Position: Home > Articles > Prokaryotic Expression and Purification of Canine β-defensin 103
Progress in Veterinary Medicine
2017
(5)
26-29
犬β-防御素cBD103的原核表达和纯化
作 者：
朱骞;包银莉;秦海斌;宋珍华;贺星亮;温海;高一龙
单 位：
公安部南京警犬研究所警犬技术公安部重点实验室;南京农业大学动物医学院
关键词：
犬β-防御素103;原核表达;纯化
摘 要：
将犬β-防御素103(canineβ-defensin 103,cBD103)基因亚克隆至原核表达载体pET-32a(+),构建重组表达质粒pET-cBD103,转化大肠埃希菌BL21感受态细胞,IPTG诱导表达后,利用Ni-NTA亲和层析纯化该蛋白,并用肠激酶酶切融合蛋白,SDS-PAGE鉴定表达产物和纯化产物。结果表明,质粒pET-cBD103经PCR及双酶切鉴定证明构建正确,大肠埃希菌中成功表达出目的蛋白,蛋白大小约为24ku,以可溶性表达为主。经过纯化和酶切,得到大小8ku的cBD103,与预期大小一致。该研究在大肠埃希菌中表达了目的蛋白,为下一步大规模制备cBD103奠定了基础。
译 名：
Prokaryotic Expression and Purification of Canine β-defensin 103
作 者：
ZHU Qian;BAO Yin-li;QIN Hai-bin;SONG zhen-hua;HE Xing-liang;WEN Hai;Gao Yi-long;Nanjing Policedog Research Institute of MPS,Policedog Technology Key Laboratory of MPS;College of Veterinary Medicine,Nanjing Agricultural University;
关键词：
canine β-defensin 103;;prokanyotic expression;;purification
摘 要：
The canineβ-defensin 103(cBD103)gene was subcloned to pET-32a(+)vector,and the constructed recombinant plasmid pET-cBD103 was transformed to competent E.coli BL21 for expression under induction of IPTG.The expressed product was purified by nickel ion affinity chromatography and identified by SDS-PAGE.Results of both PCR and enzyme digestion analysis proved that recombinant pETcBD103 was constructed correctly.The expressed protein,with a relative molecular mass of 24 ku,mainly existed in a soluble form.After purification and restriction enzyme digestion,aprotein band about 8ku was detected,and consistent with the purpose bands.The cBD103 protein was successfully expressed in E.coli,which laid a foundation for further preparation of cBD103 protein and clinical test.
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