【作者】 张崇文；

【导师】 于涟；

【作者基本信息】 浙江大学 ， 生物医学工程， 2007， 博士

【摘要】 海洋弧菌是海水环境中普遍存在的一种革兰氏阴性细菌，其中某些种类是大黄鱼等海水养殖动物细菌性溃疡病的主要病原菌。鉴于滥用抗生素所造成的药物残留、细菌耐药性等不良后果，大黄鱼弧菌病的防治不得不从以化学药物治疗为主转向免疫预防。近年来，革兰氏阴性细菌外膜蛋白作为潜在的保护性抗原已越来越引起人们的重视。本研究从患病大黄鱼上分离得到三种常见病原菌：哈维氏弧菌、副溶血弧菌和溶藻弧菌，以哈维氏弧菌为主，克隆并表达了两个外膜蛋白基因，研究了它们作为亚单位疫苗的免疫原性。利用免疫蛋白质组学方法结合基质辅助激光解析飞行时间质谱（MALDI-TOF-MS）技术，鉴定了三种弧菌具有交叉免疫反应性的外膜蛋白成分，探索开发基因工程多价疫苗的可能性。哈维氏弧菌ompK基因含有一个804bp的开放读码框（ORF），编码由267个氨基酸组成的外膜蛋白OmpK，预测的分子量约为29KDa，pI为6.764。序列测定结果用BLAST软件搜索比对，和已登录的哈维氏弧菌外膜蛋白基因ompK的同源性高达99％，该基因在GenBank登录号为DQ279075；哈维氏弧菌gapdh基因含有一个996bp的开放读码框（ORF），编码由331个氨基酸组成的三磷酸甘油醛脱氢酶GAPDH，预测的分子量约为35KDa，pI为5.187。BLAST软件搜索比对发现多种不同弧菌的gapdh是高度保守的，它们的核苷酸同源性在91％以上。该基因在GenBank登录号为DQ184650。用LipoP软件分析可知ompK基因N端存在一个由20个氨基酸组成的信号肽，但gapdh基因没有发现信号肽序列，其到达细胞外膜的机制尚不明确。将ompK基因的信号肽序列去除经重叠延伸基因拼接扩增（SOE-PCR）和gapdh基因融合成ompK-gapdh,分别构建原核表达载体pET-30a-ompK，pET-30a-gapdh和pET-30a-ompK-gapdh，在E.coliBL21中成功大量表达。重组OmpK和GAPDH经Ni-NTA亲和层析柱纯化后免疫新西兰兔，产生的多克隆抗体不仅能够分别跟各自的免疫原发生免疫反应，而且能够和提取的天然外膜蛋白产生免疫印迹，表明重组蛋白和天然外膜蛋白具有相似的免疫原性。新鲜培养的哈维氏弧菌能够分别被兔抗r-OmpK和兔抗r-GAPDH抗血清凝集，证明OmpK和GAPDH位于细胞表面。根据同源建模原理模拟了哈维氏弧菌外膜蛋白OmpK、GAPDH的三维结构。外膜蛋白OmpK是由12个反向平行的β折叠经6个表面环和5个外周回折连成的桶状结构，以单体形式穿过细胞外膜形成通道；三磷酸甘油醛脱氢酶GAPDH只是两个结构域经一短链连接而成，每个结构域各包含9个反向平行的β折叠和6个α螺旋。提取自哈维氏弧菌的天然外膜蛋白沸水浴中加热一段时间后经SDS-PAGE，分别和兔抗r-OmpK和兔抗r-GAPDH抗血清反应。由于加热使得孔蛋白的构像发生改变，包括β桶状二级结构的丧失等，兔抗r-OmpK血清反应中出现了两条反应带，表明OmpK类似于OmpA家族，具有热修饰性。而兔抗r-GAPDH血清只出现一条带，说明GAPDH无热修饰性。将r-OmpK-GAPDH，r-OmpK，r-GAPDH及二者的混合物分别免疫大黄鱼，采集血清，ELISA检测抗体效价，尽管免疫组和对照组差别显著（p＜0.05），但抗体效价不是很高，表明体液免疫在外膜蛋白引起的抗病过程中作用有限。分离试验大黄鱼的巨噬细胞，测定其对啤酒酵母的吞噬百分率和吞噬指数。统计结果表明巨噬细胞吞噬百分率和吞噬指数不仅免疫组和对照组差别显著（p＜0.05），免疫组内r-OmpK-GAPDH和其他各组的差异也很明显（p＜0.05），意味着细胞免疫在外膜蛋白的免疫保护中具有重要作用。各试验组大黄鱼用500×LD₅₀的哈维氏弧菌感染，各免疫组的相对成活率r-OmpK（37.7％），r-GAPDH（40.0％）及二者的混合物（41.9％），r-OmpK-GAPDH（69.1％）都显著高于对照组，其中r-OmpK-GAPDH（69.1％）最高，说明OmpK和GAPDH具有一定的免疫原性，二者融合后显示出明显的协同作用，可以作为亚单位疫苗的候选成分。从患病大黄鱼中分离的8株病菌，经常规生理生化方法和以HSP60基因部分序列为基础的分子生物学方法鉴定为三种病原弧菌：哈维氏弧菌、副溶血弧菌和溶藻弧菌。SDS-PAGE和Western-blots结果显示三种弧菌的外膜蛋白和哈维氏弧菌全菌多抗反应分别在45kDa，35kDa和22 kDa处出现三条大致相同的免疫条带，提示它们有可能是三种弧菌的交叉保护性抗原。利用免疫蛋白质组学方法结合基质辅助激光解析飞行时间质谱（MALDI-TOF-MS）技术，鉴定出哈维氏弧菌与副溶血弧菌和溶藻弧菌具有交叉反应性的部分外膜蛋白分别是副溶血弧菌麦芽糖孔蛋白和溶藻弧菌的一种功能未知的孔蛋白。以这些蛋白为目标抗原制备基因工程疫苗，可望能够同时抵抗不同种弧菌的感染，在基因工程多价疫苗的开发方面具有重要意义。更多还原

【Abstract】 Marine vibrios are Gram-negative bacteria ubiquitous in marine environment, someof which are main pathogenic organisms responsible to the bacterial ulcer of marineanimals. In view of the bad aftermath like the drug residue in the fish flesh and/or thedevelopment of drug-resistance in pathogens due to the misuse of antibiotics, the curepolicy to vibriosis has to change from the application of chemotherapy to theimmunotherapy. In recent years, people attached more and more importance to the outermembrane proteins （OMPs） of Gram-negative bacteria for their potential to serve asprotective