CAJ | 학술논문

据日本血吸虫菲律宾株编码卵壳前体蛋白的一段230bp 的序列设计PCR引物,以日本血吸虫中国大陆株成虫mRNA为模板,用RT-PCR方法扩增出日本血吸虫中国大陆株相应的基因片段、然后根据测序结果设计一系列引物,用5'RACE 和3'RACE 法扩增出该基因cDNA的5' 和3'端,再根据测序结果设计全长引物,用RT-PCR方法扩增出大小为423 bp的片段.经序列分析推断该基因片段为编码日本血吸虫中国大陆株卵壳前体蛋白基因的完整阅读框,对相应基因组区段测序证实该基因没有内含子(GenBank登录号:AF519182).将其克隆到表达载体pET28c(+)中,在大肠杆菌中获得表达,融合表达产物分子量约为20.9 kD.Real-time PCR结果显示该蛋白在尾蚴感染宿主后第23d即卵壳形成时高表达.利用日本血吸虫成虫抗原免疫血清对该表达产物进行Western 印迹检测,在预测位置出现了明显的识别条带,说明该编码日本血吸虫中国大陆株抱雌沟蛋白基因的表达产物具有抗原性.
거일본혈흡충비률빈주편마란각전체단백적일단230bp 적서렬설계PCR인물,이일본혈흡충중국대륙주성충mRNA위모판,용RT-PCR방법확증출일본혈흡충중국대륙주상응적기인편단、연후근거측서결과설계일계렬인물,용5'RACE 화3'RACE 법확증출해기인cDNA적5' 화3'단,재근거측서결과설계전장인물,용RT-PCR방법확증출대소위423 bp적편단.경서렬분석추단해기인편단위편마일본혈흡충중국대륙주란각전체단백기인적완정열독광,대상응기인조구단측서증실해기인몰유내함자(GenBank등록호:AF519182).장기극륭도표체재체pET28c(+)중,재대장간균중획득표체,융합표체산물분자량약위20.9 kD.Real-time PCR결과현시해단백재미유감염숙주후제23d즉란각형성시고표체.이용일본혈흡충성충항원면역혈청대해표체산물진행Western 인적검측,재예측위치출현료명현적식별조대,설명해편마일본혈흡충중국대륙주포자구단백기인적표체산물구유항원성.
The gene fragment encoding the egg-shell precursor protein of Schistosoma japonicum was amplified with RT-PCR by using PCR primer designed according to the 423 bp cDNA fragment of the Philippine strain of S.japonicum, the corresponding mDNA fragment of Chinese strain as template and then the 5' and 3' ends of this gene cDNA were amplified with 5' RACE and 3' RACE by using a series of primers designed according to the result of sequencing. Result of sequence analysis showed that this fragment, named as Sj423, contained a complete open reading frame (ORF) of gene encoding the egg-shell precursor protein of S.japonicum.(Chinese strain). As demonstrated by sequencing analysis. No intron could be detected in this gene fragment. This gene was subsequently expressed in E.coli after cloning into the expression vector pET28c(+). The molecular mass of the expressed product of this gene was 20.9 kDa as revealed by SDS-PAGE analysis, and Western blot analysis showed that the recombinant protein expressed could react well with the rabbit antiserum against the worm antigen of S.japonicum;indicating the good antigenicity of this expressed product.