人结核杆菌热休克蛋白65重组卡介苗疫苗的构建、表达及鉴定

Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium tuberculosis Heat Shock Protein 65

目的 构建人结核杆菌热休克蛋白 6 5 (HSP6 5 )重组卡介苗 (BCG)疫苗。方法 用PCR技术从BCG基因组中扩增出抗原 85B (Ag85B)的信号肽DNA序列 ,从 pCMV MTHSP 6 5质粒中扩增出HSP6 5全长基因。利用DNA重组技术将以上两个片段插入质粒 pBCG 2 10 0的人结核杆菌HSP70启动子下游 ,构建一个分泌型的原核穿梭表达质粒pBCG SP HSP6 5。利用电穿孔的方法将该质粒转入BCG中 ,经热诱导后 ,用SDS PAG电泳来观察其表达水平。利用Westernblot来检验HSP6 5的生物学活性。结果 酶切鉴定、PCR和测序分析结果表明 ,所克隆的信号肽DNA片段和热休克蛋白 6 5DNA片段与报道结果完全一致 ,重组体的连接方向正确 ,阅读框与预期完全一致。热诱导后 ,重组BCG表达的 6 5kD (1kD =0 992 1ku)蛋白占菌体总蛋白的 35 6 7% ,占裂解物上清总蛋白的 74 0 9% ,表明重组的HSP6 5基因能在BCG中高效表达 ,表达的蛋白大部分以可溶状态存在。通过Westernblot证实分泌的该蛋白能与结核杆菌HSP6 5的抗体特异性结合。结论 成功构建分泌型原核穿梭表达质粒pBCG SP HSP6 5 ,重组的HSP6 5基因能在BCG中高效表达 ,表达的HSP6 5具有生物学活性 ,重组BCG疫苗构建成功。

Objective To construct a recombinant BCG vaccine carrying heat shock protein 65 (HSP65) from human Mycobacterium tuberculosis (M.TB).Methods The signal sequence of antigen 85B amplified from BCG genome and the whole HSP65 DNA sequence of human M.TB obtained from the plasmid pCMV-MT HSP65 were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M.TB, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Recombinants were electroporated into BCG and induced by heating.Results Results of the induced expression were detected by SDS-PAGE and the biological activity of the expressed protein was tested by Western blot analysis. Results pBCG-SP-HSP65 was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and DNA sequencing analysis. After electroporated into BCG and induced by heating, the expressed 65 kD protein in rBCG detected by SDS-PAGE was up to 35.69 % in total bacterial protein and 74.09 %in the total protein of cell lysate supernatants respectively, which demonstrated the recombinant HSP65 gene could be expressed in BCG with high efficiency and the expressed proteins were mainly soluble. Western blot revealed that the secretive proteins could specially combine with antibody against human M.TB HSP65. Conclusion pBCG-SP-HSP65 was successfully constructed and HSP65 gene could be expressed in BCG with high efficiency. And the expressed proteins possess the biological activity.