应用肌肉介导基因转移制备5,10亚甲基四氢叶酸还原酶(MTHFR)抗体

５，１０亚甲基四氢叶酸还原酶（ＭＴＨＦＲ）是叶酸代谢的关键酶．为了试验肌肉介导外源基因人ＭＴＨＦＲ（ｈＭＴＨＦＲ）制备抗体和建立ＭＴＨＦＲ免疫检测的可能性，构建了ＭＴＨＦＲ基因真核表达载体（ｐｃＤＮＡ３／ＭＴＨＦＲ）；通过基因缝线法将携带ｐｃＤＮＡ３／ＭＴＨＦＲ的质粒，缝合于预先注射再生剂（丁哌卡因）的肌肉内．２个月后分离血清，所得抗体应用Ｗｅｓｔｅｒｎｂｌｏｔ，ＥＬＩＳＡ和胎肝免疫组织化学染色进行免疫鉴定．胎肝免疫组织化学显示，在肝小梁细胞浆中具有大量ＭＴＨＦＲ阳性反应颗粒；Ｗｅｓｔｅｒｎｂｌｏｔ有ＭＴＨＦＲ抗体与其抗原特异的褐色条带，分子量约为３７ｋＤ；ＥＬＩＳＡ分析表明，３种不同浓度的抗体与不同剂量的抗原反应具有剂效关系，最适抗体滴度（ＥＤ５０）为１∶４００。以上结果说明肌肉介导外源基因是获得抗体的一种简单、快捷的方法．该抗体可用于ＭＴＨＦＲ的免疫检测和有关的叶酸代谢研究工作．

The Production of Antibodies Against 5,10 Methylene Tetrahydro folate Reductase(MTHFR)by Muscle-mediated Gene Transfer

The 5,10 methylene tetrahydrofolate reductase(MTHFR) is a key enzyme in the metabolism of folic acid.To investigate the feasibility of getting the antibody against human MTHFR(hMTHFR)through skeletal muscle mediated hMTHFR gene transfer and the possibility of immune detection of MTHFR with the antibody ,the eukaryotic expression vector of hMTHFR gene (pcDNA3/MTHFR)was contructed.Gene transfer was performed by gene suture method three days after intramuscular injection of regenerative agent (bupivacaine).The antibody against hMTHFR was separated from the serum two month later and identified with Western blot,ELISA and immunohistochemistry of foetus liver cells.There were a great quantity of positive grains in cytoplasm of foetus liver cells with immunohistochemistry.And there was a positive band specially binding hMTHFR which had been produced by genetic engineering with Western blot method.The molecular weight of the antibody was around 37 kD;The ELISA assay showed that the effects of three concentrations of antibodies with antigen had relation with dosage and the approprite titer (ED50) was 1∶400.The study shows that the skeletal muscle mediated gene transfer is a simple and rapid means for production of special antibody which can be used for immune detection and the basic research on metabolism of folic acid.