antigens. This research separated three common pathogens from the infectedlarge yellow croaker: Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus. WithV. harveyi as main object, two of its OMP genes were cloned and their immunogenicity assubunit vaccines was detected. By the immunoproteomics involving 2-DE andMALDI-TOF-MS, the components of OMP that can induce cross-immunoreaction amongthree vibrios were identified, the probability of developing poly-value vaccines wasexplored.The ORF sequence of ompK from V. harveyi comprised 804 nucleotides encodingOmpK of 267 amino acids with a calculated molecular mass of 29 kDa while the ORFsequence of gapdh from K harveyi comprised 996 nucleotides encodingglyceraldehyde-3-phosphate dehydrogenase （GAPDH） of 331 amino acids with acalculated molecular mass of 35 kDa. Blastn indicates that nucleotide homology of ompKis 83-99% among the Vibrios, and gapdh is more than 91%. The nucleotide sequences ofboth ompK and gapdh were deposited in the GenBank database under the accessionnumbers of DQ279075 and DQ184650, respectively.The analysis with LipoP software showed that OmpK included a signal peptide of 20amino acids in the N-terminal domain, but it was not found in the GAPDH. It is stillunclear how GAPDH travels to the cell surface, ompK gene, of which the signal peptidesequence was omitted, was fused with gapdh by SOE-PCR into ompK-gapdh, and clonedinto pET-30a（+）. The expression systems were constructed, named pET-30a-ompK,pET-30a-gapdh and pET-30a-ompK-gapdh, respectively. The recombinant proteins wereexpressed in large scale in E.coliBL21 and purified. The anti-r-OmpK serum and theanti-r-GAPDH serum could specifically recognize the r-OmpK and the r-GAPDHrespectively. Moreover, both antisera could effectively recognize the native OMPsextracted from the fresh-cultured V. harveyi, indicating that r-OmpK and r-GAPDH can induce the same immunoreaction as the native OMPs. The anti-r-OmpK andanti-r-GAPDH can agglutinate the fresh-cultured V. harveyi, indicating that both OmpKand GAPDH all appeared on the surface of the cells.The simulation of the three-dimensional structure of OmpK showed the presence of12 anti-parallelβ-sheets, which organize theβ-cylinder, 6 surface-exposed loops and 5short periplasmic turns, indicating that OmpK structurally belonged to the porin. Themembrane-spanning anti-parallelβ-strands connected by short periplasmic turns andsurface-exposed loops made up a pore inside the cylinder. Different from OmpK, thethree-dimensional structure of GAPDH was mainly composed of two domains that wereconnected by a chain. Each domain contained 9 anti-parallelβ-strands and 6α-helices,apparently not belonging to porin family. Two bands occurred when the anti-r-OmpKserum