为比较禽类恒定链的结构和功能,应用RACE(Rapid Amplification of cDNA Ends)技术首次克隆并鉴定了鸽恒定链基因。首先用一对含高度保守的DNA片段的简并引物,从鸽脾细胞RNA扩增部分恒定链片段,接着测序并设计新引物分别从5′和3′RACE扩增延长该片段。最后根据全基因的序列设计上、下游引物,获得大小为1050bp的全长cDNA。比较核苷酸序列,鸽与鸡的Ii链同源性达到82.8%,而与人等其它动物的同源性则在52.0%以上;其中633bp的开放阅读框编码211个氨基酸残基的前体蛋白。推导和分析氨基酸序列表明,分子结构与鸡恒定链相似,其中有些氨基酸残基表现出较高的保守性。
[Objective] To prepare the monoclonal antibody against chicken major histocompatibility complex class II molecules(MHC II).[Method] The prokaryotic expression of the gene fragments of exons 2-6 encoding alpha chain of MHC II and exons 3-6 encoding beta chain of MHC II were performed based on its protein sequences.After BALB/c mice were immunized with the purified fusion proteins,the mouse spleen cells were fused with mouse myeloma cells SP2/0.Then the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay(ELISA).[Result] One hybridoma cell strain secreting monoclonal antibody against alpha chain and two strains secreting monoclonal antibody against beta chain were obtained.These three hybridoma cell strains were named as MHC II alpha-4,MHC II beta6-2 and MCH II beta6-31,respectively.Their titers of ascites in indirect ELISA were 1:256 000,1:256 000 and 1:1 280 000,respectively.These antibodies could specifically recognize MHC II alpha chain or beta chain in western blotting.[Conclusion] Three obtained hybridoma stains can stably produce the monoclonal antibody against chicken MHC class II molecules.
