氯霉素抗性融合蛋白报告系统的建立与验证

Development and Identification of a Chloramphenicol Acetyltransferase (CAT)-fusion Protein Reporting System

利用氯霉素乙酰基转移酶(CAT)N端融合了可溶性蛋白细胞所表现出的氯霉素抗性高于不溶性蛋白这一特点,建立了一种能在大肠杆菌中方便地检测到目的蛋白可溶性的报告系统。以pMalp2X高效表达载体为基础,采用PCR法扩增CAT基因,并通过引物向CAT中引入所需的多克隆位点、终止密码子及linker,将扩增得到的CAT片段插入到pMalp2X中,构建pCAR报道质粒并作测序鉴定;PCR分别扩增IGF1、IL3、Trx基因,连接到pCAR载体上,导入大肠杆菌原核表达系统中进行诱导表达,SDSPAGE分析、氯霉素抗性试验对表达目的蛋白的可溶性和表现出的氯霉素抗性的相关性加以验证。结果显示用该载体表达的重组蛋白的可溶性与氯霉素抗性具有很强的相关性。本报告系统能通过直观的平板氯霉素抗性高低正确指示位于CATN端的目的蛋白的可溶性,为其在生物工程和蛋白质构象相关疾病研究等领域的应用打下了基础。

Based on the fact that cells expressing fusions of an soluble protein with chloramphenicol aeetyhransferase （CAT） exhibit increased resistance to ehloramphenieol compared to fusions with insoluble proteins, a reporting system was developed for reporting easily the solubility of target protein in vivo in E. coli. The reporting system was constructed on the base of an efficient expression plasimd named pMal-p2X. Besides, CAT gene was isolated by PCR, during which multiple cloning site, stop eodon and linker were introduced into the CAT fragment by the primers and confirmed by DNA sequencing. The gene was then inserted into the expression plasmid pMal-p2X as a reporter and the recombinant reporting plasmid was named pCAR after identified by sequencing. IGF, IL-3 and Trx genes were amplified by PCR respectively and fused to the Nterminus of CAT in the pCAR vector to test the ability of this CAT reporting system. Recombinant plasmids were transformed into and induced to express in E. coll. SDS-PAGE and ehloramphenieol resistance test were performed to verify this system. Results showed that the solubility of the target proteins correlates well with the ehloramphenieol resistance produced by the fusions. This indicated that the CAT-fusion protein reporting system could be used to report correctly the solubility of the target protein fused to its N-terminus by simply measuring the ehloramphenieol resistance, and it is promising to be applied to bioengineering and protein-folding disease area.