小鼠β-防御素2与甲型流感病毒HA1融合基因真核载体的构建与表达

Construction and expression of mBD2/HA1 fusion gene ineukaryotic expression system

目的:构建小鼠β-防御素2(mBD2)与甲型流感病毒A/PR/8/34(H1N1)血凝素(HA1)基因融合表达的真核载体,并检测其在HEK293细胞中的表达。方法:用PCR技术分别从质粒pcDNA3.1(+)/mBD2和pcDNA3.1(+)/HA1中扩增mBD2与HA1基因片段,再用重叠延伸PCR法(overlap-PCR)将HA1与mBD2通过一段多肽接头Gly4Ser融合为mBD2-HA1。将mBD2-HA1融合基因插入真核表达载体pcDNA3.1(+),经酶切、PCR和测序鉴定正确后,用脂质体转入HEK293细胞,通过免疫荧光检测其瞬时表达。结果:融合基因mBD2-HA1的真核表达载体pcDNA3.1(+)/mBD2-HA1构建成功,并在HEK293细胞中成功表达融和蛋白。结论:融合基因mBD2-HA1真核表达载体的成功构建,为研究防御素在流感病毒核酸疫苗中的佐剂作用奠定了基础。

Objective ：To construct the eukaryotic expression vector for mouse β-defensin 2 （mBD2） and influenza A virus （A/PR/8/34） HA1 fusion gene and express it in HEK293 cells. Methods：mBD2 and HA1 gene were amplified from plasmids pcD- NA3.1 （ ＋ ）/mBD2 and pcDNA3. 1 （ ＋ ）/HA1 respectively, and a polypeptide linker Gly4Ser was used to splice mBD2 and HA1 gene by overlap-PCR for constructing the chain of mBD2-HA1 fusion gene. mBD2-HA1 fragment was inserted into eukaryotic expressing plasmid pcDNA3. 1 （ ＋ ） to construct pcDNA3. 1 （ ＋ ）/mBD2-HA1. After identification of restriction enzymes digestion, PCR and sequencing analysis, pcDNA3.1 （ ＋ ）/mBD2-HA1 was transfected into HEK293 cells by PolyFect Transfection Reagent. The transient expression of mBD2-HA1 gene was detected through immunofluorescence assay. Results：mBD2-HA1 fusion gene was gotten by overlap- PCR. Expression vector pcDNA3.1 （ ＋ ） of mBD2-HA1 fusion gene was constructed successfully. The fusion protein could be detected in HEK293 cells transfected with pcDNA3.1 （ ＋ ）/mBD2-HA1. Conclusion： The success in the construction of eukaryotic expressing plasmid for mBD2-HA1 fusion gene made a solid foundation for further exploring the function of defensin against influenza virus through DNA vaccine.