家蚕化蛹初期血液中卵黄原蛋白BmVg的纯化和鉴定  

Purification and Identification of Vitellogenin Protein in Bombyx mori Blood at Early Pupation Stage

作　　者：罗娟[1] 彭芷昕[1] 陈恩祥[1] 刘红玲[1] 杨从文[1] 沈关望[1] 张海燕[1] 侯勇[1] 林英[1] 夏庆友[1] 

机构地区：[1]家蚕基因组生物学国家重点实验室,农业部蚕桑学重点实验室,西南大学,重庆400716

出　　处：《蚕业科学》2014年第2期230-234,共5页ACTA SERICOLOGICA SINICA

基　　金：国家高技术研究发展计划"863"项目(No.2011AA-100306);国家自然科学基金青年基金项目(No.31101768);国家自然科学基金项目(No.31201853)

摘　　要：卵黄原蛋白(vitellogenin,Vg)是昆虫血液中发现最早的蛋白质,对于昆虫的繁殖至关重要。以家蚕Vg基因(BmVg)的部分序列构建原核表达载体,转化E.coil BL21(DE3)菌株,在20℃条件下用0.6 mmol/L IPTG进行诱导表达,表达产物经镍柱亲和层析纯化得到家蚕卵黄原蛋白BmVg的肽蛋白。以BmVg肽蛋白为抗原制备的兔源多克隆抗体效价>1∶512 000。采用硫酸铵分级沉淀和阴离子交换层析的纯化方法,从家蚕化蛹初期的血液中纯化获得天然的BmVg蛋白,并用制备的多克隆抗体进行Western blotting检测,鉴定此纯化的天然蛋白质为BmVg蛋白。建立的BmVg分离纯化技术将有助于进一步研究BmVg的合成、转运及利用机制。Vitellogenin is the first protein discovered in insect hemolymph and is critical for reproduction of insects. We cloned the partial sequence of Bombyx mori vitellogenin gene （ BmVg）, constructed a prokaryotic expression vector, and transformed it into E. coil BL21 （DE3）. The recombinant BmVg peptide was expressed by induction of 0.6 mmol/L IPTG at 20 ℃ and purified by Ni-NTA affinity chromatography. The purified BmVg peptide was used to prepare anti-BmVg rabbit polyclonal antibody. The obtained antibody had a titer higher than 1：512 000. Moreover, the natural BmVg protein was purified from silkworm （ Bombyx mori） hemolymph at early pupation stage by ammonium sulfate precipitation and an- ion-exchange chromatography. Western blotting analysis using the above prepared polyclonal antibody verified that the purified natural protein was BmVg protein. This established protocol for extraction and purification of BmVg will facilitate further studies on mechanisms of vitellogenin synthesis, transportation and utilization.

关 键 词：家蚕 卵黄原蛋白 原核表达 分离纯化 免疫印迹法 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]