史氏鲟(Acipenser schrenckii)两种促性腺激素β亚基的原核表达  

Expression of Two Gonadotropin β-subunits of Acipenser schrenkii in E.coli

作　　者：胡红霞[1] 贝锦新[1] 刘晓春[1] 张勇[1] 林浩然[1] 

机构地区：[1]中山大学水生经济动物研究所和广东省水生经济动物良种繁育重点实验室

出　　处：《中山大学学报（自然科学版）》2008年第2期89-92,共4页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：国家科计部十五863现代农业技术主题基金资助项目(2001AA243061,2004AA243060);北京市科学技术委员会资助项目(954830400)

摘　　要：采用遗传工程方法,重组表达史氏鲟两种促性腺激素β亚基蛋白。选用原核表达载体pET-22b(+),分别插入史氏鲟GtHⅠ&Ⅱβ亚基成熟肽cDNA序列,构建成C端含有6个组氨酸(6-His)融合蛋白标签的表达质粒;分别转化大肠杆菌表达菌株BL21(DE3)并诱导表达2个基因。SDS-聚丙烯酰胺凝胶电泳显示重组融合蛋白相对分子质量分别为:GtHⅠβ亚基大约14 000,GtHⅡβ亚基15 000左右。分别用抗6个组氨酸融合标签的单克隆抗体及兔抗鲟鱼GTH多克隆抗体对2个表达蛋白进行Western-Blot分析,结果显示重组蛋白表达正确且有较高的免疫活性。GTHⅠβ重组蛋白在2 h就有明显的表达,6 h后随着时间增加表达量不再增加;25℃诱导重组蛋白产量比37℃诱导产量低。获得的重组蛋白质将可用于建立鲟鱼GtH的放射免疫测定方法。Using the genetic engineering method, two prssed in mur sturg plasmids gonadotropin β - subunit of Acipenser schrenkii were ex- E. coli. Two cDNA Sequenes of gonadotropin （GTH） β-subunits mature peptide cDNA sequences of Amur sturgeon were inserted into pET -22b （ ＋ ） vector, using Nde I and Xho I restriction site. The recombinant of two GTH β-subunits were constructed as C-terminal histidine-tagged fusion protein. Transformants bar- boring expression vectors were induced in E. coli strain BL21 （ DE3 ）, the recombinant GtH Ⅰ -β and GTH Ⅱ-β were about 14 000 and 15 000 respectively . The results of SDS-PAGE and western-blotting with the McAb of 6- histidine showed that the recombinant protein had correct molecular weight, just as expected. They also showed high immunological activities through western-blotting with polyclonal antibody of purified sturgeon GTH. GtH Ⅰ β recombinant protein had obvious expression in 2 hours. The quantity of induced protein had no significant increase after 6 hours. The combinated protein will contribute to develop a RIA method for detection of Acipenser.

关 键 词：史氏鲟(Acipenser schrenckii) 促性腺激素 β-亚基 蛋白质 遗传工程 

分 类 号：Q78[生物学—分子生物学] S96[农业科学—水产养殖]