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目的构建mBD2与mBD3融合基因的真核表达载体,检测其在MDCK细胞中的表达情况,并探讨其抗病毒特性。方法利用PCR方法分别扩增mBD2与mBD3基因片段,通过重叠延伸PCR技术(SOE-PCR)将mBD2与mBD3通过一段多肽接头Gly4Ser连接为融合基因mBD2与mBD3。将该融合基因插入真核表达载体pcDNA3.1(+)获得重组质粒pcDNA3.1(+)/mBD2-mBD3,经酶切、PCR及测序鉴定正确后,用脂质体转染MDCK细胞,通过免疫荧光检测融合蛋白表达情况,最后采用CCID50测定并分析抗流感病毒作用。结果经鉴定后证实,构建真核表达质粒pcDNA3.1(+)/mBD2-mBD3正确,该重组质粒能在MDCK细胞中稳定表达,CCID50实验结果说明有较好的抗流感活性。结论本研究成功构建mBD2-mBD3融合基因的真核表达质粒,并证实其融合蛋白能在MDCK细胞中稳定表达,该结果为进一步研究防御素抗病毒机制奠定了坚实的基础。
Objective To construct the eukaryotic expression vector of mBD2 and mBD3 fusion gene to detect the expression of mBD2 and mBD3 in MDCK cells and to explore the antiviral properties. Methods mBD2 and mBD3 gene fragments were amplified by polymerase chain reaction (PCR). The mBD2 and mBD3 genes were fused to mBD3 by Gly4Ser through overlap extension PCR (SOE-PCR). The fusion gene was inserted into the eukaryotic expression vector pcDNA3.1 (+) to obtain the recombinant plasmid pcDNA3.1 (+) / mBD2-mBD3. After identification by restriction enzyme digestion, PCR and sequencing, MDCK cells were transfected with lipofectamine The expression of the fusion protein was detected by immunofluorescence. Finally, the CCID50 was used to determine and analyze the anti-influenza virus effect. The results confirmed that the construction of eukaryotic expression plasmid pcDNA3.1 (+) / mBD2-mBD3 correct, the recombinant plasmid can be stably expressed in MDCK cells, CCID50 results show that the better anti-influenza activity. Conclusion This study successfully constructed mBD2-mBD3 fusion gene eukaryotic expression plasmid and confirmed that the fusion protein can be stably expressed in MDCK cells, and the results provide a solid foundation for further study on the antiviral mechanism of defensins.