禽白血病劳斯肉瘤病毒衣壳蛋白P27基因的克隆、原核表达及多克隆抗体的制备

从人工感染禽白血病劳斯肉瘤病毒的SPF鸡胚成纤维细胞(CEF)培养物中提取RNA,通过RT-PCR方法扩增出720bp的gag P27基因片段,克隆至pMD18-T载体,酶切鉴定后进行序列测定和分析,表明该片段编码序列与国外RSV分离株(JO2342)的同源性达97.3%.将该片段亚克隆至pGEX-6 P-1原核表达载体,转化受体菌BL-21,经IPTG诱导表达,12%SDS-PAGE凝胶电泳,考马斯亮蓝染色,证明目的基因得到高效表达,所表达蛋白是大小为56kD的融合蛋白,占菌体总蛋白的23%.表达产物经Glutathion Spharose4 B树脂亲和层析法纯化后,每100 mL菌液最终可获得5 mg重组t27蛋白,蛋白纯度在90%以上.用兔抗自然P27蛋白阳性血清进行Western blot检测,表明重组P27蛋白有抗原反应活性.用纯化的重组P27蛋白免疫兔子制备多克隆抗体,产生的抗体与禽白血病全病毒抗原可以发生特异性反应.所制备的重组P27蛋白和多克隆抗体将为禽白血病的诊断及ELISA试剂盒的研制奠定基础.

Cloning and expression of capsid protein P27 gene of avian leucosis rous sarcroma virus and development of multiclonal antibodies against recombinant P27 protein

RNA was extracted from CEF cell infected with avian leucosis RSV and the gag-p27 gene was amplified by RT-PCR.Then the gene was cloned into plasmid pMD 18-T and sequenced.P27 gene is 720 bp in length.Sequence comparison indicated that the nucleotide homology between the P27 gene and the reportedsequence of foreign RSV(JO 2342) is 97.3 %.The whole coding redgion of p27 gene was subcloned into prokaryoticexpression vector pGEX-6p-1.The expression of recombinant plasmid carried P27 gene was transformed into BL-21 E.coli.Through induced with IPTG.The expression of recombinant protein was analyzed by SDS-PAGE.The results showed that the protein was higly expression in E.coli,and the molecular weight of the recombinant protein was 56 kD.It accounted for 23 % of the total cellular protein.After purification with Glutathion Spharose4B,5mg P27 recombinant protein was obtained from 100 ml BL-21 lysate.The immune reactivity of recombinant P27 protein was identified with positive antiserum against nature P27 protein specifically by Western-blotting analysis.Antibodies against recombinant P27 protein was obtained by subcutaneous injection of rabbit with purified P27 recombinant protein.which is specific to ALV antigen.It laid a foundation for diagnosis and development of ELISA test kit of avian leucosis.