中国人兽共患病学报
中國人獸共患病學報
중국인수공환병학보
CHINESE JOURNAL OF ZOONOSES
2010年
2期
134-139
,共6页
黄慧聪%赵巧莲%谭峰%潘长旺
黃慧聰%趙巧蓮%譚峰%潘長旺
황혜총%조교련%담봉%반장왕
隐孢子虫%p30基因%克隆%原核表达
隱孢子蟲%p30基因%剋隆%原覈錶達
은포자충%p30기인%극륭%원핵표체
Cryptosporidium%p30 gene%cloning%prokaryotic expression
目的 获取隐孢子虫Gal/GalNAc特异性凝集素p30基因,并构建原核表达系统,获得相应的重组蛋白.方法 采用聚合酶链反应(PCR)技术,从微小隐孢子虫基因组中扩增出p30基因,然后将其克隆入pMD18-T载体,转化后挑取阳性克隆进行酶切和测序鉴定,并进行生物信息学分析及预测.再将亚克隆与表达载体PET-28 a (+)分别酶切并连接,经IPTG诱导表达,表达产物用Ni-NTA亲和层析法纯化,SDS-PAGE和Western blotting检测表达效果.结果 PCR扩增得到特异的微小隐孢子虫Gal/GalNAc特异性凝集素p30基因,测得的核苷酸序列及其推导的氨基酸序列与GenBank上提交的序列同源性分别为98%～100%和99%～100%,理论等电点和分子量分别为6.4854和31 842Da,并存在9个潜在的抗原表位.经酶切、测序结果表明质粒已导入Rosetta.SDS-PAGE和Western blotting证实重组菌成功表达融合蛋白.结论 成功构建微小隐孢子虫Gal/GalNAc特异性凝集素p30原核表达系统.
目的 穫取隱孢子蟲Gal/GalNAc特異性凝集素p30基因,併構建原覈錶達繫統,穫得相應的重組蛋白.方法 採用聚閤酶鏈反應(PCR)技術,從微小隱孢子蟲基因組中擴增齣p30基因,然後將其剋隆入pMD18-T載體,轉化後挑取暘性剋隆進行酶切和測序鑒定,併進行生物信息學分析及預測.再將亞剋隆與錶達載體PET-28 a (+)分彆酶切併連接,經IPTG誘導錶達,錶達產物用Ni-NTA親和層析法純化,SDS-PAGE和Western blotting檢測錶達效果.結果 PCR擴增得到特異的微小隱孢子蟲Gal/GalNAc特異性凝集素p30基因,測得的覈苷痠序列及其推導的氨基痠序列與GenBank上提交的序列同源性分彆為98%～100%和99%～100%,理論等電點和分子量分彆為6.4854和31 842Da,併存在9箇潛在的抗原錶位.經酶切、測序結果錶明質粒已導入Rosetta.SDS-PAGE和Western blotting證實重組菌成功錶達融閤蛋白.結論 成功構建微小隱孢子蟲Gal/GalNAc特異性凝集素p30原覈錶達繫統.
목적 획취은포자충Gal/GalNAc특이성응집소p30기인,병구건원핵표체계통,획득상응적중조단백.방법 채용취합매련반응(PCR)기술,종미소은포자충기인조중확증출p30기인,연후장기극륭입pMD18-T재체,전화후도취양성극륭진행매절화측서감정,병진행생물신식학분석급예측.재장아극륭여표체재체PET-28 a (+)분별매절병련접,경IPTG유도표체,표체산물용Ni-NTA친화층석법순화,SDS-PAGE화Western blotting검측표체효과.결과 PCR확증득도특이적미소은포자충Gal/GalNAc특이성응집소p30기인,측득적핵감산서렬급기추도적안기산서렬여GenBank상제교적서렬동원성분별위98%～100%화99%～100%,이론등전점화분자량분별위6.4854화31 842Da,병존재9개잠재적항원표위.경매절、측서결과표명질립이도입Rosetta.SDS-PAGE화Western blotting증실중조균성공표체융합단백.결론 성공구건미소은포자충Gal/GalNAc특이성응집소p30원핵표체계통.
To clone and construct a prokaryotic expression system containing the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium, the p30 gene was amplified from genomic DNA of Cryptosporidium parvum by PCR and cloned into vector pMD18-T directly. The positive clones were identified by EcoR I, Xho I digestion and sequenced. The gene structure and its possible function were analyzed and predicted by using related bioinformatics softwares. The P30 gene was recombined with plamid pET-28 a (+) to construct the prokaryotic expression vector and was expressed in E. Coli with IPTG induction. Ni-NTA affinity chromatography was used to extract P30 protein and the expression effect and purification of P30 protein were determined by SDS-PAGE and Western blotting. It was demonstrated that the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum was specifically amplified, and its sequence homology of nucleotide and the deduced amino acid sequence of P30 gene with relevant sequences in GenBank were 98%-100% and 99%-100% respectively. Its theoretical iso-electric point and molecular weight were found to be 6.4854 and 31842 dalton.It was predicted to contain 9 potential epitopes. The expressed plasmid was identified by EcoR I/ Xho I digestion and sequenced and the recombinant P30 protein could be identified by SDS-PAGE and Western blotting assay. It is evident that the prokaryotic expression system for galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum has been constructed successfully.
