论文部分内容阅读
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 To design a synthetic Ascaris antibacterial peptide gene sequence and express it in Escherichia coli BL21 (DE3) to obtain the recombinant protein. Methods According to the preference of Escherichia coli for coding different codons of the same amino acid, the gene encoding Cecropin P1 was transformed. The target gene was cloned into prokaryotic vector PMD-18T and transformed into DH5α competent cells. The recombinant plasmid PET-30a-Cecropin P1 was transformed into competent cells of BL21. Positive clones were identified by double enzyme digestion. Positive strains containing the correct recombinant plasmids were induced by IPTG, and the expressed products were identified by SDS-PAGE electrophoresis. Results The two recombinant plasmids were amplified by PCR and identified by restriction enzyme digestion. All the fragments were 113 bp in length, which was consistent with the expected size. SDS-PAGE analysis showed that the recombinant plasmid PET-30a-Cecropin P1 was induced by IPTG Highly expressed under the molecular mass unit of 3.4ku protein. Conclusion The recombinant plasmid PET-30a-Cecropin P1 was successfully constructed and expressed in E. coli BL21 (DE3) as inclusion bodies, which laid the foundation for further study on the biological activity of Ascaris antimicrobial peptides.