中国农业科学Issue(19)：3922-3928,7.DOI:10.3864/j.issn.0578-1752.2014.19.022
家蚕卵黄原蛋白受体BmVgR的体外表达
Expression of Silkworm Vitellogenin Receptor in Vitro
摘要
Abstract
The objective of this study is to clone the silkworm BmVgR, to analyze its expression characteristics in insect cells and to investigate the expression patterns of the normal and mutational VgRs, thus providing a basis for its function identification. [Method] Specific primer sequences were designed according to BmVgR coding sequence, the amplified fragment was connected to the pET28a, and then transformed into E. coil BL21 (DE3), the fusion protein was obtained by IPTG induction. Ni-NTA affinity chromatography was used to purify the BmVgR protein peptide. Then the anti-BmVgR rabbit polyclonal antibody was obtained by BmVgR protein peptide as an antigen. The LBD1+EGF1 (C11D and C11V) domain of the normal and mutational VgRs was connected to the cell expression vector, the target protein was expressed with cell transfection. Immunohistochemistry was used to analyze the expression characteristics of the normal and mutational VgRs in insect cells. Western blot was used to detect the expression levels of the normal and mutational VgRs in the cell culture medium.[Result]Prokaryotic expression gained a size of about 22 kD fusion protein, which was expressed as inclusion bodies. BmVgR protein peptide was expressed and purified, the anti-BmVgR rabbit polyclonal antibody was obtained, its titer was higher than 1﹕512 000. Purity was more than 95%. The endogenous genes of BmVgR and BmVg did not express in Sf9 and Spli221. The normal and mutational BmVgR-SP+LBD1+EGF1 was successfully expressed in the Sf9 cell (the BmVgR of the vit mutant, which was mutated in the 3rd Class B region of the EGF1 domain, coding 50 amino acid residuces). Immunohistochemistry showed that vit mutant and normal BmVgR could express normally in Sf9 cytoplasm. BmVgR rabbit polyclonal antibody and Myc-tagged antibody simultaneously detected the target protein, which indicated that the VgR antibody was better. As the SP + LBD1 + EGF1 peptide existed signal peptide, the expressed protein was secreted into the cell culture medium. Western blot indicated that the presence of amino acid deletion of the mutant vit did not affect the expression of the normal protein peptide, and the expression levels of the normal and mutational VgRs were not significantly different.[Conclusion]The deletion of EGF1 domain didn’t affect the normal expression of SP+LBD1+EGF1 peptide protein, which may lead to the phenotype of the vit.关键词
家蚕/卵黄原蛋白受体/原核表达/细胞表达Key words
Bombyx mori/vitellogenin receptor/prokaryotic expression/cell expression引用本文复制引用
罗娟,夏庆友,陈恩祥,刘红玲,彭芷昕,杨从文,沈关望,张海燕,邢润苗,林英..家蚕卵黄原蛋白受体BmVgR的体外表达[J].中国农业科学,2014,(19):3922-3928,7.基金项目
国家高技术研究发展计划（“863”计划）（2011AA-100306）、国家青年基金（31101768）、国家自然科学基金 （）