reacted with the OMPs, meaning that OmpK was the heat modifiable protein likeOmpA. When membrane porins were heated at 100℃for long periods of time, thesecondary structure of the porins was relaxed and a second band appeared, probably due toconformation modifications of the protein like the lost of theβ-barrel structure. Whileonly one band occurred for anti-r-GAPDH, meaning that GAPDH was not heat modifiableprotein.The r-OmpK-GAPDH, r-OmpK, r-GAPDH and their mixture were served as antigensto immunize large yellow croaker respectively. Although the ELISA for the titers of thesera showed a significant difference （p＜0.05） between the trial groups and the controlgroup, the antibody value was not high, suggesting that the humoral immunity acted lesson the protection against viriosis in fish. Percentage of phagocytosis in cells immunizedwith r-OmpK, r-GAPDH, r-OmpK-GAPDH or the mixture of r-OmpK and r-GAPDHshowed variation in comparison with the controls and was statistically significant （P＜0.05）. Similar results were obtained for the phagocytic index. Cells treated withr-OmpK-GAPDH had significantly higher values than those in other three treated groups,but these three treated groups were not significantly different from each other. Theseresults demonstrated that the cellular immunity played more important role in theprotection against viriosis in fish. Fish in every group were challenged with the dose of500 LD₅₀ strains （5×10⁶ cfu per fish）. The fish immunized with r-OmpK-GAPDHremained high survival percentage （69.1%） compared not only with the control group, butalso with r-OmpK （37.7%） and r-GAPDH （40.0%）, even with the mixture of OmpK andGAPDH （41.9%）, suggesting a synergistic effect was created between OmpK andGAPDH when they were produced as a fusion protein. By common physiological and biochemical methods and the phylogenetic analysisbased on the partial sequences of HSP60 genes, eight strains of pathogens separated frominfected large yellow croakers were identified distinctly to the different strains whichbelonged to V. harveyi, V. parahaemolyticus and V. alginolyticus. The antiserum againstthe whole V. harveyi was prepared and used to screen OMPs from V. parahaemolyticusand V. alginolyticus that could cross-react with it. By the immunoproteomics methodinvolving 2-DE and MALDI-TOF-MS, the components of OMP that can inducecross-immunoreaction among three vibrios were identified as the maltoporin of V.parahaemolyticus and a putative porin of V. alginolyticus. Gene engineering vaccinesbased on these protein antigens are expected to protect simultaneously from the infectionof sereral different vibrios, and will give an important suggestion to the development ofpoly-value gene engineering vaccines.更多还原