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为评价嗜水气单胞菌(Aeromonas hydrophila,Ah)PDK06株外膜蛋白A(omp A)的原核表达产物是否具有抗原性及其对斑点叉尾(Ictalurus punctatus)的免疫保护力,根据Gen Bank上嗜水气单胞菌外膜蛋白omp A基因序列(Gen Bank登录号:AF146597)设计去信号肽引物,扩增获得一段约1 000 bp的序列,经鉴定正确后,构建重组表达质粒p ET-32a(+)-pomp A;然后将该质粒转入BL21(DE3)后用IPTG诱导表达,经纯化后得到pomp A重组蛋白;最后将pomp Aomp A蛋白与弗氏不完全佐剂(FIA)混合免疫斑点叉尾,评价其免疫效果。结果显示:克隆的嗜水气单胞菌pomp A基因全长为960 bp,编码319个氨基酸,融合表达的重组蛋白相对分子质量约为53,670 k Da;FIA能够提高斑点叉尾的溶菌酶活性、ACH50活性、头肾巨噬细胞呼吸暴发活性;pomp A组抗体水平在第4周达到峰值,FIA免疫组抗体水平在第5周达到峰值,且FIA能够显著提高斑点叉尾的抗体水平;攻毒后各组累计死亡率分别为93.33%(PBS)、93.33%(FIA)、50.00%(pomp A)和13.33%(FIA+pomp A),pomp A和FIA+pomp A组相对保护率分别为46.43%和85.71%;免疫组靶器官中的细菌数量显著低于对照组,且FIA+pomp A细菌含量最低。说明pomp A可以作为一个亚单位疫苗候选疫苗,而FIA佐剂能较好的增强亚单位疫苗的免疫效果。
To evaluate whether the prokaryotic expression product of omp A of Aeromonas hydrophila (Ah) PDK06 strain has antigenicity and its immunopotency against Ictalurus punctatus, according to Gen Bank The sequence of omp A gene of Aeromonas hydrophila outer membrane (GenBank accession number: AF146597) was designed to amplify a sequence of about 1 000 bp. After identified correctly, a recombinant expression plasmid p ET -32a (+) - pomp A; then the plasmid was transformed into BL21 (DE3) and induced with IPTG to obtain pomp A recombinant protein; finally, pomp Aomp A protein was incubated with Freund’s incomplete adjuvant (FIA) Mixed immunized channel catfish to assess the immune effect. The results showed that the cloned Pomp A gene of Aeromonas hydrophila was 960 bp in length and encoded a polypeptide of 319 amino acids. The molecular weight of the recombinant protein was about 53,670 kDa. FIA could improve the lysozyme activity of channel catfish, ACH50 activity and respiratory burst activity of head and kidney macrophages. Antibody levels in pomp A group reached its peak at 4 weeks, and antibody levels in FIA immunized group peaked at 5 weeks, and FIA could significantly enhance the antibody level of channel catfish; The cumulative mortality of each group was 93.33% (PBS), 93.33% (FIA), 50.00% (pomp A) and 13.33% (FIA + pomp A) respectively. The relative protection rates of pomp A and FIA + pomp A group were 46.43 % And 85.71% respectively. The number of bacteria in the target organ of immunization group was significantly lower than that of the control group, and the FIA + pomp A bacteria content was the lowest. Pomp A can be used as a subunit vaccine candidate vaccine, and FIA adjuvant can better enhance the subunit vaccine immune effect.