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目的构建细粒棘球蚴(Echinococcus granulosus,Eg)转化生长因子βⅡ型受体胞外域(Extracellular Domain of Transforming Growth Factor beta type Ⅱ receptor of Eg,EgTβRⅡ-E)原核表达载体,诱导表达融合蛋白并制备其特异性抗体。方法采集羊源Eg原头蚴,Trizol法提取总RNA,RT-PCR扩增EgTβRⅡ-E基因,构建pET28a-EgTβRⅡ-E原核表达载体,PCR、限制性内切酶双酶切和测序鉴定正确的阳性质粒转入BL21E.coli感受态细胞,IPTG诱导EgTβRⅡ-E融合蛋白表达并免疫小鼠,制备抗血清。结果成功构建pET28a-EgTβRⅡ-E原核表达载体,经0.6mmol/L IPTG诱导表达18ku的EgTβRⅡ-E融合蛋白。用融合蛋白免疫小鼠,获得高效价(1∶256 000抗血清)。结论制备的EgTβRⅡ-E融合蛋白具有抗原性,免疫小鼠获得高效价的抗血清,为研究EgTβRⅡ的生物学功能奠定了基础。
Objective To construct a prokaryotic expression vector for Echinococcus granulosus (Eg) transforming growth factor beta type Ⅱ receptor of Eg (EgTβRⅡ-E) and induce the expression of the fusion protein Its specific antibodies. Methods Echinococcus granulosus was collected by sheep source and total RNA was extracted by Trizol method. The EgTβRⅡ-E gene was amplified by RT-PCR, and the prokaryotic expression vector pET28a-EgTβRⅡ-E was constructed. PCR, restriction enzyme digestion and sequencing were used to identify the correct Positive plasmids were transformed into BL21E.coli competent cells, IPTG induced EgTβRⅡ-E fusion protein expression and immunized mice to prepare antiserum. Results The prokaryotic expression vector pET28a-EgTβRⅡ-E was successfully constructed and 18ku EgTβRⅡ-E fusion protein was induced by 0.6mmol / L IPTG. Mice were immunized with the fusion protein to obtain high titers (1: 256,000 antisera). Conclusion The prepared EgTβRⅡ-E fusion protein has antigenicity, and the immunized mice obtain high titer antiserum, which lays the foundation for studying the biological function of EgTβRⅡ.