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

Cloning and expression of reticuloendotheliosis virus(REV) and development of indirect-ELISA for detection of antibody to REV

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1.5 mg·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REVgp90蛋白,并对其在ELISA检测方面的应用作了初步探讨.

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.