目的构建家蝇天蚕素(Cecropin)Cec4原核表达质粒,表达及纯化Cec4蛋白。方法从家蝇cDNA文库中筛选家蝇天蚕素基因Cec4,设计并合成特异性引物,PCR扩增Cec4基因,连接克隆载体后转入DH5α感受态细胞,通过重组子鉴定及测序完成目的基因的克隆。用限制性内切酶BamHI和HindⅢ双酶切目的基因和质粒,构建GST表达载体pGEX 4T-1-Cec4,转化到大肠埃希菌BL-21中,利用IPTG诱导表达重组Cec4蛋白,经亲和层析纯化后进行SDS-PAGE电泳分析。结果 PCR扩增获得的Cec4基因全长为192 bp,连接到表达载体pGEX 4T-1获得重组质粒pGEX 4T-1-Cec4,转化大肠埃希菌BL-21后经30℃、0.6 mmol/L的IPTG诱导18 h,表达相对分子质量约为26×10~3上清表达的融合蛋白,亲和层析纯化后的蛋白浓度为0.117 mg/ml。结论成功构建重组质粒pGEX 4T-1-Cec4并表达和纯化获得Cec4蛋白,为其进一步研究奠定了实验基础。
Objective To express and purify the protein Cecropin 4(Cec4) from Musca domestica with a prokaryotic expression vector. Methods Cecropin gene Cec4 was screened from a housefly cDNA library. The Cec4 gene was amplified using PCR with specific primers, ligated into a cloning vector, and then transferred into DH5α competent cells. The target gene was identified using recombinant identification and sequencing. After the target gene and plasmid were double-digested with BamH I and Hind III, the digested Cec4 was ligated with pGEX 4 T-1, and the recombinant plasmid pGEX 4 T-1-Cec4 was transformed into E.coli BL-21. After expression of the plasmid was induced with IPTG, the recombinant Cec4 protein was purified using affinity chromatography and identified using SDS-PAGE. Results A Cec4 fragment 192 bp in length was double-digested and ligated into pGEX 4 T-1, resulting in the recombinant plasmid pGEX 4 T-1-Cec4. E.coli BL-21 harboring the recombinant plasmid was induced to express a protein with 0.6 mmol/L IPTG at 30 ℃, and a 26-ku soluble protein was expressed. The protein concentration after purification with affinity chromatography was 0.117 mg/ml. Conclusion The recombinant plasmid pGEX 4 T-1-Cec4 was successfully constructed and the Cec4 protein was purified. These results have laid the experimental foundation for further research.
Journal of Pathogen Biology
antimicrobial peptide Cec4