【作者】 张燕； 刘建国； 陈筑； 马欣荣； 唐琳； 吴家媛； 张剑；

【Author】 ZHANG Yan, LIU Jian-guo, Chen Zhu, MA Xi-rong, TANG Lin, WU Jia-yuan, ZHANG Jian.(Affiliated Stomatology Hospital, Zunyi Medical College. Zunyi 563003;Chengdu Biologic Research Institute,CAS, Chengdu 610041, Sichuan Province.China))

【机构】 遵义医学院附属口腔医院； 中国科学院成都生物研究所；

【摘要】 目的:构建含变形链球菌乳酸脱氢酶(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及ctxB;构建的质粒pET-ldh-ctxB经Kpn Ⅰ、Xho Ⅰ双酶切及目的基因PCR检测,均得到约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-ctxB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70KD的蛋白。结论:成功构建了变形链球菌LDH和CTB嵌合表达质粒pET-ldh-ctxB,并正确表达融合蛋白。更多还原

【Abstract】 OBJECTIVE: To construct and express the fused vector of ldh gene of Streptococcus Mutatis 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 1.4kb fused gene ldh-ctxB were obtained by restriction endonuclease digestion and PCR. The homoeology was 98% between sequence ldh in the vector pET-ldh-ctxB and Idh in GeneBank, and ctxB was 99% by sequence analysis and acomparison. The recombinant plasmid expressed the fusion protein about 70KD. CONCLUSION: The fused vector of ldh and ctxB was constructed and expressed correctly.更多还原