摘要
目的构建分泌性表达融合蛋白85B-ESAT-6重组卡介苗。方法PCR扩增结核分枝杆菌热休克蛋白60(hsp60)的调控序列和合成T4转录终止序列,分别定向克隆入pBCG2000载体的多克隆位点的上下游,得到E.coli-Mycobacterium穿梭载体质粒pBCG3000。PCR分别扩增出结核分枝杆菌分泌性抗原Ag85B的全编码序列及ESAT-6基因,然后将两基因融合插入pBCG3000载体的hsp60启动子下游得到pBCG3000-85B-ESAT-6分泌性表达质粒。用电穿孔法将pBCG3000-85B-ESAT-6质粒转化BCG细胞,得到重组卡介苗。重组卡介苗经热诱导后,SDS-PAGE电泳和Westernblotting鉴定融合蛋白85B-ESAT-6的表达及其免疫学活性。结果通过PCR、酶切以及测序鉴定,pBCG3000-85B-ESAT-6表达质粒构建完全正确,SDS-PAGE电泳未能直接观察到表达的融合蛋白条带,用ESAT-6蛋白的多抗血清通过Westernblotting证实了该蛋白主要分泌性表达于胞外并具有免疫学活性。结论分泌性表达融合蛋白85B-ESAT-6重组卡介苗构建成功。
Objective To construct a recombinant BCG capable to express the secretory form fusion protein 85B-ESAT-6. Method PCR amplified regnlatory sequence of hsp60 from the genome of H37Rv strain and the synthesized T4 transcription-termination sequence were cloned into pBCG2000 vector,yielding the E.coli-Mycobacterium shuttle vector pBCG3000. The genes encoding Ag85B and ESAT-6 protein of MTB were amplified and inserted into the multiple cloning sites downstream of the hsp60 promotor using the same cloning strategy. The resulting recombinant expression plasmid pBCG3000-85B-ESAT-6 was then transformed by electroporation into BCG, and induced by heating.The expression of the target protein was verified by SDS-PAGE and immunoblotting. Result The plasmid pBCG3000-85B-ESAT-6 was constructed successfully. The sequence was identified by PCR amplification, restriction endonuclease analysis and sequencing. The expression of the protein was verified by Western blotting. Results showed that the fusion protein could be expressed and secreted from the recombinant BCG. And the fusion protein could be recognized by the antiserum to ESAT-6 protein.Conclusion The recombinant BCG vaccine was successfully constructed.
出处
《热带医学杂志》
CAS
2006年第10期1064-1067,共4页
Journal of Tropical Medicine
