微小隐孢子虫病毒衣壳蛋白的原核表达及鉴定

Prokaryotic Expression and Identification of S-dsRNA Gene from Cryptosporidium parvum Virus

目的对微小隐孢子虫病毒衣壳蛋白S-dsRNA基因进行克隆、表达和反应原性分析。方法以微小隐孢子虫总RNA逆转录的cDNA为模板,克隆S-dsRNA基因,并转化至原核表达载体pET-28a(+)中,构建重组原核表达载体pET-28a(+)-S,转入大肠埃希菌BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察重组蛋白的表达情况,蛋白质印迹(Western blotting)分析重组蛋白与鼠抗微小隐孢子虫阳性血清的反应原性。结果 PCR和双酶切鉴定表明,重组质粒pET-28a(+)-S构建成功。SDS-PAGE结果显示,37℃下经1mmol/L IPTG诱导4h,重组蛋白表达量最大。重组蛋白主要以包涵体形式表达,相对分子质量(Mr)约为37000,与预期大小一致,重组蛋白约占蛋白总量的72.6%。Western blotting分析结果表明,重组蛋白能识别抗微小隐孢子虫阳性鼠血清。结论微小隐孢子虫病毒衣壳蛋白S-dsRNA基因表达成功,重组蛋白具有反应原性。

Objective To clone and express S-dsRNA gene of Cryptosporidium parvum virus,and investigate the reactionogenicity of the recombinant.Methods Total RNA was extracted from Cryptosporidium parvum and S-dsRNA gene was amplified by RT-PCR.The PCR product was cloned into pET-28a（＋） expression vector.The recombinant plas-mid pET-28a（＋）-S was transformed into E.coli BL21（DE3） and induced with IPTG.The expression situation of recombinant protein was analyzed by SDS-PAGE.Its reactionogenicity was examined by Western blotting analysis.Results pET-28a（＋）-S was identified by PCR and double endonuclease digestion.SDS-PAGE result showed that the recombinant protein（Mr 37 000） was expressed in the form of inclusion body.High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation at 37 ℃ for 4 h and reached up to 72.6 % of the total protein.The protein was recognized by the antisera from mice immunized with antigens from Cryptosporidium parvum oocysts.Conclusion The S-dsRNA gene of Cryptosporidium parvum virus has been expressed with adequate reactionogenicity.