禽多杀性巴氏杆菌粘附蛋白Cp39的交叉免疫保护作用

Cross-protective effect of the Cp39 adhesive proteins against avian Pasteurella multicida in mice

摘要:

摘要：【目的】比较体内外增殖禽多杀性巴氏杆菌荚膜蛋白、天然粘附蛋白Cp39和重组粘附蛋白rCp39对小鼠的交叉保护作用。【方法】用NaCl提取法制备鸡胚尿囊液和DSA培养基增殖的C48-3株荚膜蛋白，并用电洗脱方法纯化Cp39蛋白，将rCp39蛋白以可溶形式表达在大肠杆菌BL21后，用Amylose Resin亲和层析柱纯化。分别以100 μg剂量的鸡胚尿囊液增殖菌体荚膜蛋白、DSA培养基培养菌体荚膜蛋白、纯化的Cp39蛋白和rCp39蛋白通过皮下注射各试验组小鼠，生理盐水为对照组，第二次免疫后2周分别以A:1型菌C48-3株（6.7×102 cfu）和A:3型菌C51-3株（1.1×103cfu）进行攻毒试验。采集免疫后小鼠血清，用ELISA法检测抗体水平，并计算免疫保护率，来评价4种抗原对小鼠的交叉保护效果。【结果】SDS-PAGE结果显示，体内外增殖禽多杀性巴氏杆菌荚膜蛋白的条带和分子量相似，且体内外表达的Cp39蛋白的分子量相同；ELISA结果表明Cp39免疫组小鼠和rCp39免疫组小鼠血清rCp39蛋白特异性抗体的水平显著高于其他两组（P<0.05）；保护试验表明，体外增殖菌体荚膜蛋白免疫组小鼠对同源C48-3株和异源C51-3株攻毒的保护率分别为100%和60%，鸡胚尿囊液增殖菌体荚膜蛋白免疫组小鼠、Cp39免疫组小鼠和rCp39免疫组小鼠对同源C48-3株和异源C51-3株攻毒的保护率分别为100%和80%。【结论】粘附蛋白Cp39是禽多杀性巴氏杆菌荚膜蛋白中的主要交叉保护抗原，可以作为禽霍乱亚单位疫苗。

Abstract:

Abstract：[Objective] We evaluated the cross-protective effect of a 39-kDa adhesive protein (Cp39), recombinant adhesive protein (rCp39), and capsular proteins from in vitro and in vivo grown avian P. multocida in mice. [Methods] The Cp39 was purified by electroelution from capsular proteins of in vivo grown strain C48-3. The rCp39 was expressed in E. coli B21 as a soluble protein by IPTG inducing, and purified with pMALTM protein fusion and purification system. Mice of each group were subcutaneously immunized twice with 100μg of rCp39, Cp39 or capsular proteins from in vivo or in vitro grown strain C48-3 with in complete or incomplete Freund adjuvant at 2-week intervals. Five mice of each group were challenged with 100 LD50 of avian P. multocida strain C48-3 or rabbit P. multocida strain C51-3 two weeks after the second immunization, while the antibody response specific for rCp39 was determined by ELISA. [Results] SDS-PAGE indicated that the Cp39 protein was expressed in vitro and in vivo grown P. multocida. Mice immunized with rCp39, native Cp39 or capsular proteins from in vivo and in vitro grown strain C48-3 were protected completely against the challenge with the homologous strain C48-3, while 60-80% protection was demonstrated in the mice against the challenge with the heterologous strain C51-3. [Conclusion] In conclusion, rCp39 is a cross immunoprotective antigen of P. multocida capsular serogroup A strains, and might be a useful vaccine candidate against fowl cholera.