欧洲鳗鲡免疫球蛋白M重链基因的原核表达与不同组织中表达变化的定量分析

The Prokaryotic Expression and Quantitative Analysis of Immunoglobulin Heavy Chain Gene Expression Change in Different Tissues of the European Eel(Anguilla anguilla)

通过RT-PCR技术从欧鳗(Anguilla anguilla)脾脏总RNA中获得了编码成熟免疫球蛋白(Ig)重链的基因序列(GenBank accession No.GQ249838),该序列长1 686 bp,编码562个氨基酸残基,分子质量为62.2 ku。将该基因片段与pET-his载体连接构建原核表达载体,在大肠杆菌BL21中诱导表达,其表达产物经SDS-PAGE和Western-blotting分析表明新增的66 ku蛋白条带与预期值相符,且能够与兔抗欧鳗IgM血清发生强烈的特异性反应,证实了欧鳗免疫球蛋白重链(IgH)基因能够在大肠杆菌中以包涵体形式高效表达;利用荧光定量PCR检测了Ig基因在欧鳗背肌注射山羊IgG前后不同组织的表达水平,结果显示欧鳗在免疫前后其肾脏与脾脏Ig基因的表达量均明显高于其它组织,在鳃、肌肉中的表达量也较高,而在肝脏、心脏中的表达量较低。此外,对同一组织免疫前后的Ig基因表达量比较发现,除肝脏外,其它组织Ig基因表达水平在免疫后均上升,在鳃和肌肉中达到显著水平,而在脾脏中达到极显著水平。

The cDNA sequence of the European eel（Anguilla anguilla） immunoglobulin M heavy chain mature peptide was cloned using RT-PCR. The cDNA sequence （GenBank accession No. GQ249838） had 1 686 nucleotides encoding a putative protein of 562 amino acids with the calculated relative molecular mass of 62. 2 ku. The amplified DNA fragments were ligated into pET-his vector and transformed into E. coli BL21 which was then induced by IPTG. The size of the expressed protein was 66 ku estimated by SDS-PAGE electrophoresis. The recombinant proteins were strongly recognized by the serum anti-IgM of European eel detected by Western blotting. The result of SDS-PAGE and western-blot demonstrated that the fusion protein was expressed stably in E. coli BL21 with the form of inclusion bodies. The abundance of IgM transcripts in the kidney, spleen, gills, liver,muscle and heart of the European eels immuned by goat IgG and the unimmuned ones were quantified by Real-time PCR. The results showed that the levels of IgM gene expression in the kidney and spleen were constantly higher than that in other tissues. Relatively high levels of IgM gene expression were also found in the gills and the muscle whereas relatively lower abundance of IgM transcripts could be detected in the liver and the heart. Compared with the unimmuned eels, the abundance of IgM transcripts in the immuned eels increased obviously in the kidney and the heart, significantly in the gills and the muscle （P〈0.05）, extremely significantly in the spleen（P〈0.01） whereas decrease was found in the liver.