鲶鱼抗菌肽ParasinⅠ原核表达载体的构建与鉴定

Construction and Characterization of Prokaryotic Expression Vector for ParasinⅠ

目的构建鲶鱼抗菌肽(ParasinⅠ)原核表达载体。方法根据ParasinⅠ氨基酸序列及大肠埃希菌偏爱密码原则设计并合成ParasinⅠ基因寡核苷酸片段,将其与表达载pGEX-4T-1经限制性核酸内切酶双酶切后连接,转化至大肠杆菌BL21,挑取阳性菌落,抽提重组质粒并鉴定,IPTG诱导表达融合蛋白,亲和层析纯化后SDS-PAGE分析表达和纯化结果,测定抗菌活性。结果重组体插入基因序列测定证实与设计的ParasinⅠ序列一致。表达产物经SDS-PAGE蛋白电泳分析,在相对分子质量28KD处出现融合蛋白条带。抑菌试验表明,融合蛋白分子凝血酶切产物对革兰阳性的金黄色葡萄球菌ATCC25938有抑制作用。结论成功构建了ParasinⅠ成熟肽原核表达载体,为进一步研究其抗菌、抗肿瘤作用奠定了基础。

Objective To construct a prokaryotic expression vector for parasin Ⅰ . Methods Based on the amino acid sequences of parasin Ⅰ and the preference codon of E.coli in codon usage database to design and synthesize parasin Ⅰgene and insert into expression vector pGEX-4T-1. Digest the constructed recombinant plasmid with restriction endonuclease,then transform to E.coli BL21 for expression under induction of IPTG. Purify the expressed fusion protein by affinity chromatography, identify by SDS-PAGE and test for activty by bacteriostasis test. Results The amplified gene fragment was proved as parasin Ⅰ by sequencing. SDS-PAGE showed that the relative molecular mass of expressed fusion protein was 28KD, which was identical to that in theory. The fusion protein digested with thrombin inhibited the growth of S.aureus ATCC25938. Conclusion The prokaryotic expression vector for parasin Ⅰ was successfully constructed, which laid a foundation of study on antibacterial and anti-tumor effect of parasin Ⅰ .