鸡MHCⅡβ链基因的克隆和表达及其抗体制备  

Cloning and expression of the gene fragment encoding chicken MHCⅡβ chain and its antibody preparation

作　　者：徐志本[1] 余为一[1] 仲大莲[2] 李槿年[1] 周杰[1] 

机构地区：[1]安徽农业大学动物科技学院,合肥230036 [2]中国科技大学生命科学学院,合肥230026

出　　处：《安徽农业大学学报》2006年第1期51-55,共5页Journal of Anhui Agricultural University

基　　金：国家自然科学基金(30270974)资助

摘　　要：用自行设计的1对引物,通过RT-PCR从鸡脾细胞中分别克隆了鸡MHCⅡβ链基因片段,大小为798bp。核苷酸测序结果表明,与已登录的基因序列同源性为95%。将该基因片段插入含谷胱甘肽(GST)基因的质粒pGEX-4T-1,构建了pGEX-4T-1-chMHCⅡβ。经诱导表达,获得分子大小约为53 000的融合蛋白,其中chMHCⅡβ链大小约27 000。经亲和层析,获得纯化GST-β融合蛋白,进一步用此蛋白免疫小鼠,制备了鼠源抗鸡MHCⅡβ链抗体。经过琼扩和ELISA检测,表明该融合蛋白具有良好的抗原性。The gene fragment of chicken MHC class Ⅱ beta chain from spleen lymphocytes was cloned by RTPCR using a pair of primers. It was 798 bp in length and showed 95% similarity to the previously identified chicken MHC Ⅱ beta at nucleotide level. The full length chicken MHC Ⅱ beta chain cDNA was cloned into pGEX-4T-1. The recombinant plasmid, namely PGEX-4T-chMHCⅡβ, was transformed into Escherichia coli BL21 and GST-β fusion protein was induced to express. A MW of the tusion protein was about 53 000 as analyzed by SDS-PAGE. The purified fusion protein was used to immunized mice and the specific antibody, was observed, which was identified further in immunodiffusion and ELISA. The results indicated that this expressed fusion protein had favorable antigenieity.

关 键 词：MHCⅡ β链基因 克隆 原核表达 

分 类 号：S831.2[农业科学—畜牧学] S852.4[农业科学—畜牧兽医]