稳定表达鸡恒定链基因的P815细胞株的建立与鉴定

目的]获得稳定表达鸡恒定链基因（Ii）的P815细胞株。方法]利用PCR方法获取鸡Ii链基因,利用脂质体转染P815细胞,加入G418筛选阳性细胞克隆,最后利用RT-PCR进行鉴定。结果]通过PCR获得鸡Ii基因,大小为672 bp,进一步定向插入真核表达载体,构建重组质粒pEGFP-C1-Ii,经双酶切鉴定后转染P815细胞,通过多次克隆获得含鸡Ii链基因并能稳定表达的阳性细胞株,经RT-PCR再次鉴定,并命名为P815-Ii。结论]获得了稳定表达鸡Ii的P815细胞株,为进一步研究Ii的结构与功能奠定了基础。

Establishment and Identification of P815 cells of Stable Expression Chicken Stably Line Gene

Objective] The aim was to achieve the P815 cell stably expressing chicken invariant chain Ii gene.Method] PCR was used to amplification of Ii,the Ii segment was cloned into pEGFP-C1.Then the recombinant plasmids were tranfected into P815 cells and the cells were screened by G418.Finally the positive cells were identified by RT-PCR.Result] The Ii segment was achieved by 672 bp PCR;then the segment was cloned into plasmids and pEGFP-C1-Ii was reconstructed.The recombinant plasmids were identified by double digestion and then transected into P815 with liposome.After a screen with G418 and more times sub-cloning the cell strain of stable expression chicken Ii was achieved.Finally the strain was confirmed by RT-PCR and named as P815-Ii.Conclusion] The cell strain with stable expression of chicken Ii gene was established which provided basis for the research of Ii structure and function.