嗜水气单胞菌主要外膜蛋白免疫原性分析及其基因克隆与表达
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摘要
本研究选取鳗源嗜水气单胞菌L316和参考菌株嗜水气单胞菌TPS-30，分别制备了这两株菌主要外膜蛋白免疫刺激复合物(MOMP-ISCOMs)。应用这两种MOMP-ISCOMs分别免疫欧洲鳗鲡，每尾腹腔注射20ug MOMP-ISCOMs，免疫40天后，用3×10~8cfu和3×10~7cfu两个剂量的TPS-30分别腹腔注射攻毒，实验结果表明，免疫鱼能抵抗3×10~7cfu剂量TPS-30菌的攻击，TPS-30 MOMP-ISCOMs免疫鱼的相对免疫保护力为80％，而L316 MOMP-ISCOMs免疫鱼的相对免疫保护力达100％，Western-blot证实免疫过的鱼血清中含有针对嗜水气单胞菌外膜蛋白抗体，实验结果初步证实嗜水气单胞菌主要外膜蛋白免疫刺激复合物能够诱导宿主产生特异性的免疫保护作用。
根据已发表嗜水气单胞菌的外膜蛋白基因Omp的核苷酸序列设计引物，利用PCR技术，扩增、克隆了嗜水气单胞菌L316的主要外膜蛋白基因(Momp)，经T／A克隆，插入到pGEM-T系列载体上，测序分析结果表明Momp基因最长的开放阅读框(ORF)为1035nt，编码由344个氨基酸组成，分子量为36kDa的主要外膜蛋白质(MOMP)。应用Antheprot 5.0、ClustalW等分子生物学软件分析，显示主要外膜蛋白前24个氨基酸是较强的疏水性区域，可组成信号肽，其与Omp基因的同源率达96％，氨基酸的同源率高达98％。将此基因重组到表达质粒载体pGEX-4T-1上，转化到大肠杆菌BL21(DE3)中，经IPTG诱导后，可得到高效表达。SDS-PAGE电泳分析显示诱导表达的基因产物分子量约为62kDa，与预测的GST-外膜蛋白重组融合蛋白的分子量极为相似，Western-blot进一步证实，表达产物能被嗜水气单胞菌L316主要外膜蛋白特异性抗血清所识别，产生明显的染色条带，说明所表达的基因产物与天然的外膜蛋白抗原性一致。
鳗源嗜水气单胞菌L316主要外膜蛋白免疫刺激复合物的制备与应用研究，对研制鱼类疫苗学问题进行了新的初步探索；成功地克隆和表达嗜水气单胞菌L316主要外膜蛋白基因为在单因子水平上研究嗜水气单胞菌外膜蛋白的作用和免疫功能以及制备嗜水气单胞菌基因工程疫苗和亚单位疫苗奠定技术基础。
In this paper, the cloning of major outer membrane protein gene of Aeromonas hydrophila and the immunogenicity of the gene production were discussed. The major outer membrane proteins (MOMPs) of an Aeromonas hydrophila named AHL316 which isolated from diseased eel and the referential strain AhTPS-30 were purified and used for preparing Immunostimulating Complexs (MOMP-ISCOMs). The immunotrial was carried out by injecting European eel peritoneally with 20ug of MOMP-ISCOMs per eel. The immunized eels were challenged with 3 108 cfu and 3 107cfu of TPS-30 forty days post-injection respectively, and the sera from tolerant fishes were collected and analyzed. The results of the experiments showed that immunized European eel could resist to a lethal dose challenge(3 107cfu of TPS-30). The relative protection rate of the fishes immunized with the MOMP-ISCOMs of TPS-30 and L316 were 80% and 100% respectively. And the serum from immune fish could strongly bind to the outer membrane protein of Aeromonas hydrophila on We
stern-blot. The results demonstrated that the MOMPs were protective antigens and the MOMP-ISCOMs of Aeromonas hydrophila could induce the host to mount satisfied immunity.
A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene (omp) of Aeromonas hydrophila. With the specific primers, a target fragment about 1.1kb was amplified from Aeromonas hydrophila L316 via PCR .The target fragment was inserted into the linearized pGEM-T easy Vector. After enzyme restriction and sequencing analysis, The nucleotide data had been further analyzed by Antheprot 5.0 and ClutalW softwares. The analysis results showed that the cloned DNA fragment had a longest open reading frame (ORF) of 1035nt, it predicted to be encoded a 344-aa protein with the molecular weight of 36kDa. Hydrophobicity analysis suggested that the protein was highly hydrophilic, especialy at the first 24 amino-acid, this region could be function as signal peptide. The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene, and the alignment of the amino acid sequence was 98% .the recombinant plasmid was constructed with the
target gene and the expressing vector pGEX-4T-l and then was transformed into E.coli BL21 (DE3) The fusion protein was expressed under the IPTG inducing condition, and exhibited about 62kDa in size, very close to the predicted molecular weight of GST-MOMP. furthermore, the fusion protein was specifically recognized by anti-serum which raised against the major outer membrane protein of AhL316. Make all these together, it proved that the cloned gene represented the major outer membrane protein gene of AhL316, and the expressed gene products shared
identical antigenicity with the natural main outer membrane protein.
The studies on preparation and application of MOMP-ISCOMs of Ah L316 provided a new approach to fish vaccinology. The successfully cloning and expressing the major outer membrane protein gene of Ah L316 made it possible to describe this gene's function under a single factor level, and also provided technical support for developing an advanced gene engineering vaccine and subunit vaccine against Aeromonas hydrophila.
