军事医学2011，Vol.35Issue(1)：35-38,4.
人甜菜碱高半胱氨酸甲基转移酶(BHMT)表达及抗体制备
Expression and purification of human betaine-homocysteine methyltransferase (BHMT)and its antibody preparation and identification
摘要
Abstract
Objectives To prepare human BHMT protein and explore the characterization of its polyclonal antibody. Methods Total RNA was extracted from HepG2 cells by TRIzol protocol. The full-length gene of BHMT was generated by RT-PCR and spliced by EcoR I and BamH I. The recombinant expression vector pET-28a-BHMT was induced with 0. 5 mmol/L IPTG, and protein production was purified by Ni2+ affinity chromatography and to immune rabbits. Rabbits were immunized with recombinant protein HIS-BHMT, and polyclonal antibody was purified by abilitytechnique. This PcAb characterization was identified by Western blotting. Results The BHMT full-length gene confirmed by sequence analysis matched human gene BHMT in GenBank exactly. The reconstructed vector could express target protein stably in the soluble fraction of bacterial extract. The antibody became very pure following two-step purifications and could specifically react with nature BHMT protein of human liver carcinoma. Conclusion Recombinant human BHMT is successfully expressed and purified. The polyelonal antibody of BHMT could be used to detect human(BHMT). This study helps clarify the function of BHMT in high homocysteinemia.关键词
甜菜碱高半胱氨酸甲基转移酶/克隆/表达/抗体纯化/免疫印迹分类
医药卫生引用本文复制引用
冷雪,钱令嘉,王剑波,杲修杰,弓景波,赵云,武磊,战锐,王新兴,刘晓华..人甜菜碱高半胱氨酸甲基转移酶(BHMT)表达及抗体制备[J].军事医学,2011,35(1):35-38,4.基金项目
国家科技重大专项课题(2009ZX09103-629),国家自然科学基金项目(30800456) （2009ZX09103-629）
