鲮生长激素的重组表达及其多克隆抗体的制备

IPTG诱导含有鲮（Cirrhinus molitorella）生长激素基因（growth hormone,GH）的工程菌M15（pQE30-GH）重组表达鲮生长激素蛋白。SDS-PAGE凝胶电泳结果表明,在24.5kDa处有1条重组表达鲮生长激素条带,经分离纯化、复性和浓缩后得到有活性的重组鲮生长激素蛋白。以复性浓缩后的重组鲮生长激素蛋白为抗原,采用改进的方法对新西兰大白兔进行皮下多点免疫注射,获得兔抗鲮生长激素多克隆抗体。经ELISA检测该免疫血清的滴度为12800。以该多抗为一抗,Western blotting可以检测到10ng的抗原量;并且在鲮垂体组织提取液中和血清中检测到一种能与该血清作用的大小约为21.5kDa的蛋白条带。说明该试验所制备的兔抗鲮生长激素多克隆抗体具有较好的免疫活性。

Recombinant expression and antibody preparation of mud carp growth hormone

After inducement of IPTG,the recombinant protein of mud carp growth hormone（mcGH） was expressed in Escherichia coli.SDS-PAGE electrophoresis showed that there was a band of about 24.5 kDa,which size was similar to the predicted mcGH.After purification and renaturation,the recombiant mcGH was used as an antigen to inoculate the rabbit.The titer of the obtained antiserum was determined to be 1：12 800 by indirect enzyme-linked immunosorbent assay（ELISA）.Immunochemistry analysis showed that the antiserum could detect the antigen as low as 10 ng.Furthermore,western blotting analysis showed that 21.5 kDa protein in mud carp pituitary and serum could be detected using the primary antibody.These results showed that the polyclonal antiserum of rabbit anti-mcGH was effective and highly specific.