摘要
根据GenBank上旋毛虫Kazal型丝氨酸蛋白酶抑制剂(KaSPI)基因编码序列设计特异性引物,进行RT-PCR扩增,将所获PCR产物克隆至pEASY-T1载体后转入克隆感受态Tans5α,经过PCR和EcoRⅠ、XhoⅠ双酶切鉴定后进行测序。将所获目的基因与载体pET-30a(+)相连接,然后将鉴定正确的重组表达质粒pET-TsKaSPI转化到大肠杆菌BL21(DE3)中,利用IPTG诱导蛋白表达。SDS-PAGE电泳分析所得融合蛋白大小约为38 000,与预测蛋白理论值大小相符,主要以包涵体形式存在。对纯化后的重组蛋白进行Western blot鉴定,结果显示该蛋白可以被感染旋毛虫小鼠阳性血清所识别,具有良好的抗原性。将纯化的重组蛋白经腹腔注射到小鼠体内检测其对小鼠的免疫保护性,结果表明,旋毛虫肌幼虫减虫率为38.3%。通过间接ELISA法检测血清抗体水平,结果显示重组蛋白免疫小鼠血清的抗重组蛋白抗体IgG总体水平显著高于感染对照组和佐剂组。
Based on GenBank about Trichinella spiralis Kazal-type serine protease inhibitor（KaSPI）gene coding sequence,specific primers were designed,performed RT-PCR amplification,the product obtained was cloned into the vector pEASY-T1,then transformed into competent Tans5α,identified and sequenced after PCR and EcoRⅠ,XhoⅠ double digestion.The target gene was connected to expression vector pET-30a（＋）,then the correct recombinant was plasmid pET-TsKaSPI was transformed into E.coli BL21（DE3）and protein expression under inducted with IPTG.SDSPAGE analysis showed the fusion protein was about 38 000,consistent with predicted protein size,mainly in the form of inclusion bodies.The purified recombinant proteins were identified by Western blot.The results showed that the protein can be recognized by sera of mice infected with T.spiralis,with good antigenic.The purified recombinant protein was injected into mice to detect the protective immunity T.spiralis muscle larvae reduction rate was 38.3%.By indirect ELISA,recombinant protein-immunized mouse serum IgG antibodies against recombinant protein was significantly higher than that of infection control and adjuvant group.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2015年第8期1284-1289,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31372427)
教育部高等学校博士学科点专项科研基金资助项目(20132325110002)
