鸡新城疫病毒F基因片段的原核表达及其抗F蛋白单克隆抗体的制备  

Prokaryotic Expression of Fragment of Chicken NDV-F Gene and Preparation of Its Monoclonal Antibody

作　　者：孟凡涛[1] 陈芳芳[1] 余为一[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《安徽农业科学》2012年第26期12915-12916,共2页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金项目(30671537)

摘　　要：[目的]制备鸡新城疫病毒(NDV)融合蛋白(F)抗原的单克隆抗体。[方法]克隆和表达了NDV-F蛋白基因的部分片段F306,经杂交瘤技术制备了抗F蛋白的单克隆抗体。[结果]首先根据F基因和载体pGEX-4T-1的多克隆位点自行设计了1对引物,以保存的重组质粒为模板,通过PCR扩增获得长度为306 bp的片段,再将其定向插入pGEX-4T-1,构建重组质粒pGEX-4T-1-F306。其次经原核表达获得大小为35.8 kD的融合蛋白,提纯后免疫Balb/c小鼠。最后,用免疫鼠脾细胞与骨髓瘤细胞融合,经ELISA筛选和3次亚克隆获得2株稳定传代和分泌抗NDV-F的抗体的杂交瘤细胞株。连续传代10次后,细胞上清和腹水的抗体效价仍维持在1∶5 124和1∶64 000以上。[结论]获得了2株稳定分泌抗鸡NDV-F抗体的杂交瘤细胞。[ Objective ] To produce monoclonal antibody to newcastle disease virus （NDV） fusion protein F. [ Method ] The recombined fragment F306 containing NDV-F gene was cloned and expressed and its monoclonal antibody was produced with hybridoma technique. [ Result ] Firstly, a pair of specific primers were designed according to the multi-clones position of the F gene and carrier PGEX-4T-1 and a DNA fragment of 306 bp in length was amplified from a recombinant plasmid kept in our laboratory. Then this fragment was inserted into pGEX-4T-1 to restructure the plasmid pGEX-4T-1-F306. Secondly it was transformed into a strain of E. coli（ BI21 ） and expressed in prokaryotic cells. Then the purified fusion protein with 35.8kDa as antigen immunized Balb/c mice. Finally the mouse spleen cells were fused with SP2/0 cells. With ELISA and by subcloning for three times, we obtained two strains cells producing antibody to F protein. Mter cell passage for 10 times, the antibody titers kept above 1：5 124 h in cell supernatant and 1：64 000 in the ascites. [ Conclusion ] Two strains of hybridoma cells were obtained, which could stably produce the monoclonal antibody against NDV-F protein.

关 键 词：新城疫病毒 F蛋白 原核表达 单克隆抗体 

分 类 号：S858.31[农业科学—临床兽医学]