摘要
应用PCR扩增出禽多杀性巴氏杆菌C48 1成熟外膜蛋白H基因(ompmH),将ompmH片段按正确的阅读框定向插入原核表达载体pGEX 6p 1中谷胱甘肽转移酶(GST)基因的下游,获得了重组表达质粒pGEX ompmH。然后将重组质粒转化大肠埃希氏菌BL21(DE3),当转化菌的D600nm值达到0.6～0.8时,对异丙基硫代 β D 半乳糖苷(IPTG)的诱导浓度、诱导时间和诱导温度等条件进行了试验,根据聚丙烯酰胺凝胶电泳判定融合蛋白GST ompmH表达的最适条件。结果,当细菌的D600nm值为0.6～0.8时,IPTG最佳诱导浓度为0.8mmol L,最佳诱导时间为4h,最适诱导温度为25℃。并用免疫印迹法对表达产物进行检测,结果显示表达的融合蛋白GST ompmH能与C48 1外膜蛋白免疫血清发生特异性反应,证明pGEX 6p 1载体表达的融合蛋白保留了天然蛋白的抗原性。
The mature outer membrane protein(omp_(m)H) gene was amplifiedby PCR. According to the right open reading frame(ORF),the PCR product was cloned into the prokaryotic expression vector pGEX-6p-1 and obtained the recombinant plasmid pGEX-omp_(m)H. The fused protein GST-omp_(m)H was studied in detail on many factors including IPTG inducing concentration, temperature and time. The expressed product was identified by SDS-PAGE. The resltuts showed that the best concentration of IPTG was (0.8 mmol/L) and the best inducing time was 4 h and the best inducing temperature was 25 ℃.The fusion protein GST-Omp_m H could be recognized by antisera of C_(48-1) outer membrane protein using Western blotting. It was concluded that the fusion protein GST-Omp_m H possessed good antigenicity.
出处
《中国兽医科技》
CAS
CSCD
北大核心
2005年第1期45-48,共4页
Chinese Journal of Veterinary Science and Technology
基金
广东省自然科学基金资助项目(3227)
