3种截短的泡球蚴Em18基因原核表达质粒的构建、表达及鉴定

目的构建3种截短的泡球蚴18(Em18)基因的原核表达质粒,获得高效表达、有生物活性的3种重组蛋白,并对其进行初步鉴定。方法DNAman软件设计引物,PCR法扩增并构建3种截短的pET41a-Em18.1、Em18.2和Em18.3原核表达质粒,测序鉴定插入序列正确性;IPTG诱导、表达和纯化rEm18.1-GST、rEm18.2-GST和rEm18.3-GST重组蛋白,SDS-PAGE电泳及Western blot进行初步鉴定。结果成功构建了3种截短的pET41a-Em18.1、Em18.2和Em18.3重组表达质粒;SDS-PAGE检测表明rEm18.1-GST、rEm18.2-GST、rEm18.3-GST重组蛋白得到成功表达,在分子质量单位为41、45.5和45.5ku处有表达条带;Western blot显示3种重组蛋白均能被AE病人血清识别。结论成功构建了截短的pET41a-Em18.1、pET41a-Em18.2和pET41a-Em18.3原核表达质粒,表达的3种重组蛋白均具有良好的抗原性,为Em18抗原表位分析奠定了基础。

Construction, expression and identification of three truncated pET41a-Em18 prokaryotic plasmid

Objective To clone, construct and express three truncated pET41a-Em18 recombinant plasmids and to study their antigenicity. Methods The primers of three truncated Em18 were designed by DNAman biosoftware and these truncated fragments were amplified by PCR from pMD18-T/Em18, and were cloned into prokaryotic expression plasmid pET41a to construct the pET41a-Em18.1, pET41a-Em18.2, pET41a-Em18.3, respectively.The recombinant plasmids were analyzed by sequencing. The rEm18.1, rEm18.2, rEm18.3, -GST fusion proteins were expressed by induction with IPTG and purified using His-tag column, and were detected by SDS-PAGE and Western blot. Results The pET41a-Em18.1, pET41a-Em18.2, pET41a-Em18.3 positive clones were the exact recombinant plasmids and the expressed rEm18.1, rEm18.2, rEm18.3, -GST recombinant proteins can be detected as a band of 41 ku, 45.5 ku and 45.5 ku by SDS-PAGE and the results of Western blot confirmed that these three recombinant proteins could specifically react with the serum samples from patients with alveolar echinococcosis(AE), respectively. Conclusion Three truncated pET41a-Em18.1, pET41a-Em18.2, pET41a-Em18.3 are successfully constructed and the rEm18.1-GST, rEm18.2-GST, rEm18.3-GST recombinant proteins are expressed and show a good antigenicity. This work settles a foundation for epitope analysis of Em18 antigen.