嗜水气单胞菌重组Aer毒素的纯化和抗原性分析*

 对原核表达的重组嗜水气单胞菌Aer毒素进行初步纯化,比较了它与天然Aer毒素的抗原性,为建立检测该毒素的免疫学方法奠定了基础。将原核表达的重组Aer毒素经SDS-PAGE后,切下目的蛋白,利用电洗脱法将蛋白洗脱出来,后经透析、PEG8000浓缩、定量,对獭兔进行免疫,采集血清,提纯IgG,在Western blotting试验中可与天然Aer毒素起反应,说明,体外表达的重组Aer毒素保留了天然Aer毒素所具有的抗原性。同时,将嗜水气单胞菌AHCS02菌接种于兔鲜血平板,30 ℃、培养 15 h 后,将具有β-溶血的单菌落于LB液体培养基中培养后,用饱和硫酸铵法提取上清液中的天然Aer毒素,用甲醛脱毒后,以同样免疫方法免疫獭兔,免疫结束后,采集血清提取IgG,在Western blotting试验中,可以检测到重组Aer毒素。试验说明：原核表达的重组Aer毒素和天然Aer毒素诱导獭兔产生的血清IgG抗体可以互相识别重组Aer毒素和天然Aer毒素,重组Aer毒素和天然Aer毒素有相似的抗原性。

Purification of Recombinant Aer Toxin and Antigenicity Analysis

Antigencity of natural and recombinant Aer toxins of Aermonas hydrophila was analyzed by Western blotting .First,the inclusion body of recombinant Aer toxin fusion protein by prokaryotic expression was purified by SDS-PAGE and electrophoresis elution. The natural Aer toxin of Aeromonas hydrophlia strain AHCS02 was purified by ammonium sulfate pricipatition. Then both recombinant and natural Aer toxins were used to immunize rabbits to prepare anti-serum. Western blotting showed that the rabbit antibodies raised against the recombinant fusion protein could recognize the natural Aer toxin and the antibodies raised against natural Aer toxin could recognize the recombinant Aer toxin. The results indicated that natural and recombinant Aer toxins had similar antigenicity.