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

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。

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

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(BL21) 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.After 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.