225009 [2]扬州大学医学院,扬州225009

出　　处：《生物技术通报》2019年第5期85-92,共8页Biotechnology Bulletin

基　　金：政府间科技合作项目(S2016G6252)

摘　　要：设计了以hSOD1、hSOD3为编码序列,以山羊β-酪蛋白/CMV杂合启动增强子构建乳腺特异性表达载体rhSOD1、rhSOD3,共转染母山羊胎儿成纤维细胞,采用PCR和扩增产物序列分析筛选获得SOD1/3克隆细胞株,应用体细胞核移植(SCNT)制备双转基因山羊。出生小羊经PCR和扩增产物序列分析验证是否成功整合外源基因,经Western blotting、ELISA及体外活性检来验证分析表达产物。结果表明:获得SOD1/3转基因山羊胎儿成纤维细胞系6株;原代双转基因体克隆山羊1只(♀);从该转基因羊乳汁中检测到rhSOD1、rhSOD3,浓度分别为:88.81±8.36 mg/L和267.82±12.67 mg/L;转基因羊乳汁中重组人SOD酶活性为1 432±157 U/mL。研究表明,以双载体和单标记基因转染山羊胎儿成纤维细胞可获得双基因整合转基因细胞系,并且以SOD1与SOD3功能基因均可在山羊乳腺中共同表达,表达产物具有较好的生物学活性。In this study,we employed human SOD1(hSOD1)and SOD3(hSOD3)as encoding sequence along with goat beta-casein/CMV chimeric promoter to construct mammary gland-specific expression vectors rhSOD1 and rhSOD3,which then co-transfected goat fetal fibroblast cells. PCR and sequence analysis of the amplified products were applied to screen and obtain the SOD1/3 cloned lines,and somatic cell nuclear transfer(SCNT)was used to prepare bitransgenic goat. Whether or not successful integration of foreign genes in newborn goats was verified by PCR and sequence analysis of amplification product,and the expressed product was analyzed by Western blotting,ELISA and in vitro activity test. The results were as such:6 SOD1/3 transgenic goat fetal fibroblast cell lines were obtained,one original bitransgenic goat(♀)was prepared,rhSOD1 and rhSOD3 were detected in the milk from the transgenic goats,and their concentrations were 88.81± 8.36 mg/L and 267.82± 12.67 mg/L,respectively. The biological activity of recombinant human SOD in the goat milk was 1 432±1 57 U/mL. This study implies that two expression vectors and single marker gene can simultaneously transfect goat fetal fibroblast cells to obtain the transgenic cell line with double gene integration,the SOD1 and SOD3 functional genes may co-express in the mammary gland,and the expressed products show fine biological activity.

关 键 词：重组人超氧化物歧化酶 乳腺特异性表达 转基因山羊 共表达 

分 类 号：S827[农业科学—畜牧学]