摘要
目的构建Beclin1真核表达载体,检测该基因在真核细胞内的表达,为进一步研究其在弓形虫感染致病中的作用打下基础。方法 PCR扩增目的基因Beclin1后插入到真核表达载体pEGFP中,双酶切鉴定并进行序列测定;通过脂质体介导的方法用重组质粒转染293T细胞,再以Western blot方法检测该基因编码蛋白在真核细胞内的表达。结果 PCR扩增出目的基因Beclin1片段,大小为1 353bp,与预期值一致。构建了pEGFP-Beclin1真核表达载体,经双酶切鉴定插入片段大小正确;DNA测序后与GenBank中的Beclin1基因序列比对,同源性为100%;Western blot检测该蛋白在真核细胞内表达。结论成功构建了pEGFP-Beclin1真核表达载体,该基因编码蛋白可以在真核细胞内表达,为进一步研究该基因在弓形虫感染致病中的作用打下了基础。
Objectives To construct a eukaryotic expression vector for Beclin 1 and to detect its expression in eukaryotic cells for further research into its role in the pathogenesis of toxoplasmosis. Methods PCR was used to amplify the Bec- lin 1 gene, and the target gene was then inserted into the eukaryotic expression vector pEGFP. The recombinant vector was verified with double digestion and sequencing. The recombinant plasmid pEGFP-Beclinl was transfected into 293T cells and then Western blotting was used to detect the expression of the protein that the gene coded for in eukaryotic cells. Results Amplification with PCR yielded a Beclin 1 gene fragment 1,353 bp in length, which was consistent with ex- pectations. The eukaryotic expression vector pEGFP-Beclinl was constructed, and double digestion and identification in dicated that a fragment of the correct size was inserted. The sequence yielded by DNA sequencing was compared to the se quence registered in GenBank to determine their similarity, which was 100%. Expression of Beclin 1 protein was success fully detected in eukaryotic cells with Western blotting. Conclusion Findings suggested that the eukaryotic expression vector pEGFP Beclinl was successfully constructed and that the protein would be expressed in eukaryotic cells. This work has provided the basis for further study of the pathogenesis of toxoplasmosis.
出处
《中国病原生物学杂志》
CSCD
北大核心
2015年第11期986-988,993,共4页
Journal of Pathogen Biology
基金
安徽省教育厅高校省级自然科学研究重点项目(No.KJ2013A149)
