西农萨能羊MAT基因的原核表达及多克隆抗体制备

Prokaryotic expression and polyclonal antibody preparation of malonyl/acetyl transacylase gene of Xinong Saanen Goat

【目的】制备高特异性、高效价的乙酰/丙二酸单酰转移酶(MAT)多克隆抗体,为研究山羊MAT在脂肪酸代谢中的功能奠定基础。【方法】采用PCR方法扩增奶山羊的MAT基因,将其亚克隆到pET32a(+)载体得到pET32a(+)-MAT原核表达载体,并转化到大肠杆菌BL21(DE3)中诱导表达。使用Ni-NTA琼脂糖亲和层析柱纯化融合蛋白,纯化的MAT蛋白经复性后进行活性检测。采用皮下免疫法将纯化的MAT蛋白免疫兔子制备多克隆抗体,用ELISA和Western blot检测血清多克隆抗体的效价和特异性。【结果】PCR扩增得到981bp的MAT基因编码区;成功构建了pET32a(+)-MAT原核表达载体,诱导表达获得了56ku的重组蛋白。活性检测表明,复性后的MAT具有转移酶活性,ELISA检测显示抗体效价达1∶128 000,经Western blot鉴定,制备的多克隆抗体能特异性检测原核表达的MAT蛋白以及在HEK-293细胞中表达的MAT蛋白。【结论】获得了具有酶活性的MAT蛋白以及高特异性、高效价的兔抗MAT多克隆抗体,为研究MAT在脂肪酸代谢中的功能提供了重要的试验工具。

【Objective】This study prepared polyclonal antibody with high affinity and specificity to improve the study of biological function of malonyl/acetyl transacylase gene(MAT)in fatty acid metabolism.【Method】The goat MATgene was cloned by PCR before being ligated with pET32a(+)to construct prokaryotic expression vector pET32a(+)-MAT.pET32a(+)-MAT was then transformed into E.coli BL21(DE3)competent cells for induction expression.Recombinant protein was purified by Ni-NTA column and used as antigen to immune rabbit to prepare polyclonal antibody.Titer of the polyclonal antibody and specificity were analyzed using ELISA and Western bolt at last.【Result】The coding sequence of MAT gene with length of 981 bp was cloned by PCR and the prokaryotic expression vector pET32a(+)-MAT was constructed successfully.The recombinant protein with the length of 56 ku was obtained.The enzymatic activity assay of the purified recombinant protein showed that the refolded protein retained transferase activity.ELISA analysis showed that the titer of the obtained antibody was 1∶128 000.Western blot showed that the antibody could specifically combine with the MAT protein expressed in both prokaryotic cell andHEK-293 cell.【Conclusion】The recombinant MAT protein with transferase activity and the MAT polyclonal antibody with high affinity and specificity was generated successfully,which would improve the study of biological function of MAT in fatty acid metabolism.