【摘要】 根据哈维氏弧菌外膜蛋白OmpK的基因序列设计一对引物,应用聚合酶链式反应(PCR)方法,从分离自患病大黄鱼的哈维氏弧菌基因组中扩增获得一段约800bp的序列。将其克隆到pGEMTeasy载体,测序结果证明该序列是哈维氏弧菌外膜蛋白OmpK基因。用PCR方法去除其信号肽序列,定向克隆到原核表达载体pGEX4T2构建重组表达质粒pGEX4TOmpK。IPTG诱导后能够在大肠杆菌BL21中高效表达分子量约为53kD的GSTOmpK融合蛋白。用纯化后的融合蛋白免疫新西兰兔获得了高效价的抗血清。Westernblotting分析表明,它与从哈维氏弧菌中提取的约27kD的外膜蛋白能够发生特异反应,提示外膜蛋白OmpK可能是哈维氏弧菌的重要保护性抗原之一。更多还原

【Abstract】 Vibrio harveyi is a causative agent of vibriosis that infects large yellow croaker(Pseudosciaena crocea)as well as some other marine fish and brings severe loss to their culture. The development of vaccines against vibrio is recognized as a priority to the complement of existing control measures. Recent studies emphasized the role of the outer membrane protein (OMP) of pathogenic bacteria in protective antigenicity. A target of protective immunity, OmpK, is the receptor for KVP40 which is a broad-host-range vibriophage. To investigate the possibility of OmpK as vaccine candidate, primes were designed according to OmpK gene sequence published in GenBank, and a piece of DNA sequence about 800 bp was amplified by PCR from genomic DNA of V. harveyi isolated from infected P. crocea. The gene was cloned into pGEM-Teasy vector and sequenced. The Blast alignment result indicated that it was indeed the outer membrane protein(OmpK) gene of V. harveyi. After its signal peptide sequence was discarded by PCR and the remains was inserted into E.coliexpression vector pGEX-4T-2, a fusion expression vector pGEX-4T-OmpK was constructed, which can amply the expression of a 53 kD fusion protein GST-OmpK in E.coli BL21 when induced by IPTG. The fusion protein was purified and used as antigen to immunize the New Zealand rabbit, the antiserum with high antibody value was acquired. The Western-blotting shows that the anti-recombinant OmpK serum can specifically react to the 27 kD component of natural Omps extracted from V. harveyi. This result indicates that the OmpK may be one of the important protective antigens of V. harveyi, and may play a role in protecting fish from infection of V. harveyi.更多还原