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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2011, Vol. 37 Issue (4): 363-370    DOI: 10.3785/j.issn.1008-9209.2011.04.002
生物科学与技术     
禽网状内皮增生病毒囊膜基因gp90的克隆表达及ELISA方法的初步建立
余 冲1,黄 勇1,2,田明星1,石 敏1,李 敏1,赵芳芳1
1 .四川农业大学动物医学院,四川 雅安 ６２５０１４ ;  2.四川农业大学动物疫病与人类健康四川省重点实验室,四川 雅安 ６２５０１４)
Cloning and expression of reticuloendotheliosis virus (REV) and development of indirectELISA for detection ofantibody to REV
YU Chong１ , HUANG Yong１,２ , TIAN Ming-xing１ ,SHI Min１ ,LI Min１ ,ZHAO Fang-fang１
1 . College of Veterinary Medicine , Sichuan A gricultural University , Ya,an , Sichuan 625014 , China ; 2 . Key Laboratory of Animal Disease and Human Health of Sichuan Province , Sichuan Agricultural University , Y a,an , Sichuan 625014 , China
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摘要： 根据禽网状内皮组织增生症病毒( reticuloendotheliosis virus ,REV) SNV 株前基因组cDNA 序列,经生物信息学分析,设计并合成引物,以四川分离株REV-SC１ 为模板,扩增囊膜基因gp90 抗原性、亲水性较高的１２１ ～ １ ０２３ bp 基因片段;将目的基因克隆至pMD-19T 载体,进行测序和比对;将基因片段按正确的阅读框架定向克隆至pET-３２a( ＋ ) ,转化大肠杆菌BL２１(DE３) ,以IPTG 诱导表达,包涵体洗涤纯化,探讨以重组gp90 蛋白为包被抗原初步建立检测REV 的间接酶联免疫吸附试验( ELISA)方法.结果表明:１)REV-SC１ 株gp90 核苷酸序列与在GenBank 上已发表序列的同源性最高为99% ,于２３１ 位、８７０ 位发生了２ 个碱基突变;２)SDS-PAGE 分析表明表达蛋白分子质量约为４５ ku ,与预期分子质量大小相符;免疫印迹(Western-blot)结果表明重组蛋白具有生物学活性;３) 间接ELISA 检测方法的最佳抗原包被质量浓度为１ .５mg.mL -１ ,一抗稀释度为１ ∶ ３２０ ,临界值为0.126 ;特异性、敏感性试验结果良好.综上,本试验成功表达了REV gp９０ 蛋白,并对其在ELISA 检测方面的应用作了初步探讨.
Abstract: Based on the nucleotide sequence of avian reticuloendotheliosis virus ( REV ) SNV strain ,Sichuan isolates REV-SC１ were used as a template , and a pair of primers was designed to amplify the １２１-１ ０２３ bp of gp90 gene fragment encoding high antigenic and hydrophilic domain of C‐terminal protein . The gp90 gene was inserted into pMD-１９T vector and sequenced , and the online software of NCBI blast was used to analyze the sequencing result . The gp90 gene was inserted to expression plasmid pET-３２a( ＋ ) , and the recombinant plasmid was transformed into Escherichia coli BL２１ (DE３ ) , then using IPTG to induce expression of protein , at last , an indirect enzyme‐linked immunosorbent assay (ELISA) method was established for detecting the antibody to REV . The results showed that :１) Compared with other REV strain nucleotide sequences , the maximum matching rate was ９９％ , and there was a mutation separately at the ２３１ and ８７０ sites . ２) SDS‐PAGE analysis showed that the expected protein was got with molecular mass of approximately ４５ ku , and the Western-blot result indicated good reactogenicity of the target protein . ３) An indirect ELISA was established by using the recombinant protein . The optimal antigen coating concentration was set as １ .５ mg/mL -１ , and the best anti-serum dilute degree was at １ ∶ ３２０ , and the cut-off value ( OD４５０ nm ) was 0.126 . The detection results revealed that the method had good diagnostic sensitivity and specificity .
出版日期: 2011-07-20
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余 冲
黄 勇
田明星
石 敏
李 敏
赵芳芳

引用本文:

余 冲,黄 勇,田明星,石 敏,李 敏,赵芳芳. 禽网状内皮增生病毒囊膜基因gp90的克隆表达及ELISA方法的初步建立[J]. 浙江大学学报(农业与生命科学版), 2011, 37(4): 363-370.

YU Chong, HUANG Yong, TIAN Ming-xing,SHI Min,LI Min,ZHAO Fang-fang. Cloning and expression of reticuloendotheliosis virus (REV) and development of indirectELISA for detection ofantibody to REV. Journal of Zhejiang University: Agric. & Life Sci., 2011, 37(4): 363-370.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2011.04.002    或    http://www.zjujournals.com/agr/CN/Y2011/V37/I4/363

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