非融合绿色荧光蛋白标记EV71型人肠道病毒的构建

发布时间：2018-01-21 22:17

本文关键词： 人肠道病毒肠道病毒型绿色荧光蛋白荧光标记基因重组　出处：《山东医药》2017年42期 　论文类型：期刊论文

【摘要】：目的构建非融合绿色荧光蛋白(EGFP)标记的肠道病毒71型(EV71),为EV71抗病毒机制研究及抗病毒药物的筛选提供方法。方法取野生型病毒EV71,在EV71基因组的3D蛋白编码区后添加HBV的link序列,将EGFP基因连入link与EV71型病毒3'非翻译区之间,构建EGFP标记的EV71病毒基因组,并转染横纹肌瘤RD细胞以获得EV71-link-EGFP重组病毒。采用PCR方法鉴定重组病毒中link、EGFP位置。感染RD细胞后24、48、72、96、120、144和168 h时收获病毒,采用组织半数感染量法测定病毒滴度,绘制病毒生长曲线,比较重组病毒与野生型病毒的生长动力学。RT-PCR测定EGFP转录表达。将重组病毒感染RD细胞后72 h裂解,采用Western blotting法检测RD细胞中GFP、P1蛋白的表达情况。重组病毒感染RD细胞后,经过13轮传代,Western blotting法检测EGFP基因稳定性。结果 RD细胞内表达较强的GFP荧光,病毒感染细胞后病毒的mRNA正常表达,重组病毒中link序列、EGFP基因链接位置正常,证实EV71-link-EGFP重组病毒拯救成功。重组病毒滴度的曲线峰值与野生型病毒滴度的峰值相同。重组病毒中EGFP的表达在48 h时最高。重组病毒中GFP和P1蛋白表达正常稳定。经过13轮传代,第13代重组病毒表达的GFP蛋白与第1代无明显差别。结论通过添加link序列成功构建了非融合表达的EV71-link-EGFP重组病毒,可在RD细胞中良好生长并高效表达EGFP,且具有稳定的遗传性。
[Abstract]:Objective to construct a non-fusion green fluorescent protein (EGFP) labeled enterovirus 71 (EV71). Methods the wild-type virus EV71 was selected and the link sequence of HBV was added to the 3D protein coding region of EV71 genome. The EGFP gene was inserted into the 3 'untranslated region between link and EV71 virus type 3 to construct the EV71 virus genome labeled by EGFP. The recombinant EV71-link-EGFP virus was obtained by transfection of Rd cells of striated leiomyoma. The location of Linker-EGFP in the recombinant virus was identified by PCR method. 24% of the recombinant virus was infected with Rd cells. Virus titer was measured by tissue half infection method and virus growth curve was drawn after harvesting virus at 144th and 168h. The growth kinetics of recombinant virus and wild-type virus were compared. RT-PCR was used to detect the transcription of EGFP. 72 h after the recombinant virus was infected with Rd cells, the recombinant virus was lysed. The expression of GFPP 1 protein in Rd cells was detected by Western blotting method. After the recombinant virus was infected with Rd cells, it was subcultured for 13 rounds. Results the stability of EGFP gene was detected by Western blotting. Results GFP fluorescence was strongly expressed in Rd cells, and mRNA expression was normal after virus infection. The link sequence of the recombinant virus was normal. It was confirmed that the EV71-link-EGFP recombinant virus was successfully rescued. The peak value of the recombinant virus titer was the same as that of the wild-type virus. The expression of EGFP in the recombinant virus was expressed at 48. The expression of GFP and P1 protein in the recombinant virus was normal and stable. There was no significant difference between the GFP protein expressed by the 13th generation recombinant virus and the first generation. Conclusion the non-fusion expressed EV71-link-EGFP recombinant virus was successfully constructed by adding the link sequence. EGFP can grow well and express EGFPin efficiently in Rd cells, and it has stable heredity.
【作者单位】：齐鲁医药学院;
【基金】：山东省高校中医药抗病毒协同创新中心基金资助项目(XTCX2014B01-07)
【分类号】：R373
【正文快照】： 肠道病毒属于小RNA病毒科,分为肠道病毒A、B、C、D共4种类型。肠道病毒71型(EV71)属于HEV-A型,其感染后主要引起手足口病,还能引起无菌性脑膜炎、脑干脑炎和麻痹型脊髓灰质炎等多种与神经系统相关的疾病。自1957年首次被报道以来,EV71已引发全球范围内的10多次爆发与流行[1,2]

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