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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。
Objective To recombinantly express and purify human betaine homocysteine methyltransferase (BHMT) protein and prepare the polyclonal antibody against BHMT. Methods Total RNA was extracted from human HepG2 cells by TRizol method. The BHMT gene was obtained by RT-PCR. The recombinant plasmid pET-28a-BHMT was digested with EcoRⅠand BamHⅠ, and induced by 0.5 mmol / L IPTG. And purified to obtain the recombinant protein; the rabbit was immunized with the recombinant BHMT protein to obtain the rabbit polyclonal antibody, and the antibody was purified, and its ability to recognize human natural BHMT protein was analyzed by Western blotting. Results The sequencing proved that the human BHMT gene was identical to the human BHMT gene in Gen-Bank by RT-PCR. The recombinant BHMT prokaryotic expression vector could stably express soluble BHMT protein. After the recombinant BHMT protein was immunized with rabbit serum antibody Purified to obtain high purity BHMT antibody, this antibody can specifically recognize the natural human liver cancer BHMT protein. Conclusion The recombinant BHMT protein can be successfully expressed and purified. The obtained BHMT polyclonal antibody can be used in the detection of human BHMT protein and lay a foundation for studying the mechanism of BHMT in hyperhomocysteinemia.