嗜水气单胞菌外膜蛋白ompA基因的原核表达及免疫保护性研究

Prokaryotic expression of outer membrane protein A(omp A) gene of Aeromonas hydrophila and its immunoprotection

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。

The expression of outer membrane protein A （OmpA） from Aeromonas hydrophila （ PDK06 ） in Escheriehia coli, the antigenicity and immunoprotection in Ictalurus punctatus were analyzed. According to ompA gene sequence published in GenBank （AF146597）, no signal peptide primers were designed and the DNA fragment of about 1 000 bp was amplified by Polymerase Chain Reaction （PCR） from genomic DNA of A. hydrophila PDK06, isolated from disease I. punctatus. The sequence was sub - cloned into pET - 32a （ ＋ ） and the recombinant plasmid was named pET - 32a （ ＋ ） - pompA which was further transferred into host bacteria E. coli BL21 （ DE3 ） with IPTG induction for expression. The immune effect of the pompA with adjuvants （FIA） against I. punctatus infected A. hydrophila were detected. The results showed the no signal peptide ompA gene of A. hydrophila was 960 bp in length, encoding a protein comprising 319 amino acids, of which the putative relative molecular mass was 53.67 kDa. The FIA can improve the serum lysozyme activity, ACH50 activity and head kidney macrophage respiratory burst activity. The antibody level of the vaccinated fish without FIA were the highest at 4 weeks p. v. , while the antibody level of the vaccinated fish with FIA peaked at 5 weeks p. v. , which indicated FIA can significantly improve serum antibody levels of I. punctatus. The cumulative mortalities of the vaccinated fish were 93.33% （PBS）, 93.33% （FIA）, 50.00% （pompA）, and 13.33% （FIA ＋ pompA）, respectively, which correspond to an RPS of 46. 43% and 85.71% for pompA and FIA ＋ pompA, respectively. The result of serum bactericidal activity sugges- ted that the survival rates of A. hydrophila of the vaccinated fish were significantly lower than the survival rate of the control, and the bacterial survival rate of the FIA ＋ pompA vaccinated fish was the lowest. These indicated that pompA can be a subunit vaccine candidate vaccine, and FIA was a suitable adjuvant to enhanced the performance of pompA.