论文部分内容阅读
背景:中枢神经系统髓鞘中蛋白部分的1/3为髓鞘碱性蛋白。作为一个蛋白质家族在人脑中有4种分子形式。目的:探讨重组人脑脊髓碱性蛋白表达及其免疫学的功能。设计:单一样本研究。单位:四川大学华西基础医学与法医学院生物化学与分子生物学教研室。材料:实验于2003-08/2004-03在四川大学华西基础医学与法医学院生物化学与分子生物学教研室完成。人脑hMBP相对分子质量为21500,T4DNALigase,小牛肠碱性磷酸酶,RNase,PEG8000等,硝酸纤维滤膜,抗人脑髓鞘碱性蛋白ELISA试剂盒。干预:①采用EcoR1和BamH1酶切人脑髓鞘碱性蛋白基因cDNA克隆片段。②重组表达载体p5TMP的构建及转化。③重组表达载体转化菌的生长及其诱导表达。④重组hMBP抗体制备主要观察指标:①蛋白表达产物的检测与鉴定。②人脑hMBP抗体检测与鉴定。结果:①分别有4999bppGEX-5TDNA和557bp人脑髓鞘碱性蛋白插入片段。②原对照质粒一条浓区带消失,而重组体有特异蛋白质区带出现。相对分子量为42000。③5次注射人脑髓鞘碱性蛋白后抗体效价达到1/16。并证实其hMBP抗原特异性。结论:本文构建了含相对分子质量为21500人脑髓鞘碱性蛋白外显子Ⅰ-Ⅶ编码序列的表达载体,还成功制备了重组人脑髓鞘碱性蛋白抗体。
Background: One-third of the protein portion of the central nervous system myelin is myelin basic protein. As a family of proteins, there are 4 molecular forms in the human brain. Objective: To investigate the expression of recombinant human brain spinal cord alkaline phosphatase and its immunological function. Design: Single Sample Study. Unit: Department of Biochemistry and Molecular Biology, West China School of Fundamental Medicine and Forensic Medicine, Sichuan University. MATERIALS: The experiment was performed at the Department of Biochemistry and Molecular Biology, West China School of Fundamental Medicine and Forensic Medicine, Sichuan University from August 2003 to March 2004. Human brain hMBP relative molecular mass of 21500, T4DNALigase, calf intestinal alkaline phosphatase, RNase, PEG8000, nitric oxide membrane, anti-human brain myelin alkaline protein ELISA kit. Intervention: (1) EcoR1 and BamH1 enzyme digestion of human myelin basic protein gene cDNA clone fragment. Construction and transformation of recombinant expression vector p5TMP. ③ recombinant expression vector transformed bacteria growth and induced expression. ④ recombinant hMBP antibody preparation of the main observation indicators: ① protein expression product detection and identification. ② human brain hMBP antibody detection and identification. Results: ① There were 4999bppGEX-5T DNA and 557bp human MBP insert, respectively. ② a control zone of the original plasmid disappears, and recombinants with specific protein bands appear. The relative molecular weight of 42,000. ③ 5 times injected human brain myelin basic protein antibody titer reached 1/16. And confirmed its hMBP antigen specificity. CONCLUSION: In this paper, we constructed an expression vector containing 21500 human MBP exon Ⅰ-Ⅶ coding sequence and successfully prepared recombinant human MBP antibody.