CTP-Ub-HBcAg原核表达载体的构建及其表达

目的构建CTP-Ub-HBcAg原核表达载体并诱导和鉴定其融合蛋白的表达。方法以质粒pcDNA3.1(-)-Ub-HBcAg中的Ub-HBcAg融合基因为模板设计引物,上游引物带有CTP基因序列,进行PCR扩增,PCR产物克隆到原核表达质粒pMAL-c2x中,菌落PCR阳性克隆测序鉴定,鉴定正确的质粒转化大肠杆菌BL21(DE3)诱导表达,并进一步通过直链淀粉树脂亲和层析法纯化融合蛋白,Western blotting鉴定。结果 PCR扩增得到820 bp大小的条带,为CTP-Ub-HBcAg融合序列。该序列被克隆至表达质粒pMAL-c2x中,阳性克隆菌落测序正确,并在DE3中获得诱导表达。Western blotting鉴定为70 KD的CTP-Ub-HBcA融合蛋白。结论构建CTP-Ub-HBcAg原核表达载体并成功表达,为进一步研究该融合蛋白的功能提供了实验基础。

Construction and expression of prokaryotic plasmid of CTP-Ub-HBcAg

Objective To construct a prokaryotic expression vector of CTP-Ub-HBcAg and identify its recombinant protein expression.Methods A pair of primers were designed based on fusion gene Ub-HBcAg in pcDNA3.1 （-）-Ub-HBcAg with the forward primer having CTP gene.The fusion gene Ub-HBcAg was amplified by PCR,then the product was cloned into pMAL-c2x,a prokaryotic expression vector.The correct vector was transformed into E.coli BL21 （DE3） to express the fusion protein after identification by colony PCR and sequence test.The fusion protein was purified by dextrin sepharose high performance and identified by Western blotting.Results 820 bp band was obtained by PCR,then the fusion gene was cloned into the pMAL-c2x plasmid which was transfected into DE3 and expressed successfully.The colony PCR and sequencing were confirmed correctly.Finally,70 KD protein was obtained and identified to be fusion protein CTP-Ub-HBcAg by Western blotting.Conclusion The prokaryotic expression vector of CTP-Ub-HBcAg was successfully constructed,and it may provid a way for the further research on the function of fusion protein CTP-Ub-HBcAg.