鸡pcdh 8基因部分片段的克隆、表达及多克隆抗体的制备  

Cloning and expression of the partial gene fragments and preparation of the polyclonal antibody in chicken protocadherin 8

作　　者：李永海[1] 李萌[1] 赵兴华[1] 林俊堂[1] 

机构地区：[1]新乡医学院生命科学技术学院河南省高校遗传性疾病与分子靶向药物重点实验室培育基地,河南新乡453003

出　　处：《黑龙江畜牧兽医》2013年第8期20-23,192,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31000475);新乡医学院2011重点领域招标课题项目(ZD2011-24)

摘　　要：为了制作鸡原钙黏蛋白8(protocadherin 8,pcdh 8)的多克隆抗体,试验设计了针对pcdh 8膜外肽段相应基因的1对引物,从鸡胚的脑组织中提取总RNA,通过RT-PCR合成cDNA;然后以cD-NA为模板扩增膜内肽段对应的基因片段,测序正确后将其克隆至原核表达载体pGEX-2TK中;在大肠杆菌BL21中,以IPTG诱导表达GST-pcdh 802融合蛋白;利用Glutathione-sepharose 4B柱亲和层析纯化GST-pcdh 802融合蛋白,并采用足垫免疫的方法免疫大鼠制作多克隆抗体。结果表明:成功纯化了pcdh 8的融合蛋白,并制备出多克隆抗体,经Western-blot检测该抗体可用于鸡胚脑组织pcdh 8蛋白的表达检测。To prepare the polyclonal antibody against chicken protocadherin 8 （ pcdh 8）, a pair of primers was designed from the corresponding gene in a peptide fragment of the outer membrane. The total RNA was extracted from the brain tissues of the chicken embryo, and then was used to synthesize cDNA using RT- PCR; the corresponding gent fragments were amplified taking the cDNA as a template, and then cloned in the prokaryotic expression vector pGEX -2TK after sequencing; the GST -pcdh8 fusion protein was expressed in E. coli BI21 by IPTG induction; GST - pcdh8 fusion protein was purified using glutathione - sepharose 4B affinity column chromatography, and then the rats were immunized u- sing footpad immunization for preparation of polyclonal antibody. The results showed that the podh8 fusion protein was successfully purified, and the poly antibody was also successfully prepared. The results indicate that the antibody can he used for the detection of pcdh8 protein expression in brain tissues of the chicken embryo through Western - blot detection.

关 键 词：原钙黏蛋白 异源表达 鸡胚 

分 类 号：S831[农业科学—畜牧学]