变异链球菌乳酸脱氢酶与霍乱毒素B亚单位嵌合表达质粒的构建和表达

目的:构建含变异链球菌乳酸脱氢酶(LDH)和霍乱毒素B亚单位(CTB)嵌合原核表达质粒,并诱导表达融合蛋白。方法:应用PCR技术扩增LDH编码基因ldh和CT编码基因ctxB,定向克隆至原核表达质粒pET32a(+)上,通过限制性内酶切、PCR和序列测定分析鉴定后转化大肠杆菌BL21(DE3),并经IPTG诱导表达融合蛋白。结果:PCR扩增得到了ldh和ctxB;构建的质粒pET-LDH/CTB经KpnⅠ、XhoⅠ双酶切和目的基因PCR检测,均得到1.4 kb大小的片段,与预计目的基因片段大小相同;质粒pET-LDH/CTB中插入的ldh序列与GeneBank中ldh比较同源性为98%,ctxB的同源性达99%,插入的相位正确;经IPTG诱导表达了约70×103的蛋白。结论:成功构建了变异链球菌LDH和CTB嵌合表达质粒pET-LDH/CTB,并正确表达融合蛋白。

Construction and expression of the fused Vector of Lactate dehydrogenase of Streptococcus Mutans and cholera toxin B subunit

AIM: To construct and express the fused vector of ldh gene of Streptococcus Mutans and the encoding gene ctxB of cholera toxin B subunit.METHODS: The gene ldh and ctxB were amplified by PCR,and integrated into the vector pET32a(+).The recombinant plasmids were identified by restriction endonuclease digestion,PCR and sequencing.Plasmids were transformed into E.coli BL21(DE3)and induced by IPTG.RESULTS: The 1.4 kb fused gene ldh-ctxB was obtained by restriction endonuclease digestion and PCR.The homoeology was 98% between sequence ldh in the vector pET-LDH/CTB and ldh in GeneBank,and ctxB was 99% by sequence analysis and acomparison.The recombinant plasmid expressed the fusion protein of 70 KD.CONCLUSION: The fused vector of ldh and ctxB was constructed and expressed correctly.