根据已发表嗜水气单胞菌的外膜蛋白基因Omp的核苷酸序列设计引物，利用PCR技术，扩增、克隆了嗜水气单胞菌L316的主要外膜蛋白基因(Momp)，经T／A克隆，插入到pGEM-T系列载体上，测序分析结果表明Momp基因最长的开放阅读框(ORF)为1035nt，编码由344个氨基酸组成，分子量为36kDa的主要外膜蛋白质(MOMP)。应用Antheprot 5.0、ClustalW等分子生物学软件分析，显示主要外膜蛋白前24个氨基酸是较强的疏水性区域，可组成信号肽，其与Omp基因的同源率达96％，氨基酸的同源率高达98％。将此基因重组到表达质粒载体pGEX-4T-1上，转化到大肠杆菌BL21(DE3)中，经IPTG诱导后，可得到高效表达。SDS-PAGE电泳分析显示诱导表达的基因产物分子量约为62kDa，与预测的GST-外膜蛋白重组融合蛋白的分子量极为相似，Western-blot进一步证实，表达产物能被嗜水气单胞菌L316主要外膜蛋白特异性抗血清所识别，产生明显的染色条带，说明所表达的基因产物与天然的外膜蛋白抗原性一致。
鳗源嗜水气单胞菌L316主要外膜蛋白免疫刺激复合物的制备与应用研究，对研制鱼类疫苗学问题进行了新的初步探索；成功地克隆和表达嗜水气单胞菌L316主要外膜蛋白基因为在单因子水平上研究嗜水气单胞菌外膜蛋白的作用和免疫功能以及制备嗜水气单胞菌基因工程疫苗和亚单位疫苗奠定技术基础。
In this paper, the cloning of major outer membrane protein gene of Aeromonas hydrophila and the immunogenicity of the gene production were discussed. The major outer membrane proteins (MOMPs) of an Aeromonas hydrophila named AHL316 which isolated from diseased eel and the referential strain AhTPS-30 were purified and used for preparing Immunostimulating Complexs (MOMP-ISCOMs). The immunotrial was carried out by injecting European eel peritoneally with 20ug of MOMP-ISCOMs per eel. The immunized eels were challenged with 3 108 cfu and 3 107cfu of TPS-30 forty days post-injection respectively, and the sera from tolerant fishes were collected and analyzed. The results of the experiments showed that immunized European eel could resist to a lethal dose challenge(3 107cfu of TPS-30). The relative protection rate of the fishes immunized with the MOMP-ISCOMs of TPS-30 and L316 were 80% and 100% respectively. And the serum from immune fish could strongly bind to the outer membrane protein of Aeromonas hydrophila on We
stern-blot. The results demonstrated that the MOMPs were protective antigens and the MOMP-ISCOMs of Aeromonas hydrophila could induce the host to mount satisfied immunity.
A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene (omp) of Aeromonas hydrophila. With the specific primers, a target fragment about 1.1kb was amplified from Aeromonas hydrophila L316 via PCR .The target fragment was inserted into the linearized pGEM-T easy Vector. After enzyme restriction and sequencing analysis, The nucleotide data had been further analyzed by Antheprot 5.0 and ClutalW softwares. The analysis results showed that the cloned DNA fragment had a longest open reading frame (ORF) of 1035nt, it predicted to be encoded a 344-aa protein with the molecular weight of 36kDa. Hydrophobicity analysis suggested that the protein was highly hydrophilic, especialy at the first 24 amino-acid, this region could be function as signal peptide. The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene, and the alignment of the amino acid sequence was 98% .the recombinant plasmid was constructed with the
target gene and the expressing vector pGEX-4T-l and then was transformed into E.coli BL21 (DE3) The fusion protein was expressed under the IPTG inducing condition, and exhibited about 62kDa in size, very close to the predicted molecular weight of GST-MOMP. furthermore, the fusion protein was specifically recognized by anti-serum which raised against the major outer membrane protein of AhL316. Make all these together, it proved that the cloned gene represented the major outer membrane protein gene of AhL316, and the expressed gene products shared
identical antigenicity with the natural main outer membrane protein.
The studies on preparation and application of MOMP-ISCOMs of Ah L316 provided a new approach to fish vaccinology. The successfully cloning and expressing the major outer membrane protein gene of Ah L316 made it possible to describe this gene's function under a single factor level, and also provided technical support for developing an advanced gene engineering vaccine and subunit vaccine against Aeromonas hydrophila.
引文
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84. Matsui K Protecive immunity by porin in experimental mouse sahnonellosis [J]. Microbiol Immunol, 1989,33(9):699.
85. Merino S, Nogueras M M, Aguilar A, Rubires X, Alberti S, Benedi VJ, Tomas JM.Activation of the complement classical pathway (C1q binding) by mesophilic Aeromonas hydrophila outer membrane protein [J]. Infect &Immun, 1998, 66(8):3825-31.
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87. Morein, S.Belak, et al. ISCOMs of Viral Envelope Proteins Against Aujeszky's Disease[J]. Veterinary Microbiology, 1989,20:143-154.
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89. Plumb A, Neutralization of channel catfish virus by serum of channel catfish [J]. Wild.Dia., 1990,9:324-330.
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93. Rodriguez L A Fernandez A I, Nieto. Production of the lethal acetylcholiesterase toxin by different Aeronmonas hydrophila strains[J]. Fish Diseases 1993,16:73-78.
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