氯霉素乙酰基转移酶双抗体夹心ELISA检测方法的建立

Establishment of a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase gene

目的 建立氯霉素乙酰基转移酶 (chloramphenicolacetyltransferase ,CAT)双抗体夹心ELISA的实验室检测方法。方法 从质粒pBLCAT6经PCR扩增CAT基因序列 ,插入到原核表达质粒pGEX 2T中 ,在大肠埃希菌DH5α中诱导表达 ;以纯化的融合蛋白GST CAT为抗原免疫实验用兔 ,得到的CAT抗血清进行生物素标记 ,并在此基础上利用生物素链和亲和素放大系统建立双抗体夹心ELISA法。构建不同YY1结合位点突变的HPV16LCRCAT报道质粒 ,体外瞬时转染真核细胞 ,提取胞质蛋白 ,以建立的方法进行CAT表达检测。结果 SDS PAGE显示表达的GST CAT融合蛋白相对分子质量约为 5 40 0 0 ;制备的CAT抗血清可有效地识别原核及哺乳动物细胞表达的CAT蛋白 ;在不同启动子及调节序列的控制下 ,利用建立的CAT检测技术证明在瞬时转染的细胞中可诱导 2～ 8倍的CAT表达增强。结论 建立的双抗体夹心ELISA方法可敏感地反映上游序列的启动子活性 ,以及启动子调节序列变化对启动子活性的影响 。

Objective To establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene.Methods The full-length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template,and inserted into the prokaryotic-expression plasmid pGEX-2T. The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits. The antibody was labeled with biotin,and a sandwich ELISA technique with biotin-streptavidin amplify system was established. Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro. The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay.Results SDS-PAGE displayed that the molecular-weight of the expressed fusion protein was about 54 000. The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells. Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method. Conclusion The established method could sensitively reflect the activities of the upstream promoters,as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities. Therefore,it proposes a convenient assay for the studies using CAT as the reporter gene.