AEG-1肺归巢域与Flagellin融合基因的杆状病毒制备及鉴定

Preparation and characterization of the baculovirus containing AEG-1 lung homing domain and Flagellin fusion gene

目的通过重叠PCR方法获得AEG-1C-Flic融合基因,采用Bac-To-Bac杆状病毒表达系统获得重组AEG-1C-Flic杆状病毒,为后续AEG-1C-Flic病毒样颗粒疫苗的制备奠定基础。方法从鼠伤寒沙门菌的基因组中PCR扩增细菌鞭毛蛋白Flagellin,采用重叠PCR的方法将AEG1肺归巢域段基因与Flagellin融合,并添加猴免疫缺陷病毒(simian immunodeficiency virus,SIV)包膜蛋白gp41信号肽与跨膜区,构建AEG-1C-Flic pFastBacTM重组表达载体,转座大肠杆菌E.coliDH10Bac感受态细胞后获得重组杆粒,重组杆粒转染昆虫细胞Tn5,获得AEG-1CFlic重组杆状病毒,并采用Western Blot方法对该病毒进行鉴定。结果构建的AEG-1C-Flic杆状病毒表达载体经双酶切及测序正确,通过转化E.coli DH10Bac感受态细胞获得重组Bacmid,经PCR鉴定大小正确,Western Blot结果显示Bacmid转染Tn5细胞后成功获得重组杆状病毒rBv AEG-1C-Flic。结论成功构建的AEG-1C-Flic杆状病毒可用于新型AEG1-病毒样颗粒疫苗的制备。

Objective Obtain AEG-1 C-Flic fusion gene by overlapping PCR method,then using Bac-To-Bac expression system to obtain the recombinant baculovirus AEG-1 C-Flic,which will lay the foundation for producing subsequent AEG-1 C-Flic virus like particle.Methods Bacterial flagellin（C）was obtained by PCR amplification from Salmonella typhimurium genome,the fusion gene containing the lung homing domain of AEG1 gene and Flagellin gene was amplified using overlapping PCR method,the signal peptide and transmembrane region of simian immunodeficiency virus（Simian immunodeficiency,virus,SIV）envelope protein gp41 was also added at the 3＇and 5＇end of the fusion gene,respectively.AEG-1 C-Flic fragment was inserted into pFastBacTM to construct the recombinant expression vector pF/AEG-1 CFlic,pF/AEG-1 C-Flic was then transformed into E.coliDH10 Bac competent cells to obtain recombinant bacmid,which was then transfected into Tn5 insect cells to generate AEG-1 C-Flic baculovirus,Western Blot method was then performed to detect the baculovirus.Results pF/AEG-1 C-Flic was successfully constructed by overlapping PCR,and the recombinant baculovirus AEG-1 C-Flic was successfully packaged and the expression of AEG-1 was detected by Western Blot.Conclusion The results showed the recombinant AEG-1 C-Flic baculovirus was successfully rescued and this can be further used for producing the novel AEG1-virus like particles.