IBDV VP2与Gallus Akirin2真核双表达载体的构建及应用

Constructionand application of eukaryotic co-expression vector carrying IBDV VP2 and Gallus Akirin2

为提高传染性法氏囊病(IBD)VP2DNA疫苗的免疫效力,将优化合成的IBDV VP2与Gallus Akirin2串联基因定向克隆至真核表达载体pTriEx-4,获得重组质粒pVP2-Akirin2。体外转染293T细胞,提取表达产物进行SDSPAGE和Western blot分析,结果显示:在58 000和35 000附近各有一蛋白条带,分别能与鸡IBD阳性血清和兔Akirin2多克隆抗体发生特异性反应。将pVP2-Akirin2与同期构建的pVP2对照质粒分别免疫SPF鸡,加强免疫2周后,攻IBDV BC6-85强毒,4d后统计保护率,并监测免疫过程中血清IBD抗体滴度和IBDV中和抗体滴度的动态变化,结果显示:SPF鸡二免后14d,pVP2-Akirin2组血清IBD抗体滴度和IBDV中和抗体滴度均极显著高于pVP2组(P<0.01),且对IBDV强毒株BC6-85攻击的保护率为75%,高于pVP2对照组(55%)。结果表明:Gallus Akirin2基因对IBD VP2DNA疫苗具有明显的免疫增强作用。

To improve the immunogenicity of the VP2 DNA vaccine of infectious bursal disease （IBD） ,the optimized and synthesized IBDV VP2 and Gallus Akirin2 tandem gene was cloned into eukaryotic expression vector pTriEx-4 to obtain the recombinant plasmid pVP2-Akirin2. Then the plasmid was used to transfect into 293T cells and analyzed by SDS-PAGE and Western blot. As ex- pected, the two calculated proteins were approximately 58 000 and 35 000, could be specifically rec- ognized by positive sera of IBD and rabbit polyclonal anti-Akirin2, respectively. Then, the SPF chickens were immunized with pVP2-Akirin2 and pVP2 respectively,followed by challenging with IBDV BC6-85 at 2 weeks post the boost vaccination and calculated protective rate 4 days later. Mo- reover, the antibody titer of IBD and neutralizing antibody titer of IBDV were monitored during the immunologic process. The results showed that the specific antibody was dramatically higher in the group immunized with pVP2-Akirin2 than that of the pVP2 immunized group after boosting （P〈 0.05）,and 75% of the chickens were protected after challenged with IBDV BC6-85 strain, while the protection rate was 55% in pVP2 group. These provide a evidence that Gallus Akirin2 could enhance the immune efficacy of VP2 DNA vaccine.