DT40细胞sIgMλ轻链基因的克隆鉴定及原核表达

Prokaryotic expression of the λ light chain of sIgM cloned from DT40 cells

本研究利用RT-PCR技术,从鸡法氏囊淋巴瘤细胞系DT40中扩增sIgMλ轻链基因并在E.coli中进行了表达。序列分析结果表明,sIgMλ轻链基因全长852nt,含有一个长度为657nt的ORF及长度为195nt的3'-UTR,该基因与鸡Igλ基因的序列同源性为95%,差异位点主要存在于λ轻链的可变区。氨基酸序列预测结果表明,DT40sIgMλ蛋白与鸡Igλ蛋白的序列同源性为92.6%。构建的原核表达载体pET-λ在E.coliBL21(DE3)中成功的获得大量表达。本实验为进一步研究sIgM在IBDV感染法氏囊B淋巴细胞中的作用奠定了基础。

The λ light chain of slgM was cloned by RT-PCR from the total cellular RNAs of DT40 cells. DNA sequencing showed that the λ light chain of slgM was 852 nt long including an 657 nt open reading frame （ORF） and 195 nt 3＇-UTR. The sequence homology between DT40 sIgM λ light chain and chicken Ig λ light chain was 95 % at nucleotide level and 92.6 % in deduced amino acids. The PCR product was cloned into prokaryotic expression vector pET-28a for expression. SDS-PAGE analysis showed that the λ light chain of slgM was abundantly expressed in E.coli BL21 （DE3）. The present study provided foundation for further investigation on the role of sIgM in the infection process of IBDV to B-lymphocytes.