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红笛鲷免疫球蛋白重链M(IgM)基因的克隆与表达分析

发表时间:2014-11-15  浏览量:1881  下载量:520
全部作者: 张新中,吴灶和
作者单位: 南通大学生命科学学院;广东海洋大学广东省水产经济动物病原生物学及流行病学重点实验室
摘 要: 研究应用cDNA末端快速克隆技术(rapid-amplification of cDNA ends,RACE)成功克隆了红笛鲷(Lutjanus sanguineus)免疫球蛋白(immunoglobulin, Ig)重链M基因(IgM)的全长cDNA序列。IgM的全长序列为2 046 bp,编码595个氨基酸残基。BLAST分析显示红笛鲷IgM与其他已知物种IgM基因的最高同源性为80%. 构建的系统进化树显示,红笛鲷IgM与大黄鱼(Larimichthys crocea)的等IgM亲缘关系较近。实时定量PCR (real-time PCR,RT-PCR)分析表明,IgM的表达上调开始发生在哈氏弧菌ZJ0706感染后的6 h内,且随着感染时间的延长而呈现持续上调的趋势。构建的重组表达质粒pET28a-IgM经异丙基硫代半乳糖苷(IPTG)诱导后在大肠杆菌(Escherichia coli)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了多克隆抗体。ELISA检测结果显示,所获得的兔抗血清效价约为1∶40 000. Western blotting检测发现,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。
关 键 词: 水产生物学;红笛鲷;IgM;实时定量PCR;原核表达
Title: Cloning and expression analysis of immunoglobulin M (IgM) gene from humphead snapper Lutjanus sanguineus
Author: ZHANG Xinzhong, WU Zaohe
Organization: School of Life Sciences, Nantong University; Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Guangdong Ocean University
Abstract: In the present study, full-length cDNA sequences of immunoglobulin (Ig) M (IgM) gene was cloned by rapid-amplification of cDNA ends (RACE) technique from humphead snapper (Lutjanus sanguineus). Full-length cDNA sequence of IgM is 2046 bp, encoding 595 amino acids. BLAST analysis revealed that the IgM amino acid sequences of humphead snapper shared high identity (80%) with that of others. Phylogenetic tree was constructed by the neighbor-joining method, and the results suggested that IgM of humphead sanpper shared the closest genetic relationship with the IgM of Larimichthys crocea. The results of fluorescent real-time quantitative PCR (RT-PCR) showed that the expression of IgM can be detected in head kidney, and increased continuously as time goes on in 6 h post ZJ0706 infection. In addition, IgM is subcloned into pET28a(+) with ZPTG induction to construct expression plasmids pET28a-IgM. Western blotting 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 immuning 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 blotting revealed that specific antigen-antibody reaction was occurred between the antiserum and the recombinant proteins.
Key words: aquatic biology; Lutjanus sanguineus; IgM; real-time PCR; prokaryotic expression
发表期数: 2014年11月第21期
引用格式: 张新中,吴灶和. 红笛鲷免疫球蛋白重链M(IgM)基因的克隆与表达分析[J]. 中国科技论文在线精品论文,2014,7(21):2195-2205.
 
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