摘要
采用RT-PCR扩增文昌鸡热休克蛋白70(Heat shock protein 70,HSP70)c DNA编码蛋白区,将扩增产物与p ET-28a(+)载体连接,重组质粒经PCR、酶切鉴定后测序并进行生物信息学分析;构建p ET-28a-c HSP70表达载体,经IPTG诱导表达后,进行SDS-PAGE和Western blot分析。结果显示,文昌鸡HSP70 c DNA编码蛋白区全长为1 905 bp,编码634个氨基酸。经IPTG诱导表达后,得到一个带组氨酸标签约80 ku的融合蛋白,采用抗His单克隆抗体进行Western blot得到一条约80 ku特异性抗体结合带,表明文昌鸡HSP70原核表达载体成功构建并表达。
Heat shock protein 70 （HSP70） cDNA of Wenchang chickens was amplified by RT-PCR and cloned into pET-28a（ ＋ ）vector. The recombinant plasmid was identified by endonuclease digestion and sequencing after PCR and analyzed by bioinformatics. The recombinant plasmid was transformed into E. coli host cells. Protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blot. The results showed that the HSP70 cDNA was 1 905 bp in length and encoded 634 amino acids. A 80 ku fusion protein with a His-tag was induced by IPTG and identified by Western blot using anti-his-tag monoclonal antibody,getting a special antibody binding band, which indicated that prokaryotic expression vector of HSP70 from Wenchang chickens was constructed and expressed successfully.
出处
《中国家禽》
北大核心
2015年第3期5-8,共4页
China Poultry
关键词
文昌鸡
HSP70
克隆
表达
Wenchang chickens
HSPT0
cloning
expression
