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目的将恶性疟原虫红细胞膜表面蛋白1(Plasmodium falciparum erythrocyte membrane protein 1,Pf EMP1)N-末端区段(N-terminal segment,NTS)共价偶联到重组铜绿假单胞菌去毒外毒素(recombinant exoprotein A,r EPA)上,构建NTS-r EPA偶联蛋白,以提升NTS的免疫原性。方法用偶联试剂Sulfo-EMCS将马来酰亚胺基团加到r EPA上,利用NTS所携带的1个自由巯基将NTS共价连接到马来酰亚胺化的r EPA上,形成NTS-r EPA偶联蛋白,通过分子排阻层析从偶联反应产物中去除未偶联的蛋白。采用Ellman反应测定r EPA上所携带的马来酰亚胺基团数量以及NTS-r EPA偶联蛋白的偶联比,SDS-PAGE鉴定纯化的偶联蛋白。结果通过偶联试剂Sulfo-EMCS在每摩尔r EPA上加上了4.3摩尔的马来酰亚胺基团;NTS与马来酰亚胺化的r EPA反应生成了NTS-r EPA偶联蛋白,偶联比为4.1;通过分子排阻层析去除了未偶联的蛋白,得到了纯化的偶联蛋白。结论成功构建了NTS-r EPA偶联蛋白,为今后针对NTS的研究奠定了基础。
Objective To covalently couple the N-terminal segment (NTS) of Plasmodium falciparum erythrocyte membrane protein 1 (Pf EMP1) to the recombinant P. aeruginosa detoxified exotoxin recombinant exoprotein A, r EPA) to construct an NTS-r EPA-coupled protein to enhance the immunogenicity of NTS. Methods Maleimide groups were added to r EPA using the coupling reagent Sulfo-EMCS and NTS was covalently attached to maleimidated r EPA using one free thiol carried by NTS to form NTS -r EPA-conjugated protein, uncoupled protein is removed from the coupled reaction product by size exclusion chromatography. The Ellman reaction was used to determine the amount of maleimide groups carried on r EPA and the coupling ratio of NTS-r EPA-conjugated proteins, and the purified coupled protein was identified by SDS-PAGE. As a result, 4.3 moles of maleimide groups were added per mole of r EPA by coupling reagent Sulfo-EMCS; NTS reacted with maleimidated r EPA to produce NTS-r EPA-coupled protein, The coupling ratio was 4.1; the unconjugated protein was removed by size exclusion chromatography to obtain the purified coupling protein. Conclusion The NTS-r EPA-conjugated protein was successfully constructed and laid the foundation for future research on NTS.