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为研究苏云金芽胞杆菌(Bacillus thuringiensis)杀虫晶体蛋白与其受体钙粘蛋白的相互作用,本研究利用苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus,Ac MNPV)囊膜蛋白GP64细胞膜锚定的特点,将172 bp的N端信号肽(GP64 signal peptide,gp64sp)和135 bp的C端膜锚定区域(GP64 C-terminal transmembrane domain,gp64ctd)连接,并在中间添加了一段含有6个酶切位点的序列,再连接到表达载体p IZ/V5-His,成功构建了一个使外源基因在细胞膜上表达的重组载体p IZ/V5-gp64。将扩增的3 882 bp的棉铃虫钙粘蛋白(Helicoverpa armigera cadherin,HaCAD)基因重复区和近膜区片段与重组载体p IZ/V5-gp64进行酶切和连接,构建重组载体p IZ/V5-gp64-HaCAD,将其转染棉铃虫细胞系Ha-E-1,通过筛选、细胞克隆和PCR验证,获得了重组钙粘蛋白基因的转基因细胞系Ha-T-CAD。免疫荧光法检测证实,钙粘蛋白在转基因细胞系Ha-T-CAD中成功表达。Western blot检测发现,在Ha-T-CAD细胞的膜蛋白中有140 k D大小的钙粘蛋白。转基因细胞系Ha-T-CAD的构建将为Bt杀虫晶体蛋白作用机理以及昆虫对Bt毒素的抗性机制研究提供基础资料。
In order to study the interaction between the Bacillus thuringiensis insecticidal crystal protein and its receptor cadherin, we used the membrane protein of Ac MNPV (AcMNPV) The 172 bp N-terminal signal peptide (gp64sp) and the 135 bp C-terminal transmembrane domain (gp64ctd) were ligated with a linker containing 6 A restriction enzyme site, and then ligated into the expression vector pIZ / V5-His. A recombinant vector pIZ / V5-gp64 was successfully constructed to express the foreign gene on the cell membrane. The amplified 3 882 bp repeat region and proximal membrane region of Helicoverpa armigera cadherin (HaCAD) gene were digested and ligated with the recombinant vector pIZ / V5-gp64 to construct the recombinant vector pIZ / V5 -Gp64-HaCAD, which was transfected into the Helicoverpa armigera cell line Ha-E-1. The recombinant cell line Ha-T-CAD of cadherin gene was obtained by screening, cell cloning and PCR. Immunofluorescence assay confirmed that cadherin was successfully expressed in the transgenic cell line Ha-T-CAD. Western blot showed 140 kD-sized cadherin in the membrane proteins of Ha-T-CAD cells. The construction of transgenic cell line Ha-T-CAD will provide the basic data for the mechanism of Bt insecticidal crystal protein and the mechanism of insect resistance to Bt toxin.