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目的进一步研究EB病毒诱导基因3(EBI3)蛋白在病原生物感染宿主中的功能,探讨原核表达小鼠EBI3蛋白并初步鉴定。方法收集日本血吸虫感染的C57/BL6小鼠脾细胞后提取总RNA,利用所合成引物经RT-PCR获得EBI3的cDNA,将该基因亚克隆入原核表达载体pET32a(+),转化至大肠埃希菌(E.coli)BL21株,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,对包涵体进行变性、纯化和复性后获得可溶性的小鼠重组EBI3蛋白,用Western blot对其鉴定。用纯化的重组蛋白免疫BALB/c小鼠获取血清,经饱和硫酸铵法和DEAE-Sephadex A-50柱纯化得到抗小鼠重组EBI3蛋白多克隆抗体,ELISA检测其免疫活性。结果基因工程获得目的蛋白变性复性成功后经Western blot鉴定证实为小鼠重组EBI3蛋白,免疫小鼠后取血清,分离纯化获得抗小鼠重组EBI3蛋白多克隆抗体,ELISA结果表明该蛋白具有免疫原性。结论成功地表达了小鼠重组EBI3蛋白,并制备了抗小鼠重组EBI3蛋白多克隆抗体,为进一步研究EBI3蛋白在病原生物感染宿主中的功能奠定了基础。
Objective To further study the function of EBV3 gene in the pathogen-infected host and to explore the prokaryotic expression of mouse EBI3 protein and preliminary identification. Methods Total RNA was extracted from splenocytes of C57 / BL6 mice infected with Schistosoma japonicum. The cDNA of EBI3 was obtained by RT-PCR using the synthesized primers. The cDNA was subcloned into prokaryotic expression vector pET32a (+) and transformed into E. coli E. coli strain BL21 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the inclusion body was denatured, purified and refolded to obtain soluble mouse recombinant EBI3 protein. Western blot identification. The purified recombinant protein was used to immunize BALB / c mice to obtain the serum. The purified recombinant protein was purified by ammonium sulfate column and DEAE-Sephadex A-50 column to obtain the polyclonal antibody against mouse recombinant EBI3 protein. The immunological activity was detected by ELISA. Results The genetically engineered protein was denatured and renatured successfully. After identification by Western blot, recombinant mouse EBV3 protein was confirmed. After being immunized, the serum was taken and the polyclonal antibody against mouse recombinant EBI3 protein was isolated and purified. ELISA showed that the protein was immunized Original. Conclusion The mouse recombinant EBI3 protein was successfully expressed and the anti-mouse recombinant EBI3 protein polyclonal antibody was prepared, which laid the foundation for further study on the function of EBI3 protein in the pathogen-infected host.