减毒鸡白痢沙门氏菌ΔcrpC79-13株的构建及其对雏鸡的免疫活性

为构建鸡白痢沙门氏菌C79-13株Δcrp基因缺失突变株,并初步观察ΔcrpC79-13缺失菌株作为活疫苗对雏鸡的免疫活性,将含缺失320bp crp基因的重组自杀性质粒pREΔcrp与C79-13进行接合转移,两步法筛选出无抗性标记的ΔcrpC79-13缺失菌株;通过半数致死量测定其毒力;给4日龄雏鸡口服免疫缺失菌株,在不同时间点根据胸腺、法氏囊、脾等免疫器官发育和平均日增重、外周血淋巴细胞转化试验、Griess法NO测定及血清IgG动态观察免疫水平。结果显示ΔcrpC79-13的毒力较C79-13降低约99.6%(LD50>5.0×109CFU);雏鸡接种ΔcrpC79-13(1.0×109 CFU/只)后不影响雏鸡生长,第14～21天体内特异性细胞和体液免疫水平达到最高。表明成功构建了减毒鸡白痢沙门氏菌ΔcrpC79-13株,其毒力显著降低,免疫雏鸡安全,具有良好的免疫活性。

Construction of an attenuated Salmonella pullorum ΔcrpC79-13 mutant and its immunogenicity in chicks

To construct an Δcrp mutant C79-13 strain of Salmonella pullorum and determine immunogenicity of the mutant in chicks,the recombinant suicide vector pREΔcrp with 320 bp-deleted crp(cAMP receptor protein) gene was conjugated with C79-13.The unmarked crp deleted strain without resistance was selected by the two-step method introduced by the transduction of recombinant suicide plasmid.The virulence of ΔcrpC79-13 was evaluated by median lethal dose(LD50).The immune organ index and average daily body weight gain of 4-day-old chicks immunized with ΔcrpC79-13 were measured at different time.The proliferation activity of T-lymphocytes and NO levels of macrophages in peripheral blood were determined by MTT assay and method of Griess,respectively.The serum IgG antibody was examined by ELISA.The LD50 test showed that the virulence of the mutant was approximately 99.6% lower than C79-13.The weight gain of the thymus,spleen and bursa and average daily body weight gain of chicken immunized with ΔcrpC79-13(1.0×109 CFU per chick) were no significant difference to the control group(P>0.05).The results of lymphocyte transformation test(MTT),NO of lymphocyte secretion and serum IgG antibody showed that immunological reaction in vivo could achieve the highest level on the 14th to 21st day after immunization.In conclusion,the attenuated ΔcrpC79-13 mutant with good immunogenicity was constructed successfully.