细粒棘球绦虫硫氧还蛋白过氧化物酶基因的克隆、表达及鉴定

目的克隆细粒棘球绦虫(Echinococcus granulosus,Eg)硫氧还蛋白过氧化物酶(thioredoxin peroxidase TPx)基因,构建原核表达载体,通过诱导表达,纯化融合蛋白抗原,免疫小鼠制备抗血清。方法从包囊中分离原头蚴,抽提总RNA,采用RT-PCR的方法扩增EgTPx基因,克隆到原核表达载体pET-41b中,转化大肠杆菌BL21,经过IPTG诱导目的基因表达,SDS-PAGE对表达蛋白进行分析,通过谷胱甘肽Sepharose4B层析柱分离纯化获得重组蛋白并免疫小鼠,制备抗血清。Western blot鉴定抗血清的特异性,ELISA测定抗体的效价。结果用PCR方法扩增获得的EgTPx基因长度为582bp,经酶切鉴定和序列测定,插入到原核表达载体的基因片段为EgTPx基因,SDS-PAGE结果显示表达融合蛋白相对分子质量约为54kDa,鼠抗EgTPx抗体能与融合蛋白和天然原头蚴抗原发生特异性反应;间接ELISA法测定该抗体效价为1∶256000。结论成功地制备了鼠抗EgTPx抗体,并能够特异识别原核表达的融合蛋白EgTPx和天然原头蚴抗原,为研制有效的包虫病疫苗及相关诊断试剂盒奠定了基础。

Cloning, fusion expression and identification of gene encoding the thioredoxin peroxidase from Echinococcus granulosus

The total RNA was extract from hydatid cyst protoscoleces(PSC)and was used as template.A cDNA sequence(EgTPx)of the thioredoxin peroxidase gene(TPx)from PSC of Echinococcus granulosa(Eg)was cloned into the expression vector pET-41b by RT-PCR,and the positive recombinants were transformed to E.coli BL21,and then identified by restriction enzyme digestion.Finally,the genetic engineered bacteria pET-41b-EgTPx were induced with IPTG and the expressed products were analyzed by SDS-PAGE.The soluble expressed fusion protein was purified through glutathione sepharose 4B affinity column.Meanwhile,Western blot assay was used to determine the specificity of antiserum and ELISA was applied to detect the titers of antiserum.It was demonstrated that the length of the EgTPx gene amplified with PCR was 582 bps and the gene fragment inserted into the prokaryotic expression vector was proved to be the EgTPx gene after enzyme digestion and sequencing.As demonstrated by SDS-PAGE,the relative molecular mass(Mr)of the expressed fusion protein was approximately 54 000.The specific mouse antiserum against the purified EgTPx protein could bind to the fusion protein and natural protoscoleces antigen with a binding titer of 1∶256 000 in ELISA assay.It is evident the EgTPX fusion protein with satisfactory immunoreactivity and antigenicity has been obtained through genetic engineering technique and it can be used as an antigen in the development of the related vaccine as well as preparation of the laboratory diagnostic kits.