乙肝HBsAg和结核杆菌HSP70融合表达载体的构建

Construction of an eukaryotic expression vector pcDNA3.1-S-HSP70

目的：构建乙肝HBsAg和结核杆菌HSP70融合表达质粒。方法：用 PCR方法从结核杆菌H37Rv基因组中扩增出结核杆菌HSP70 基因序列，应用T-A克隆技术，克隆到质粒 pGEM-T vector，经 DNA测序证实基因碱基无误后，亚克隆到真核表达质粒 pcDNA3.1（+），构建成pcDNA3.1-HSP70重组质粒。然后，再用PCR方法，从质粒pEcob6中扩增出HBsAg基因，并克隆到质粒pcDNA3.1-HSP70，将HBsAg基因的C端与HSP70基因N端连接，构建 HBsAg和 HSP70融合表达质粒 pcDNA3.1-S-HSP70。结果：经酶切鉴定和DNA测序证实重组质粒构建正确。结论：乙肝HBsAg和结核杆菌HSP70融合表达载体构建成功。

Objective: To construct an eukaryotic expression plasmid containing HBsAg and TB.Hsp70. Methods: By PCR and T-A cloning techniques, the full length gene of TB.Hsp70 from genome of Mycobacterium tuberculosis H37Rv was amplified and cloned directly into pGEM-T vector ,then the recombinant plasmid was digested with BamHⅠand EcoRⅠ, and was subcloned into plasmid pcDNA3.1(+) ( pcDNA3.1-HSP70).The HBsAg gene (without stop codon) was amplified from plasmid pEcob6 by PCR . After digested with HindⅢ and BamHⅠ, the PCR product was cloned into plasmid pcDNA3.1-HSP70. Results: Restriction enzyme digesting and DNA sequencing confirmed that the recombinant eukaryotic expression plasmid containing HBsAg and mycobacterium tuberculosis HSP70 (pcDNA3.1-S-HSP70) had been constructed correctly. Conclusion: An eukaryotic expression plasmid pcDNA3.1-S-Hsp70 has been constructed successfully.