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  

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

作　　者：乔彩霞[1] 关云涛[1] 陈洪岩[1] 刘怀然[1] 蔡雪辉[1] 李昌文[1] 葛俊伟[1] 刘家森[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2004年第3期169-173,共5页Chinese Journal of Preventive Veterinary Medicine

摘　　要：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gagP27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＿6P＿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GlutathionSpharose4B树脂亲和层析法纯化后,每100mL菌液最终可获得5mg重组P27蛋白,蛋白纯度在90%以上。用兔抗自然P27蛋白阳性血清进行Westernblot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。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.

关 键 词：禽白血病 禽劳斯肉瘤病毒 P27基因 克隆 表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学] Q785[农业科学—兽医学]