传染性法氏囊病病毒非结构蛋白VP5的原核表达及多克隆抗体的制备  

Prokaryotic Expression and Preparation of Polyclonal Antibody for IBDV Non-structure Protein VP5 Gene

作　　者：张宁[1] 高宏雷[1] 高玉龙[1] 李俊山[1] 冉多良[2] 王笑梅[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽传染病研究室,哈尔滨150001 [2]新疆农业大学,乌鲁木齐830052

出　　处：《中国生物工程杂志》2006年第10期30-35,共6页China Biotechnology

基　　金：国家"973"计划资助项目(2005CB523202)

摘　　要：利用PCR技术,从传染性法氏囊病病毒(IBDV)Gx、Gt毒株中分别扩增出VP5基因,将其克隆到表达载体pET30a、pET28a中。经PCR、酶切和序列分析鉴定获得重组质粒命名为pET28a-GtVP5、pET30a-GxVP5。将pET30a-GxVP5、pET28a-GtVP5分别转化宿主菌BL21(DE3),在IPTG诱导下成功表达了约24kDa的Gx-VP5及23kDa的Gt-VP5融合蛋白,它们都以包涵体形式存在。将Gx-VP5纯化后的蛋白免疫8周龄BALB/c雌鼠,ELISA分析表明制备的抗血清效价在1∶25600以上,Westernblot分析VP5表达产物能与抗6×HismAb及抗IBDV多克隆抗血清发生反应,具有良好的免疫反应特异性。With a pair of primers designed according to the sequence of infectious bursal disease virus （IBDV）, the VP5 gene of IBDV was amplified from vvlBDV-Gx, IBDV-Gt, respectively. Then IBDV VP5 was cloned into expressing vector pET30a and pET28a. The recombinant plasmicl was identified by restrictive digestion, PCR and sequence analysis, then named pET30a-GxVP5, pET28a-GtVP5, respectively. The recombinant plasmid was transformed into E. coli BL21 （DE3） and induced by IPFG. SDS-PAGE results indicated that Gx-VP5, Gt-VP5 were about 24kDa, 23kDa, respectively. These recombinant fusion proteins mainly existed as inclusion bodies. High titer anti-VP5 serum was also prepared in BALB/c mice immunized with purified Gx-VP5 fusion protein inclusion. ELISA results showed that titer was above 1 ： 25600 and Western blot analysis showed that the expressed protein Gx-VP5 reacted with polyclonal antibody and 6 ×His monoclonal antibody. It explained that the expressed protein Gx-VP5 possess specific satisfactory immunological reaction.

关 键 词：传染性法氏囊病病毒 非结构蛋白 VP5 克隆表达 多克隆抗体 

分 类 号：Q786[生物学—分子生物学]