红笛鲷主要组织相容性复合物Ⅰα抗原基因的克隆与表达分析

摘要

In the present study, full-length cDNA sequences of histocompatibility complex Ⅰ a antigen (MHC Ⅰ α was cloned by rapid amplification of cDNA ends technique(RACE)from humphead snapper( Lutjanus sanguineus). Full-length cDNA sequence of MHC Ⅰ α is 1 369 bp, encoding 354 amino acids. BLAST analysis revealed that the amino acids sequence of MHC Ⅰ a shared high identity (84% ) with other MHC I . Phylogenetic tree was constructed by the Neighbor-Joining method, and the results suggested that MHC I a of humphead snapper shared the closest genetic relationship with the MHC Ⅰ of Epinephelus coioides. The results of fluorescent real-time quantitative RT-PCR showed that the expression of MHC Ⅰ a mRNA could be detected in head kidney, and the maximum expression appeared in 6 - 15 h post infection. MHC I a was subcloned into pET32a( + )to construct expression plasmids pET32-MHC Ⅰ α. SDS-PAGE and Western blot analysis indicated that the recombinant proteins were successfully expressed in Escherichia coli BL21 ( DE3). Then the recombinant proteins were purified and the antiserum was obtained by immunizing rabbits with the purified recombinant proteins emulsified with adjuvant. ELISA analysis showed that the titer of the antiserum prepared in this study was 1:40 000. The results of the Western blot revealed that specific antigen-antibody reaction occurred between the antiserum and the recombinant proteins.%为研究红笛鲷免疫防御相关基因的作用机理和调控机制,实验应用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)成功克隆了红笛鲷组织相容性复合物Ⅰ α(MHC Ⅰ α)抗原基因的全长cDNA序列,MHC Ⅰα的全长序列为1 369 bp,编码354个氨基酸残基.BLAST分析显示,红笛鲷MHC Ⅰ α与其他已知物种MHC Ⅰ α基因的最高同源性为84％.构建的系统进化树显示,红笛鲷MHC Ⅰα与石斑鱼等MHC Ⅰ α亲缘关系较近.Real-time PCR分析表明,MHC Ⅰ α在头肾中的最大表达量出现在哈氏弧菌ZJ0706诱导后6～15 h内；构建的重组表达质粒pET32a-MHC Ⅰα经IPTG诱导后在大肠杆菌BL21( DE3)中获得了正确表达.将纯化后的重组蛋白与佐剂混合后免疫新西兰纯种大白兔制备多克隆抗体.ELISA检测显示,所获得的兔抗血清效价约为1:40 000.Western blot检测发现,本实验制备的抗血清能特异性地与重组蛋白发生抗原抗体反应.