论文部分内容阅读
目的构建狂犬病病毒(Rabies virus,RV)SRV9株糖蛋白(Glycoprotein)基因的重组伪狂犬病病毒(Pseudorabiesvirus,PRV),并进行鉴定。方法将SRV9株糖蛋白基因表达盒和报告基因Lac Z表达盒克隆至转移载体p8AA中,构建转移质粒p8AA-LacZ-G,与PRV Bartha-K61株基因组共转染BHK-21细胞,待细胞发生病变后,收集病毒液,进行多轮蓝色噬斑筛选,获得重组病毒rPRV-LacZ-G,对其进行电镜观察、PCR及Western blot鉴定,并测定重组病毒的滴度。结果酶切及测序鉴定证实,转移质粒p8AA-LacZ-G构建正确;电镜观察显示,重组病毒rPRV-LacZ-G呈典型的PRV特征结构;PCR分析显示可见RV 1 790 bp的特异性条带;Western blot显示,重组病毒可与SRV9株糖蛋白单抗发生反应,形成特异条带;经测定,重组病毒的滴度为106.25TCID50/ml,较Bartha-K61亲本株的滴度(107TCID50/ml)下降。结论成功构建了RV SRV9株糖蛋白基因重组PRV rPRV-LacZ-G,为其应用于兽用狂犬病疫苗的开发奠定了基础。
Objective To construct and identify Pseudorabiesvirus (PRV) of Glycoprotein gene of rabies virus (SRV9) strain. Methods The glycoprotein gene expression cassette and reporter gene Lac Z expression cassette of SRV9 strain were cloned into the transfer vector p8AA. The transfer plasmid p8AA-LacZ-G was constructed and transfected into BHK-21 cells with the PRV Bartha-K61 genome. After the lesions were collected, the virus solution was collected and screened by multiple rounds of blue plaques. The recombinant virus rPRV-LacZ-G was obtained and observed by electron microscopy, PCR and Western blot, and the titer of the recombinant virus was determined. Results The restriction endonuclease digestion and sequencing proved that the transfer plasmid p8AA-LacZ-G was constructed correctly. Electron microscopy showed that the recombinant virus rPRV-LacZ-G showed a characteristic PRV structure. PCR analysis showed a specific band of RV 1 790 bp. Western blot showed that the recombinant virus reacted with glycoprotein mAb of SRV9 strain to form specific bands. The titer of the recombinant virus was 106.25 TCID50 / ml, which was higher than that of Bartha-K61 parent strain (107 TCID50 / ml) decline. Conclusion The recombinant PRV rPRV-LacZ-G, a glycoprotein gene of RV SRV9 strain, was successfully constructed and laid the foundation for its application in the development of rabies vaccine for veterinary use.