表达绿色荧光蛋白重组鸭肠炎病毒构建

发布时间：2018-04-02 15:26

本文选题：鸭肠炎病毒　切入点：UL基因　出处：《中国农业科学》2016年14期

【摘要】：【目的】鸭肠炎病毒(duck enteritis virus,DEV)不同毒株间存在明显差异,DEV疫苗株的UL2基因在195bp后连续缺失528bp,导致第65位氨基酸后连续缺失176aa~([1])。将绿色荧光蛋白(GFP)基因插入DEV UL2基因中,获得表达绿色荧光蛋白的重组病毒,以研究UL2基因对DEV生物特性的影响和探讨DEV作为载体表达外源基因的可行性。【方法】以实验室保存的DEV细胞适应株DNA为模板,利用PCR技术扩增出病毒UL2基因上下游序列并克隆入pMD-18T载体;以UL2基因作为外源基因插入靶点及同源重组臂,将CMV启动子控制的含有GFP-gpt基因表达盒克隆入DEV UL2基因中,构建含GFP基因的转移质粒载体pT-UL2-GFP-gpt;用脂质体将其与DEV细胞适应株共转染CEF细胞,待80%细胞出现病变后,冻融3次,接种到新鲜CEF细胞单层的6孔培养板中,用含5%血清、1%双抗、1%琼脂的M199培养液覆盖,在荧光显微镜下挑取单个有绿色荧光的蚀斑,再接到新的细胞上,重复蚀斑筛选、纯化表达绿色荧光蛋白的重组病毒;利用PCR、基因测序技术鉴定重组病毒;重组病毒接种CEF(moi=0.01),每12h取出1瓶接毒细胞,分别收集上清和细胞,测量其病毒含量,绘制一步生长曲线;重组病毒在CEF中连续传代20次,在荧光显微镜下观察绿色荧光蛋白表达情况,并用PCR检测GFP的传代稳定性;重组病毒免疫4周龄SPF鸭后14d,肌肉注射接种DEV强毒(CVCC AV1221),观察免疫保护情况。【结果】经双酶切鉴定,成功构建了含绿色荧光蛋白报告基因的转移质粒载体p T-UL2-GFP-gpt,将其与DEV共转染CEF细胞后8h,即可见转染细胞中有带有绿色荧光的梭形细胞,经过8轮蚀斑筛选,获得纯化的重组病毒rDEV-△UL2-GFP-gpt;PCR鉴定及基因测序结果显示,GFP标记基因成功地插入到DEV基因组中,替换了DEV UL2基因的196—723位核苷酸;一步生长曲线结果显示,重组病毒在细胞和上清中的病毒含量分别在36h和72h达到峰值,为10~(6.2)TCID_(50)/0.1m L、10~(5.5)TCID_(50)/0.1m L,与亲本毒无明显差异;重组病毒在CEF中连续传代,1—5代可以稳定表达GFP基因,第6代起,开始出现少量没有荧光的细胞病变,15—20代中绝大部分细胞病变无绿色荧光,GFP在细胞连续传代过程中容易出现突变;重组病毒以10~(3.0)TCID_(50)/只免疫麻鸭,免疫后14d能完全抵抗DEV强毒株的攻击,与亲本毒免疫原性一致。【结论】成功构建了表达绿色荧光蛋白的DEV,首次证实UL2基因缺失不影响其在细胞中的复制,也不影响其免疫原性。荧光重组病毒的构建为DEV UL2基因功能、活载体疫苗研究奠定了基础。
[Abstract]:[objective] there were significant differences among different strains of duck enteritis virus (DEAV). The UL2 gene of DEV vaccine strain was deleted 528 BP after 195bp, which led to the deletion of 176aaAU after 65th amino acid.The green fluorescent protein (GFP) gene was inserted into the DEV UL2 gene to obtain the recombinant virus expressing green fluorescent protein.In order to study the effect of UL2 gene on the biological characteristics of DEV and explore the feasibility of expressing exogenous gene with DEV as a vector, [methods] DNA, an adaptive cell line of DEV cells, was used as a template.The upstream and downstream sequences of the virus UL2 gene were amplified by PCR and cloned into the pMD-18T vector. The CMV promoter controlled GFP-gpt gene expression box was inserted into the DEV UL2 gene with UL2 gene as the target and homologous recombination arm.A single green fluorescent plaque was selected under a fluorescence microscope and then was inserted into a new cell. The recombinant virus expressing green fluorescent protein was purified by repeated plaque screening.The results of PCR identification and gene sequencing of purified recombinant virus rDEVUL2-GFP-gptHN showed that the GFP-labeled gene was successfully inserted into the DEV genome and replaced the 196-723 nucleotides of the DEV UL2 gene, and the results of one-step growth curve showed that the GFP-labeled gene was successfully inserted into the DEV genome and replaced the 196-723 nucleotides of the DEV UL2 gene.The virus content of recombinant virus in cell and supernatant reached its peak at 36h and 72h, respectively, which was 10~(6.2)TCID_(50)/0.1m L10, 5.5TCIDD / 0.1 L, which had no significant difference with parental virus, and the recombinant virus could express GFP gene stably in 1-5 passages of CEF, and the sixth generation.In the 15-20 passage, most of the cytopathic diseases were easy to mutate in the continuous passage of cells, and the recombinant virus was only immunized with 10~(3.0)TCID_(50)/.On the 14th day after immunization, DEV could completely resist the attack of DEV virulent strain, which was consistent with the immunogenicity of parent virus. [conclusion] the expression of green fluorescent protein was successfully constructed. It was proved for the first time that the deletion of UL2 gene did not affect its replication in cells.Nor does it affect its immunogenicity.The construction of fluorescent recombinant virus lays a foundation for the study of DEV UL2 gene function and live vector vaccine.
【作者单位】：中国兽医药品监察所;
【基金】：北京市自然科学基金(6162025)
【分类号】：S852.65

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