摘要
用含丹参柯巴基焦磷酸合酶(Salvia miltiorrhizacopalyl diphosphate synthase,SmCPS)基因的重组质粒pET32CPS转化大肠杆菌BL21trxB(DE3)进行诱导表达,SDS-PAGE结果表明SmCPS基因在大肠杆菌中获得了表达;对影响可溶性表达的4个因素,即诱导温度、IPTG诱导浓度、诱导时宿主菌的密度(A600)和诱导时间进行了优化。结果发现在宿主菌A600达到1.0时加入0.4 mmol.L-1IPTG,在20℃诱导培养8 h表达的可溶性蛋白量最高,约占细胞总蛋白的35.6%。用Ni2+亲和色谱法纯化表达产物,纯化蛋白产率达到12.3 mg.L-1。将纯化蛋白免疫制备兔源SmCPS抗血清,ELISA测定效价为1∶24 300,且经Western blotting鉴定该抗体可特异性识别SmCPS抗原,获得了效价高、免疫反应性好的SmCPS多克隆抗体,为进一步从蛋白水平研究SmCPS表达与丹参酮类有效成分生物合成相关性提供了物质基础。
The expression plasmid pET32CPS harboring SmCPS gene was transiormed into E. coli BI21 trxB （DE3） resulting in recombinant strain E. coli [ pET32CPS ]. The induction of E. coli [ pET32CPS ] in different temperatures, induction time, IPTG concentrations and A600 values of E. coli were performed. The optimal expression conditions of SmCPS were characterized according to the orthogonal analysis, and the ratio of the interest protein to total proteins reached to 35.6%. The recombinant SmCPS protein purified by Ni^2＋ affinity chromatography column was identified by SDS-PAGE and Western blotting, and then used for rabbit immunization. The titer of the rabbit antiserum against SmCPS was about 1 ： 24 300 after the third immunization, and could specifically recognize the antigen of SmCPS protein by Western blotting analysis. The successful preparation of polyclonal antibody against SmCPS laid a foundation for further correlative study between expression of SrnCPS and the production of tanshinones in protein level.
出处
《药学学报》
CAS
CSCD
北大核心
2008年第7期766-772,共7页
Acta Pharmaceutica Sinica
基金
国家重点基础研究发展计划资助项目(2006CB504700)
国家高科技研究发展计划资助项目(2007AA02Z104)
中国中医科学院基本科研业务费自主选题项目(ZZ2006098)
关键词
丹参
柯巴基焦磷酸合酶
优化表达
抗体制备
Salvia miltiorrhiza
copalyl diphosphate synthase
optimizing expression
antibody preparation
