重组人脑髓鞘碱性蛋白及其抗体的制备与研究

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。

Recombinant human brain myelin basic protein and its antibody preparation

BACKGROUND: Central nervous system (CNS) myelin is made up of 70% lipids and 30% proteins with human brain myelin basic protein (hMBP) constituting 1/3 of the proteins. MBP is a family of proteinswith four isoforms only in human brain. OBJECTIVE: To investigate the expression of MBP and its immunological function. DESIGN: Single sample study. SETTING: Department of Biochemistry and Molecule Biology, Huaxi College of Priclinical Medicine and Forensic Medicine, Sichuan University. MATERIALS: This experiment was conducted at Department of Bio chemistry and Molecule Biology, Huaxi College of Priclinical Medicine and Forensic Medicine, Sichuan University from August 2003 to March 2004. Relative molecular weight was 215000 hMBP cDNA clone pGEMP,prokaryotic expression recombinant vector pGEX-5T, T4 DNA Ligase,calf intestine alkaline phosphates, X-gal, IPTG, 123 Ladder (BRL), nitrocellu lose ,4-chloro-1-Naphthol;MBP and Anti- MBP ELISA kit. INTERVENTIONS: ①hBMP gene cDNA clone segment was digested with EcoR1 and BamH1; ②Construction and transformation of the recombinant expression vector p5TMP; ③ The growth and induced expression of the recombinant expression vector transformed bacteria; ④ Preparation of the antibody of recombinant hMBP. MAIN OUTCOME MEASURES: ① Detection and identification of the expressed protein product; ② Detection and identification of hMBP anti body. RESULTS: ① Screening and identification of recombinant MBP: The 4 999 bp pGEX-5T DNA and 557 bp MBP inserted fragment were obtained, indicating that the white colonies contained recombinant expression plasmid of exogenous DNA; ② A dense band disappeared from the control plasmid while a new special polypeptide band with apparent molecular weight 42 000 was detected in recombinant cell lysate; ③After 5 subcutaneous injections, antibody was obtained at 1:16 titer. The specificity of the antibody to MBP was confirmed.CONCLUSION: Compared with the original expression vector, the new constructed expression vector containing 215 000 MBP exons Ⅰ-Ⅶ coding sequence was not only without the coding area defects of 34 bp in 5' sequence but also expressed more highly in prokaryote obviously. In addition, the recombinant MBP antibody prepared successfully.