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

目的重组表达及纯化人甜菜碱高半胱氨酸甲基转移酶(betaine-homocysteine methyltransferase,BHMT)蛋白,制备BHMT多克隆抗体并对其性质进行研究。方法 TRizol法提取人HepG2细胞总RNA,RT-PCR获得BHMT基因,经EcoRⅠ和BamHⅠ双酶切,构建重组表达载体pET-28a-BHMT,经0.5 mmol/L IPTG诱导表达,表达产物通过Ni柱亲和纯化获得重组蛋白;以重组BHMT蛋白免疫兔,获得兔多克隆抗体,并对抗体进行纯化,应用Western印迹方法分析其识别人天然BHMT蛋白的能力。结果经测序证明,RT-PCR得到的人BHMT基因与Gen-Bank中人的BHMT基因完全一致;构建的BHMT原核表达载体可稳定地表达可溶性人BHMT蛋白;重组BHMT蛋白免疫兔后,兔血清抗体纯化获得高纯度BHMT抗体,此抗体可以特异性地识别人肝癌组织中的天然BHMT蛋白。结论成功重组表达并纯化人BHMT蛋白,所获得的BHMT多克隆抗体可以应用于人BHMT蛋白的检测,为研究BHMT在高同型半胱氨酸血症中的作用机制奠定基础。

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 EcoRⅠand BamHⅠ.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 ability technique.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 polyclonal antibody of BHMT could be used to detect human（BHMT）.This study helps clarify the function of BHMT in high homocysteinemia.