摘要
目的设计合成蛔虫抗菌肽基因序列,并在大肠埃希菌BL21(DE3)中表达,以获得重组蛋白。方法根据大肠埃希菌对编码同种氨基酸不同密码子的偏嗜性,对蛔虫抗菌肽Cecropin P1编码基因进行改造,人工合成目的基因,克隆到原核载体PMD-18T,并转化DH5α感受态菌株;构建重组质粒PET-30a-Cecropin P1,并转化BL21感受态菌株;用双酶切鉴定阳性克隆;含有正确重组质粒的阳性菌株经IPTG诱导表达,SDS-PAGE电泳鉴定表达产物。结果PCR扩增及构建的两个重组质粒双酶切鉴定,均获得113bp的基因片段,与预期大小一致;SDS-PAGE电泳分析显示,含有重组质粒PET-30a-Cecropin P1的阳性菌株在IPTG诱导下高效表达了分子质量单位为3.4ku的蛋白质。结论成功构建了重组质粒PET-30a-Cecropin P1,并在大肠埃希菌BL21(DE3)中以包涵体形式高效表达,为进一步研究蛔虫抗菌肽的生物学活性奠定了基础。
Objective The complete gene sequence of Asearis antibacterial peptide was cloned, acquired and expressed in Escherichia coli BL21 （DE3） in order to obtain recombinant protein. Methods The obtained gene （Cecropin P1） sequence of the antibacterial peptide from Ascaris was modified according to E. coli preferred codons, then subeloned into the prokaryotic expression vector pET30a（-k ） and expressed in E. coli BL21 （DE3） after the induction of IPTG. The expressed product was analyzed by SDS-PAGE. Results All products of PCR and the double restricted enzyme digestion of the two reconstructed plasmids were 113 bp in length, which was consistent with what was expected. The analysis of SDS-PAGE indicated that the reconstructed plasmid pET30a（ ＋ ）-Cecropin P1 could express positively the protein with molecular weight of 3.4 ku after the induction of IPTG. Conclusion The gene plasmid pET30a（ ＋ ）-Cecropin P1 was correctly constructed and the Cecropin P1 was expressed effectively in E. coli in the form of inclusion bodies, which has significance in investigating the function of the Cecropin P1 further.
出处
《中国病原生物学杂志》
CSCD
2009年第7期532-534,551,共4页
Journal of Pathogen Biology
