摘要
利用亲和层析和Ｗｅｓｔｅｒｎ－ｂｌｏｔ等分子生物学技术，表达、纯化、鉴定了重组日本血吸虫菲律宾株２６ｋＤａ抗原（ｒＳｊｐ２６ＧＳＴ），并比较了其与日本血吸虫大陆株２６ｋＤａ抗原（ｒＳｊｃ２６ＧＳＴ）基因ｃＤＮＡ序列的异同，分析了其应用前景。分别以质粒ｐＧＥＸ和血吸虫大陆株总ＲＮＡ为模板，经ＰＣＲ或逆转录ＰＣＲ（ＲＴ－ＰＣＲ）扩增出的Ｓｊｐ２６ＧＳＴ和Ｓｊｃ２６ＧＳＴｃＤＮＡ，其片段长度均为６５４ｂｐ。通过双脱氧测序结果显示两者碱基排列顺序相同。用大肠杆菌（Ｅ．ｃｏｌｉ）表达载体在Ｅ．ｃｏｌｉ中高效表达Ｓｊｐ２６ＧＳＴ，ｒＳｊｐ２６ＧＳＴ占菌体总蛋白的２５％。用谷胱甘肽（ＧＳＨ）亲和层析柱一步纯化法，得到纯品Ｓｊｐ２６ＧＳＴ，将其作为靶抗原用Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测血吸虫大陆株感染者和感染小鼠血清及用Ｓｊｃ２６ＧＳＴ免疫的小鼠血清，反应均为阳性。本研究提示Ｓｊｐ２６ＧＳＴ与Ｓｊｃ２６ＧＳＴ在ＤＮＡ序列、氨基酸顺序和抗原性等方面高度同源。
Employing molecular biological technology such as affinity chromatography and western blot, the recombinant Philippine Schistosoma japonicum 26 kDa antigen (rSjp26GST) was expressed, purified and identified ,and compared it with Chinese Schistosoma japonicum 26 kDa antigen (Sjc26GST) to evaluate its application prospect. The double strand cDNA gene of Sjp26GST and Sjc26GST amplified by PCR or reverse transcript PCR (RT PCR) using plasmid pGEX and total RNA of Chinese Schistosoma japonicum as templet, respectively,was both 654 bp.The two species cDNA sequence determined by dideoxy nucleotide method were identical. The level of rSjp26GST highly expressed in E. coli containing E. coli expression plasmid is 25% of the total bacterial protein. The rSj26GST purified by affinity chromatography on immobilized glutathione from bacteria in a one step procedure was taken as target antigen to detect the infected human and mice serum and the mice serum from mice immunized with Sjc26GST by Western blot. All these results were positive. This investigation suggusted that rSjp26GST have potential practical value to be developed as a candidate vaccine against Chinese Schistosoma japonicum because of its highly homologue with Sj26cGST in cDNA sequence and immunogenicity.
出处
《中国寄生虫病防治杂志》
CSCD
1998年第2期130-134,共5页
Chinese Journal of Parasitic Disease Control
基金
国家自然科学基金
总理专项基金
关键词
日本血吸虫
26kDa抗原
表达
纯化
RT-PCR
Schistosoma japonicum 26 kDa antigen, expression, purification, Western blot, RT PCR
