摘要
目的克隆、表达幽门螺杆菌中性粒细胞激活蛋白napA与霍乱毒素B亚单位ctxB融合基因napA-ctxB(nctB),为制备预防H.pylori感染的疫苗奠定基础。方法用PCR方法扩增ctxB目的基因片段,克隆至pQE30-napA质粒的napA基因上游,构建含双基因的表达质粒pQE30-napA-ctxB(pQE30-nctB),经测序分析确认后转化E.coliDH5α,经IPTG诱导表达融合蛋白NCTB,融合蛋白NCTB经镍离子柱纯化。结果PCR扩增出807bp的目的基因片段nctB。工程菌pQE30-nctB-DH5α经IPTG诱导后,SDS-PAGE显示有新生的蛋白表达条带,Mr为30000,与预期的一致,约占菌体总蛋白的27%,重组蛋白用Ni2+-NTA树脂提纯,纯化后的蛋白质经SDS-PAGE分析可见单一条带,图象软件分析表明纯度可达94%以上。Westernblot显示重组蛋白质有良好的抗原性。结论构建含双基因的表达质粒pQE30-nctB成功,并在大肠杆菌DH5α中高效的表达。
Objective To construct the fusion gene of H. pylori neutrophil activating protein （napA） and cholera toxin subunit B （ctxB） and expressed it in E. coli DH5α. Methods The ctxB gene was amplified by PCR and cloned into plasmld pQE30-napA. The recombinant plasmid was identified by sequencing, and then transformed into E. coli DH5α. The transfonnant colony was induced with IPTG and the expressed protein was purified by Ni^2＋ -NTA column chromatography. Expression of the fusion protein was analyzed by SDS-PAGE and Western blotting. Results The gene fragment at length of 807 bp was amplified and cloned into plasmid pQE30 successfully. SDS-PAGE showed a protein band with relative molecular weight of 30 000, which was consistent with the expectation. The expressed product contained about 27% of total somatic protein, and reached a purity of 94% after Ni^2＋ -NTA column chromatograph. Western blot method showed good antigenicity of the recombinant protein. Conclusion The recombinant plasmid of neutrophil activating protein（napA） and cholera toxin subunit B （ctxB） is suecessfully construeted and the napA-ctxB gene is highly expressed in E. coli DH5α. The fusion protein could be used in development of Helicobacter pylori vaccine.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第2期172-175,共4页
Immunological Journal
