胞嘧啶脱氨酶的原核表达和多克隆抗体的制备-东南大学学报医学版

胞嘧啶脱氨酶的原核表达和多克隆抗体的制备

单位：1.东南大学基础医学院,微生物与免疫学系,江苏,南京,210009； 2.南京大学,生理学系,江苏,南京,210093； 3.第二军医大学南京军医学院,分子医学研究所,江苏,南京,210099

分类号：R392.11

出版年·卷·期（页码）：2002·21·第三期（224-226）

摘要：

目的:在原核系统中表达并纯化大肠杆菌胞嘧啶脱氨酶(cytosine deaminase,CD),制备鼠抗大肠杆菌CD多克隆抗体.方法:亚克隆CD基因到原核表达载体pMAL-c2和pBV222中,并转化入大肠杆菌DH5α内,诱导表达并纯化MBP-CD和6his-CD融合蛋白,用纯化的MBP-CD融合蛋白免疫小鼠制备多克隆抗体.结果:通过重组质粒的酶切筛选出重组阳性克隆,成功地表达和纯化出MBP-CD和6his-CD融合蛋白,用纯化的MBP-CD成功制备了鼠抗CD多克隆抗体,并用6his-CD和GST-CD重组蛋白进行Western印迹分析,证实了抗体的正确性.结论:应用多克隆抗体可以检测体内外CD基因的表达,为临床前和临床上深入开展CD基因的生物治疗研究提供重要的实验材料.

Objective To express and purify the recombinant protein of cytosine deaminase(CD) in prokaryotic system, preparing the mouse anti E.coli CD polyclonal antibody.Methods CD gene was subcloned into vector pMAL?c2 and pBV222,and transformed into the E.coli DH 5α .MBP?CD and 6his?CD recombinant protein was expressed and purified,MBP?CD recombinant was used to immune the mouse for preparing polyclonal antibody.Result The recombinant clones were picked out by the methods of restrictional enzyme cut analysis.MBP?CD and 6his?CD recombinant protein was successfully purified.The polyclonal antibody was also successfully prepared by MBP?CD recombinant protein and was proved by the methods of Western blot analysis of 6his?CD and GST?CD recombinant protein.Conclusion With the polyclonal antibody,CD expression can now be monitored throughout in vitro and in vivo gene transfer studies with the polyclonal antibody.The polyclonal antibody can be used in further biological gene therapy of CD gene.

参考文献：

[1] Austin E A, HUBER B E. A first step in the development of gene therapy for colorectal carcinoma:cloning sequencing and expression of escherichia coli cytosine deaminase, 1993
[2] Polak A, SCHOLER H J. Mode of action of 5-fluorocytosine and mechanisms of resistance. 1975. doi:10.1159/000221854
[3] Anderson L, KILSTRUP M, NUHARD J. Pyrimidine purine and nitrogen control of cytosine deaminase synthesis in Eschericina coli K12.Involvement of the glnLG and purr genes in the regulation of codA expression. 1989. doi:10.1007/BF00456087
[4] Haack K, MOEBIUS U, KNEBEL M V. Detection of cytosine deaminase in genetically mosified tumor cells by specific antibodies. 1997(8). doi:10.1089/hum.1997.8.11-1395
[5] Daniel A, HAMSTERA, David J. Enzyme/Prodrog therapy for head and neck cancer using a catalytically superior cytosine deaminase, 1999(10)
[6] Senter P D, SU P C D, KATSURAGI T. Generation of 5-fluorouraicil from 5-fluorocytosine by monoclonal antibody cytosine deaminase conjugates. 1991. doi:10.1021/bc00012a012

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