摘要
目的构建日本血吸虫重组质粒pET28α-Sj32,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增Sj32抗原编码基因;将该基因克隆至原核表达载体pET28α,构建重组质粒pET28α-Sj32并转化大肠埃希菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果RT-PCR扩增出1270bp的Sj32基因;双酶切和PCR鉴定证实Sj32基因成功插入pET28α中,SDS-PAGE分析显示表达产物为分子质量单位约为42ku的重组蛋白,与预期结果一致,表达的蛋白约占菌体总蛋白的24%;Western blot鉴定重组蛋白能被日本血吸虫感染的兔血清识别。结论成功构建了日本血吸虫重组质粒pET28α-Sj32,该质粒在大肠埃希菌BL21中获得了高效融合表达,表达的融合蛋白具有抗原特异性。
Objective To construct and express the recombinant plasmid pET28α-Sj32 of Schistosoma japonicum（Sj）in Escherichia coli BL21（DE3）.Methods Total RNA was extracted from Sj adult worms by ultrasonic disruption and the antigen genes Sj32 were amplified by RT-PCR from total RNA,and then cloned into prokaryotic expression plasmid pET28α to construct pET28α-Sj32.The recombinant plasmid was transformed into E.coli BL2（DE3）,and rBL21（pET28α-Sj32）was induced with isopropyl-β-D-thiogalactopyranoside（IPTG）.The expressed products were analyzed and identified by SDS-PAGE and Western blot.Results The 1 270 bp Sj32 fusion gene was successfully amplified by RT-PCR and cloned into pET28α by restriction analysis and PCR identification.The recombinant plasmid pET28α-Sj32 was successfully constructed.The relative molecular mass of the expressed recombinant protein was approximately 42 ku according to SDS-PAGE,and the amount of the expressed protein was 24% of total bacterial proteins.According to Western blot,the fusion protein was recognized by sera from rabbits infected with Sj.Conclusion The recombinant plasmid pET28α-Sj32 was successfully constructed and is highly expressed in E.coli in a fused form with His-tag,and the expressed fusion protein shows specific antigenicity.
出处
《中国病原生物学杂志》
CSCD
2010年第4期286-289,共4页
Journal of Pathogen Biology
基金
重庆市科委地方病重大专项(No.2008AB5055
2008AA5008和2008AB5054)
