利用Bac-to-Bac杆状病毒表达系统在家蚕中表达人生长素  

Expression of Human Growth Hormone in Bombyx mori Using Bac-to-Bac Baculovirus Expression System

作　　者：付凡[1] 陈蔚[1] 唐顺明[1,2] 汪生鹏[1,2] 黄金山[1,2] 赵巧玲[1,2] 沈兴家[1,2] 

机构地区：[1]江苏科技大学,江苏镇江212003 [2]中国农业科学院蚕业研究所,农业部蚕桑遗传改良重点开放实验室,江苏镇江212018

出　　处：《蚕业科学》2011年第2期320-324,共5页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.31072083);国家高技术研究发展计划“863”项目(No.2007AA100504)

摘　　要：人生长素是由腺垂体分泌的一种在人体生长、发育及代谢中发挥重要作用的激素,具有广泛的生理作用。利用家蚕核型多角体病毒(BmNPV)Bac-to-Bac表达系统,将人生长素基因(hgh)克隆到杆状病毒转移载体pFastBacHTb,通过细菌转座子原理,转化BmDH10Bac细胞,获得重组穿梭载体质粒Bacmid-hgh,并将其转染家蚕BmN细胞,获得含有hgh的重组杆状病毒。将此重组病毒感染家蚕BmN细胞,收集重组病毒液并穿刺接种家蚕5龄起蚕,3 d后观察到家蚕感染了杆状病毒的症状,并通过SDS-PAGE和Western blotting方法在家蚕血液中检测到分子质量约20 kD的目的蛋白,表明人生长素通过Bac-to-Bac系统在家蚕体内获得了表达。Human growth hormone（hGH） secreted by adenohypophysis plays an important role in human growth,development and metabolism,showing extensive physiological functions.Using Bac-to-Bac expression system of Bombyx mori nuclepolyhedrovirus（BmNPV）,a hgh gene was cloned into a baculovirus transfer vector pFastBacHTb which was used to transform BmDH10Bac to prepare recombinant shuttle vector Bacmid-hgh through bacterial transposition.The recombinant plasmid Bacmid-hgh was used to transfect BmN cells to obtain the recombinant baculovirus containing human growth gene hgh.The recombinant baculovirus was used to infect BmN cells and the recombinant virus was collected and used to inoculate newly exuviated Bombyx mori larvae of the 5th instar.After 3 d,the larvae showed symptom of infection by baculovirus.SDS-PAGE and Western blotting detected target protein with molecular weight of approximately 20 kD in hemolymph of the larvae,indicating that human growth hormone had been successfully expressed in Bombyx mori larvae through Bac-to-Bac system.

关 键 词：Bac-to-Bac杆状病毒表达系统 家蚕核型多角体病毒 家蚕 人生长素基因 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]