本文關(guān)鍵詞： 人腸道病毒 腸道病毒型 綠色熒光蛋白 熒光標記 基因重組　出處：《山東醫(yī)藥》2017年42期 　論文類型：期刊論文

【摘要】：目的構(gòu)建非融合綠色熒光蛋白(EGFP)標記的腸道病毒71型(EV71),為EV71抗病毒機制研究及抗病毒藥物的篩選提供方法。方法取野生型病毒EV71,在EV71基因組的3D蛋白編碼區(qū)后添加HBV的link序列,將EGFP基因連入link與EV71型病毒3'非翻譯區(qū)之間,構(gòu)建EGFP標記的EV71病毒基因組,并轉(zhuǎn)染橫紋肌瘤RD細胞以獲得EV71-link-EGFP重組病毒。采用PCR方法鑒定重組病毒中l(wèi)ink、EGFP位置。感染RD細胞后24、48、72、96、120、144和168 h時收獲病毒,采用組織半數(shù)感染量法測定病毒滴度,繪制病毒生長曲線,比較重組病毒與野生型病毒的生長動力學。RT-PCR測定EGFP轉(zhuǎn)錄表達。將重組病毒感染RD細胞后72 h裂解,采用Western blotting法檢測RD細胞中GFP、P1蛋白的表達情況。重組病毒感染RD細胞后,經(jīng)過13輪傳代,Western blotting法檢測EGFP基因穩(wěn)定性。結(jié)果 RD細胞內(nèi)表達較強的GFP熒光,病毒感染細胞后病毒的mRNA正常表達,重組病毒中l(wèi)ink序列、EGFP基因鏈接位置正常,證實EV71-link-EGFP重組病毒拯救成功。重組病毒滴度的曲線峰值與野生型病毒滴度的峰值相同。重組病毒中EGFP的表達在48 h時最高。重組病毒中GFP和P1蛋白表達正常穩(wěn)定。經(jīng)過13輪傳代,第13代重組病毒表達的GFP蛋白與第1代無明顯差別。結(jié)論通過添加link序列成功構(gòu)建了非融合表達的EV71-link-EGFP重組病毒,可在RD細胞中良好生長并高效表達EGFP,且具有穩(wěn)定的遺傳性。
[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.
【作者單位】： 齊魯醫(yī)藥學院;
【基金】：山東省高校中醫(yī)藥抗病毒協(xié)同創(chuàng)新中心基金資助項目(XTCX2014B01-07)
【分類號】：R373
【正文快照】： 腸道病毒屬于小RNA病毒科,分為腸道病毒A、B、C、D共4種類型。腸道病毒71型(EV71)屬于HEV-A型,其感染后主要引起手足口病,還能引起無菌性腦膜炎、腦干腦炎和麻痹型脊髓灰質(zhì)炎等多種與神經(jīng)系統(tǒng)相關(guān)的疾病。自1957年首次被報道以來,EV71已引發(fā)全球范圍內(nèi)的10多次爆發(fā)與流行[1,2]

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