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  

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

作　　者：张燕[1,2] 刘建国[1] 陈筑[1] 马欣荣[3] 唐琳[1] 吴家媛[1] 张剑[1] 管晓燕[1] 

机构地区：[1]遵义医学院附属口腔医院,贵州遵义563003 [2]滨州医学院附属医院口腔内科,山东滨州256603 [3]中国科学院成都分院生物研究所,四川成都610041

出　　处：《牙体牙髓牙周病学杂志》2012年第7期371-374,406,共5页Chinese Journal of Conservative Dentistry

基　　金：国家自然科学基金资助项目(30160086);贵州省优秀教育科技人才省长专项基金项目[(2001)6号]

摘　　要：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。AIM ： To construct and express the fused vector of ldh gene of Streptococcus Mutans and the en- coding gene ctxB of cholera toxin B subunit. METHODS： The gene ldh and ctxB were amplified by PCR, and inte- grated into the vector pE332a （ ＋ ）. 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.

关 键 词：变异链球菌 乳酸脱氢酶 霍乱毒素B亚单位 原核表达质粒 

分 类 号：R780.2[医药卫生—口腔医学]