CAJ | 학술논문

为评价嗜水气单胞菌(Aeromonas hydrophila, Ah)PDK06株外膜蛋白A(ompA)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GenBank上嗜水气单胞菌外膜蛋白ompA基因序列(GenBank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pET-32a( +) -pompA; 然后将该质粒转入BL21 (DE3)后用IPTG诱导表达, 经纯化后得到pompA重组蛋白; 最后将pompAompA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pompA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 kDa;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; pompA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%( pompA)和13. 33%( FIA+pompA) , pompA和FIA+pompA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+pompA细菌含量最低. 说明pompA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.
위평개기수기단포균(Aeromonas hydrophila, Ah)PDK06주외막단백A(ompA)적원핵표체산물시부구유항원성급기대반점차미 ( Ictalurus punctatus)적면역보호력, 근거GenBank상기수기단포균외막단백ompA기인서렬(GenBank등록호: AF146597)설계거신호태인물, 확증획득일단약1 000 bp적서렬, 경감정정학후, 구건중조표체질립pET-32a( +) -pompA; 연후장해질립전입BL21 (DE3)후용IPTG유도표체, 경순화후득도pompA중조단백; 최후장pompAompA단백여불씨불완전좌제( FIA)혼합면역반점차미 , 평개기면역효과. 결과현시:극륭적기수기단포균pompA기인전장위960 bp, 편마319개안기산, 융합표체적중조단백상대분자질량약위53, 670 kDa;FIA능구제고반점차미 적용균매활성、 ACH50활성、 두신거서세포호흡폭발활성; pompA조항체수평재제4주체도봉치, FIA면역조항체수평재제5주체도봉치, 차FIA능구현저제고반점차미 적항체수평; 공독후각조루계사망솔분별위93. 33%( PBS)、 93. 33%( FIA)、 50. 00%( pompA)화13. 33%( FIA+pompA) , pompA화FIA+pompA조상대보호솔분별위46. 43%화85. 71%; 면역조파기관중적세균수량현저저우대조조, 차FIA+pompA세균함량최저. 설명pompA가이작위일개아단위역묘후선역묘, 이FIA좌제능교호적증강아단위역묘적면역효과.
The expression of outer membrane protein A (OmpA) from Aeromonas hydrophila (PDK06) in Escherichia 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 se-quence 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 sub-unit vaccine candidate vaccine, and FIA was a suitable adjuvant to enhanced the performance of pompA.