EnMIC2重组减毒沙门氏菌的构建及免疫保护效果研究

成功构建了表达鸡毒害艾美耳球虫(Eimeria necatrix)广东株(GD)微线蛋白-2基因(EnMIC2)的重组减毒鼠伤寒沙门氏菌，在IPTG诱导下获得了EnMIC2的高效表达，SDS-PAGE分析表明，表达产物约占菌体总蛋白的8．6％，分子量大小约为35ku，与抗E．necatrix的高免血清进行Western Blotting，结果为阳性。将重组减毒沙门氏菌以10^8和10^9 CFU／鸡免疫4日龄岭南黄肉鸡，免疫后2周血清中可检测到特异性IgG，3周时抗体水平达到峰值，同时可在肠道检测到特异性IgA的分泌。免疫接种后3周，试验鸡分别经口攻击感染500个E．necatrix(GD)孢子化卵囊，结果显示免疫组与非免疫组有相似的排卵囊曲线；但10^8和10^9CFU免疫组的卵囊总产量分别能降低26．62％和48．92％。

Immunogenic Characterization of the Recombinant Attenuated Salmonella typhimurium Expressing MIC2 Gene of Eimeria necatrix

The ORF sequence of micronem protein-2(MIC2) gene from Eimeria necatrix(GD) was cloned by PCR amplification using specific primers designed according to the sequence of EnMIC2(GD), and ligated to vector pYA3342 to construct recombinant plasmid pYA3342-EnMIC2 for transforming E.coli x6212. Then the positive recombinant plasmid identified by PCR amplification and endonuclease digestion was electroporated into attenuated S. typhimurium X4550. The recombinant EnMIC2 expressed in Salmonella by inducing of 1 mmol/L IPTG was approximately 35 ku in size and accounting for 8.6% in total bacterial proteins and could be detected in western blotting using hyperimmune serum against E.necatrix. The specific serum IgG against EnMIC2 in chickens post-immunized(PI) with recombinant Salmonella at the dosage of 10~8 or 10~9 CFU per bird was probed, and reached to the peak 3 weeks PI. Specific IgA in intestine was also detected 3 weeks PI. These immunized chicken were challenged with 500 E. necatrix sporulated oocysts per bird 3 weeks PI and can reduce the oocyst production by 26.62% and 48.92%, respectively in low or high vaccinating dosage compared to the control groups.