小鼠β防御素2、3融合基因载体构建及其抗流感病毒作用

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。

Expression of mBD2-mBD3 fusion gene in eukaryotic expression system and its activity of anti-influenza A virus

Objective To construct the eukaryotic expression vector for mouseβ-defensin 2 （mBD2） andβ-defensin 3 （mBD3） fusion gene, study the expression of fused protein mBD2-mBD3 in MDCK cells,and observe its activity against influenza A virus. Methods mBD2 and mBD3 genes were amplified from plasmids pcDNA3.1 （＋）/mBD2 and pcD- NA3. 1 （＋）/mBD3 respectively, and a polypeptide linker Gly4Ser was used to splice mBD2 and mBD3 gene by SOE-PCR for constructing mBD2-mBD3 fusion gene. mBD2-mBD3 fragment was inserted into eukaryotic expressing plasmid pcDNA3. 1（＋） to construct pcDNA3.1 （＋）/mBD2-mBD3. The recombinant plasmid was identified by restriction enzymes digestion, PCR and sequencing analysis. Then pcDNA3. 1（＋）/mBD2-mBD3 was transfected into MDCK cells by Poly- Fect Transfection Reagent. The expression of mBD2-mBD3 gene was detected through immunofluorescence assay and the activity against influenza virus with the plasmid was also determined by CCIDS0. Results Expression vector pcDNA3.1 （＋）/mBD2-mBD3 was constructed successfully. The fused protein could be detected in MDCK cells transfected with pcDNA3.1（＋）/mBD2-mBD3 and the experiments of CCIDS0 shows a better activity of anti-influenza. Conelusion The construction of the eukaryotic expression vectors for mBD2-mBD3 fusion gene, peDNA3.1 （＋）/mBD2-mBD3, was completely successful, mBD2-mBD3 fusion protein could be expressed in MDCK cells stably. This result established a solid foundation for further exploring anti-virus mechanism of defensin.