摘要
为表达、获取日本血吸虫谷胱甘肽转移酶 (SjGST)基因工程重组蛋白 ,以日本血吸虫 (中国大陆株 )cDNA为模板 ,设计、合成特定寡核苷酸引物 ,RT—PCR法扩增GST编码基因序列 ,将扩增产物连接 pGEM-T克隆载体 ,再亚克隆到真、原核表达质粒pBK -CMV中 ,转染大肠杆菌XL1 -blue ,经IPTG诱导后用SDS—PAGE分析表达效果。结果RT—PCR法特异性扩增出日本血吸虫GST编码区基因片段 ,其大小约为 670bp。重组 pGEM -T克隆载体和重组 -GST表达质粒经双酶切及以相应质粒为模板的PCR法均证实其中含有插入的GST目的基因。重组菌经IPTG诱导后以大小约 2 9KD融合蛋白形式表达 ,诱导后 6小时表达产物约占菌体蛋白 3 0 %。结论 pBK -SjcGST重组质粒的成功构建和高效表达 ,为进一步纯化提取基因工程重组日本血吸虫GST蛋白 ,分析其在血吸虫病免疫诊断、免疫预防中的作用提供了必要的条件。
To clone and expression of the gene encoding 26KD Glutathion-S-Transferase of Schistosoma japonicum(Chinese strain),Methods total RNA was isolated from adult worms using TRIzol reagent.The cDNA encoding 26KD Glutathion-S-Transferase was amplified by RT-PCR and cloned into pGEM-T vector,then,subcloned into the expressive plasmid vector pBK-CMV.The recombinants were identified by restriction enzyme analysis and PCR technique.The positive recombinant E.coli XL1-blue was induced by IPTG.SDS-polyacrylamide gel electrophoresis of total cellular protein from the bacteria was performed to detect the 26KD SjGST gene product.Results The objective gene was amplified and subcloned into expressive vector.The positive recombinant could be induced to express SjGST fusion protein with a molecular weight of 29KD.Induction 6 hours latter,the amount of exogenous proteins was about 30% of the total bacterial proteins.Conclusions The gene encoding 26KD SjGST was amplified and pBK-SjGST recombinants was successfully constructed.The recombinant 26KD SjGST protein was expressed in high efficiency and will be used to further study in immunodignosis and immunoprophyaxis of Schistomiasis japonica.
出处
《生物学杂志》
CAS
CSCD
2000年第3期13-15,共3页
Journal of Biology
基金
安徽省自然科学基金!(95-医- 0 2 8)
安徽省科委 1 999科研基金
关键词
日本血吸虫病
SjGST
基因工程疫苗
免疫诊断
Schistosoma japonicum
Glutathion-S-Transferase
polymerase china reaction
gene cloning
gene expression.
