文档介绍：
红笛鲷免疫球蛋白重链 M 基因的克隆与表
达分析#
张新中1,吴灶和2,3,简纪常3,鲁义善3*
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(1. 南通大学生命科学学院;
2. 仲恺农业工程学院;
3. 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:本研究应用 RACE 技术成功克隆了红笛鲷(Lutjanus sanguineus)免疫球蛋白重链 M
基因(IgM)的全长 cDNA 序列。IgM 的全长序列为 2046 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。
Real-time RT-PCR 分析表明,IgH 的表达上调开始发生在哈氏弧菌 ZJ0706 感染后的 6h
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-IgH 经
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 检测显示,所获
得的兔抗血清效价约为 1 : 40000。Western blot 检测发现,本实验制备的抗血清能特异性的
与重组蛋白发生抗原抗体反应。
关键词:红笛鲷;IgM;RACE;Real-time RT-PCR;原核表达
中图分类号:S968
Cloning and expression analysis of immunoglobulin M (IgM)
from humphead snapper Lutjanus sanguineus
ZHANG XinZhong1, WU Zaohe2,3, JIAN Jichang3, LU Yishan3
(1. School of life sciences, Nantong university;
2. Zhongkai University of Agriculture and Engineering;
3. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic
Economic Animals)
Abstract: In the present study, full-length cDNA sequences of immunoglobulin M (IgM) was
cloned by rapid amplification of cDNA ends technique (RACE) from humphead snapper
(Lutjanus sanguineus). Full-length cDNA sequence of IgM is 2045 bp, encoding 595 amino acids.
BLAST analysis revealed that the IgM amino acids sequence of humphead snapper shared high
identity (80%) with that of others. ic tree was constructed by the Neighbor-Joining
method, and the results suggested that IgM of humphead sanpper shared the closest ic
relationship with the IgM of Larimichthys crocea. The results of fluorescent real-time quantitative
RT-PCR showed that the expression of IgM mRNA could be detected in head kidney, and
increased continuously as time goes on in 48 h post infection. In addition, IgM was subcloned into
pET28a(+) to construct expression plasmids pET28a-IgM. SDS-PAGE and western blot analysis
indicated that the bin