表面展示棉铃虫钙粘蛋白基因细胞系的建立  

Establishment of a Novel Transgenic Insect Cell Line Displaying Cotton Bollworm(Helicoverpa armigera) Cadherin Protein on Membrane Surface

作　　者：颜晓[1] 苏蕊[1] 郑桂玲[1] 梁革梅[2] 张杰[2] 万方浩[1,2] 李长友[1] 

机构地区：[1]青岛农业大学农学与植物保护学院/山东省植物病虫害综合防控重点实验室,青岛266109 [2]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室,北京100094

出　　处：《农业生物技术学报》2016年第5期755-763,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31272376);植物病虫害生物学国家重点实验室开放基金(No.SKLOF201315);山东省"泰山学者"建设工程专项

摘　　要：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。The purpose of this study was to establish a transgenic insect cell line containing Helicoverpa armigera cadherin （HaCAD） for subsequent study on the interactions between Bacillus thuringiensis （Bt） insecticidal crystal protein and its receptor cadherin. Based on the specific membrane-adhering characteristics ofAutographa californica multiple nucleopolyhedrovirus （AcMNPV） envelope protein GP64, PCR primers were designed based on AcMNPV gp64 gene sequence. A 172 bp DNA fragment of gp64 signal peptide （gp64sp） and a 135 bp fragment of the gp64 C-terminal transmembrane domain （gp64ctd） were amplified by PCR, respectively. Both gp64sp and gp64ctd fragments were ligated and an additional DNA sequence containing 6 restriction digestion sites was inserted. This 301 bp fragment was replaced into the multiple cloning sites of expression vector pIZ/VS-His. A surface display vector named pIZ/V5-gp64 that allowed the exogenous gene to be expressed on the cell membrane was successfully constructed. The amplified 3 882 bp of the cadherin repeat （CR） and the membrane proximal region （MPR） of HaCAD and recombinant plasmid pIZ/VS-gp64 were digested with restriction enzymes, respectively, and then ligated. A recombinant vector named pIZ/VS- gp64-HaCAD was constructed. This vector was transfected into cotton bollworm cell line Ha-E-1 via liposome- mediated transfection. The transfected cells were screened on the selective culture medium containing zeocin and the selected cells were further cloned. A transgenic cell line Ha-T-CAD containing the recombinant HaCAD gene was obtained. A majority of transgenic cells were circular in shape while a few cells exhibited short spindle shape. Compared with the original parent Ha-E-1 cells, no obvious changes in the both morphology and size of the transgenic cells Ha-T-CAD were seen. PCR verification results showed that 3 882 bp fragment of HaCAD gene was successfully integrated into these cells. The results of immunofluorescent visualization revealed that in the tra

关 键 词：钙粘蛋白 棉铃虫 转基因细胞系 GP64蛋白 蛋白表达 

分 类 号：Q962[生物学—昆虫学]