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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。
Objective To construct Beclin1 eukaryotic expression vector to detect the expression of this gene in eukaryotic cells and lay the foundation for further study on its role in the pathogenesis of Toxoplasma gondii infection. Methods Beclin1 gene was inserted into eukaryotic expression vector pEGFP by PCR and identified by double enzyme digestion. The recombinant plasmid was transfected into 293T cells by lipofectamine 2000, and then was detected by Western blot The expression of the encoded protein in eukaryotic cells. Results The Beclin1 gene was amplified by PCR and its size was 1 353bp, which was consistent with the expected value. The eukaryotic expression vector pEGFP-Beclin1 was constructed. The size of the inserted fragment was confirmed by double enzyme digestion. The sequence of Beclin1 gene in GenBank was 100% homology after DNA sequencing. The protein was detected by Western blot in eukaryotic cells expression. Conclusion The eukaryotic expression vector pEGFP-Beclin1 has been successfully constructed. The gene encoding protein can be expressed in eukaryotic cells, which lays the foundation for further study on the role of pEGFP-Beclin1 in pathogenicity of Toxoplasma gondii infection.