禽多杀性巴氏杆菌基因组表达文库的构建及其免疫效果

Construction and detection of the immune efficacy from genomic expression library vaccines against avian Pasteurella multocida

以限制性内切酶Sau3AⅠ酶切禽多杀性巴氏杆菌基因组DNA,回收500～3000bp的DNA片段,连接真核表达载体pcDNA3.1(+),电转化于大肠杆菌感受态细胞TG1中,构建禽多杀性巴氏杆菌的基因组表达文库。将文库随机分为5个子文库(子文库Ⅰ-子文库Ⅴ),分别提取各子文库重组质粒,以pcDNA3.1(+)和PBS为对照进行动物试验,每组16只BALB/c小鼠,各子文库组和pcDNA3.1(+)组以100μg/只的剂量肌注免疫,PBS组每只小鼠肌注100μL 1×PBS,各组均免疫3次,每次间隔2周。间接ELISA检测免疫小鼠的血清抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖情况,三免2周后检测脾淋巴细胞IFN-γ的分泌情况。强毒攻击,计算小鼠的相对保护率。结果显示,动物免疫后子文库Ⅰ组质粒免疫的小鼠血清抗体水平及淋巴细胞增殖水平和IFN-γ分泌水平均持续上升,明显高于其他各组(P<0.05)。动物攻毒试验表明,各子文库组的重组质粒均可为免疫小鼠提供一定的保护,其中第Ⅰ组的保护效果最好,说明子文库Ⅰ组中含有较好的保护性抗原基因,这为进一步筛选相关的免疫原基因和研制新型疫苗奠定了的基础。

The random DNA fragments （500-3 000 bp）, which obtained from genomic of avian Pasteurella multocida CVCC474 with restriction enzyme Sau3A ]; were cloned into the BamH I site of the eukaryotic expression vector pcDNA3.1 （＋） to construct the genomic expression library. The library was subdivided into five clone pools （pool I-V ）and the recombinant plasmids （namely vaccines） were extracted from each pool. The Balb/c mice named group I -V were vaccinated with these vaccines, respectively, and the mice injected with pcDNA3. 1 （＋） and PBS were negative control. The immune responses were evaluated based on serum antibody titers,lymphocyte proliferation assay and levels of the cytokines IFN-7. The protective efficacy following challenge with a virulent avian Pasteurella multocida was evaluated based on relative protection rate. The results showed the levels of antibodies,lymphoCyte proliferation （SI value） and the levels of IFN-γ were higher in mice immunized with pool I vaccine than in those vaccinated with pool II-V vaccines （P d0.05）. Furthermore, the pool I vaccine provided better protection than that other vaccines after challenge with avian Pasteurella multocida. The results indicted the pool I may be contain some of the protective antigen genes and lay the foundation for the development of novel vaccines against avian Pasteurella multocida.