表达绿色荧光蛋白重组鸭肠炎病毒构建

doi:10.3864/j.issn.0578-1752.2016.14.014

摘要/Abstract

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pT-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10^6.2TCID₅₀/0.1mL、10^5.5TCID₅₀/0.1mL，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10^3.0TCID₅₀/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，为DEV UL2基因功能、活载体疫苗研究奠定了基础。

关键词: 鸭肠炎病毒, UL2基因, 绿色荧光蛋白, 重组病毒

Abstract: 【Objective】Compared with duck enteritis virus(DEV) virulent strain, the vaccine strain has a 528 bp deletions at the UL2, resulting to a 176 aa deletion after amino acids 65. To study the effect of UL2 gene on virus biological properties and explore the feasibility of the DEV as a carrier to express foreign gene, a recombinant DEV expressing the green fluorescent protein (GFP) were constructed;【Method】In this study, the UL2 gene of DEV was chosen as a target site and homologous arm for recombination. Two fragments of UL2 gene were amplified by polymerase chain reaction (PCR) with DNA of DEV cell-adapted strain as template, and were cloned into the pMD-18T vector. The expression cassette including GFP gene and gpt gene controlled by CMV promoter was cloned into UL2 gene as a transfer vector pT-UL2-GFP-gpt. Confluent CEF monolayers were transfected with DEV and Lipofectamine 2000 was used as the transfer vector. When the cytopathic effect (CPE) was observed, the total supernatant and cells were harvested. The infected virus was diluted and then plated on the fresh CEF, and overlaid with M199-FBS containing 1% agarose. When green fluorescent plaques were observed, plaque-purification was carried out to obtain a green fluorescent plaque population termed rDEV-△UL2-GFP-gpt, PCR and sequencing assay were used to identify the recombinant virus. CEF cells cultured in 25cm2 flasks were inoculated with recombinant virus at an MOI of 0.01. The cells and supernatants were harvested respectively every 12 hours, the titer of virus were measured and the one-step growth analyses was performed; To evaluate the genetic stability of GFP gene in the recombinant virus, the virus was passaged in primary CEF 20 times. Four-week-old specific- pathogen-free (SPF) ducks were inoculated intramuscularly with the recombinant virus, and the ducks were challenged with lethal DEV (CVCC AV1221) by intramuscular injection at 14 days post vaccination, then the ducks were observed for symptom of disease and death.【Result】The recombinant expression vector pT-UL2-GFP-gpt was correctly constructed, identified by double-enzyme digestion. After 8 hours of transfection, spindle cells with green fluorescent were appeared. After 8 rounds of plaque-purification, the purified rDEV-△UL2-GFP-gpt were obtained. The results of the PCR and sequencing indicated that the GFP expression cassette has already successfully insert into the DEV genome, which replaced 196-723 nucleotide of UL2. The recombinant virus possessed growth kinetics were similar to that of the parental virus, the cell titer peaked at 36 hours with the peak titer 106.2TCID50/0.1mL, and the supernatant titer peaked at 72 hours with the peak titer 105.5TCID50/0.1mL. The virus were passaged in CEF cells 20 times, the GFP gene was stably maintained in 1st to 5th passages, however, from the 6th passage, there was little CPE without green fluorescent, and in 15th to 20th passages, most CPE had no green fluorescent, GFP mutated during subculture. All immunized animals were protected against subsequent challenge with lethal DEV, the insertion of the GFP gene did not alter the protective efficacy of parental virus. 【Conclusion】In this research, the recombinant DEV expressing the green fluorescent protein were successfully constructed, and firstly has confirmed that the deletion of UL2 gene has no effect on virus replication in cells and the immunogenicity in ducks. This study laid a foundation for the research of the function of the DEV UL2 gene and the DEV vector vaccine.

Key words: duck enteritis virus, UL2 gene , GFP, recombinant virus

孙莹，李俊平，黄小洁，李岭，曹明慧，李启红，李慧姣，杨承槐. 表达绿色荧光蛋白重组鸭肠炎病毒构建[J]. 中国农业科学, 2016, 49(14): 2805-2812.

SUN Ying, LI Jun-ping, HUANG Xiao-jie, LI Ling, CAO Ming-hui, LI Qi-hong, LI Hui-jiao, YANG Cheng-huai. Construction and Characterization of Recombinant Duck Enteritis Virus Expressing the Green Fluorescent Protein[J]. Scientia Agricultura Sinica, 2016, 49(14): 2805-2812.

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