【作者】 毛芝娟；

【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】 副溶血弧菌Vibrio parahaemolyticus是我国海水养殖鱼、虾、贝类的重要病原，每年由此菌引起的弧菌病给养殖业造成严重的损失。在浙江省沿海网箱养殖中，该菌与哈维氏弧菌、溶藻弧菌等重要病原弧菌一起，主要危害大黄鱼幼鱼，每年的高温季节可引起大量的死亡。前期研究中以福尔马林灭活的副溶血弧菌全菌苗免疫大黄鱼，获得了较好的保护效果。本研究开展该菌主要保护性抗原成份之一—外膜蛋白的克隆、表达和免疫原性研究，为高效疫苗的研制提供依据。以饱和硫酸铵梯度盐析法结合琼脂糖柱sephadex G200凝胶过滤法初步纯化了大黄鱼血清免疫球蛋白，再经割胶回收法得到进一步纯化的重链片段，分子量约为75kDa，免疫新西兰兔后获得兔抗大黄鱼免疫球蛋白血清，为ELISA和Western blotting检测技术奠定了基础。采用十二烷基肌氨酸钠法(Sarkosyl)提取不同来源的4株副溶血弧菌分离株的主要外膜蛋白，通过SDS-PAGE比较分析主要条带和分子量组成。SDS-PAGE显示4株菌的主要外膜蛋白由5-6条组成，分子量分布在21-54kDa，其中，21kDa、28 kDa和35 kDa的条带是所有菌株共有的，表明副溶血弧菌中部分主要外膜蛋白条带可能是保守的。制备了不同抗血清，对4菌株的外膜蛋白进行免疫印迹分析表明，副溶血弧菌的主要外膜蛋白具有免疫原性，全菌兔抗血清主要与一分子量为21kDa的主要外膜蛋白条带发生印迹反应；感染后恢复期大黄鱼血清识别分子量为21kDa、28 kDa、35 kDa和44 kDa的蛋白条带，可能与抗感染免疫应答有关；其中21 kDa的条带是所有抗血清均识别的，提示了该蛋白良好的免疫原性。以N端氨基酸测序法初步确定了21kDa的主要免疫原性外膜蛋白为ompW，设计引物从浙江省养殖大黄鱼分离株zj2003株基因组DNA克隆到该蛋白成熟肽的编码序列，基因登录号为DQ425109。亚克隆到原核表达载体pET30a中获得重组载体pET30a-ompW，转染大肠杆菌Escherichia coli BL21，在IPTG诱导下表达出预期分子量(27kDa)的重组蛋白，重组蛋白以包涵体形式存在。以镍柱亲合层析法获得纯化的重组蛋白，免疫新西兰兔获得高免血清，该血清在免疫印迹中识别重组蛋白，提示了重组蛋白得到正确表达。重组蛋白同时被全菌兔抗血清和感染恢复期大黄鱼血清所识别，进一步验证了与21kDa外膜蛋白免疫反应性的一致。研究结果显示了OmpW具有良好的免疫原性，可能应用于种的快速鉴定和疫苗开发。克隆了副溶血弧菌zj2003株的4种外膜蛋白ompV、ompK、ompU和TolC及两种铁调外膜蛋白psuA和pvuA，基因登录号分别为DQ016303、DQ016304、DQ006287、DQ141606、DQ141607和DQ141608。并亚克隆到原核表达载体pET30a，获得重组质粒pET30a-OMP，转化到大肠杆菌BL21，在IPTG诱导下实现大量表达，重组蛋白均以包涵体形式表达。除重组蛋白TolC在SDS-PAGE中显示的分子量(47kDa)大于预期分子量(39.6kDa)，其他重组蛋白的分子量与预期相符，分别为32kDa、33 kDa、48 kDa、80kDa和78 kDa。以镍柱Ni-IDA亲和层析法纯化得到重组蛋白，结合不同抗血清进行免疫印迹，分析了各蛋白的免疫反应性及在体外培养中的表达情况。常规培养条件下提取的外膜蛋白制备的兔抗血清存针识别ompK、ompU和YolC三种重组蛋白的抗体，发生了印迹反应，显示了此三种蛋白具有免疫反应性，并且对应天然蛋白在菌体中得到表达；但未与重组的ompV、pvuA和psuA发生识别反应；提示了对应的三种外膜蛋白在本研究培养条件下未得到表达或未能引起抗体应答，有必要进一步摸索合适的培养条件使更多的抗原蛋白得到表达。副溶血弧菌ompK与哈维氏弧菌ompK多抗发生交叉识别反应，提示了两种蛋白具有同源性。纯化的7种副溶血弧菌重组外膜蛋白ompW、ompV、ompK、ompU、TolC、psuA和pvuA依生物功能的不同分为四组：ompW和ompV组，ompK组，ompU和TolC组，及psuA和pvuA组，采用腹腔注射的方式免疫1~+龄大黄鱼，以间接ELISA法检测免疫后4-8周内特异性抗体水平；在免疫后第28天进行活菌攻击测试各组的免疫保护率；通过免疫印迹法检测了感染后恢复期大黄鱼血清对重组外膜蛋白的抗体产生情况，分析了各重组外膜蛋白的免疫原性。免疫后4-8周，重组蛋白免疫鱼血清抗体效价持续升高，在28天后的攻击试验中获得80％以上的免疫保护率；感染后恢复期大黄鱼血清识别除TolC外的所有重组蛋白。研究结果显示了6种副溶血弧菌外膜蛋白ompW、ompV、ompK、ompU、psuA和pvuA具有良好的免疫原性，是宿主免疫应答的保护性抗原；尤其是ompK单个蛋白免疫组获得90％的保护率，由于该蛋白是弧菌中广泛存在的保守性蛋白，极有可能作为制备亚单位疫苗等高效弧菌疫苗的候选抗原。将副溶血弧菌重要的免疫原性铁调外膜蛋白pvuA的部分编码片段pvuA1与绿色荧光蛋白EGFP基因一起，构建入真核表达载体pCI，获得重组载体pCI-pvuA1-EGFP，其中pvuA1与下游EGFP形成一完整的1551bp的ORF，预期编码一分子量为57.16kDa的融合蛋白；纯化质粒转化Vero细胞后观察到绿色荧光的表达。以20μg／尾的剂量肌肉注射均重为75g的大黄鱼，4周后以活菌攻击，获得了中等程度的保护；免疫印迹法检测到了重组质粒免疫鱼血清内存在pvuA重组蛋白的抗体，指示了DNA疫苗免疫后鱼体表达了目的蛋白，并诱导产生了相应抗体。更多还原

【Abstract】 Vibrio parahaemolyticus has been listed one of the pathogenic vibrios endangeringnet-cage cultured large yellow croaker(Pseudosciaena crocea) in coastal areas of China.Immunoglobulins of large yellow croaker was purified after salting out with gradientconcentration of saturate ammonium sulfate and filtering with Sephadex G200, thenrecovered from SDS-PAGE gel. And polycolonal antibody was prepared with Newzealand rabbits for ELISA and Western blotting detection. Outer membrane proteins(OMP) were extracted from four strains of V. parahaemolyticus by method of detergenttreatment with Sarkosyl and separated by SDS-PAGE. Three different antiserum, rabbitantiserum against formalin-killed-cells (FKC) of V. parahaemolyticus zj2003 (apathogenic strain isolated from diseased large yellow croaker, from Xiangshan bay,Zhejiang province), rabbit antiserum against OMP of the bacteria and large yellowcroaker convalescent antiserum were prepared for western blotting of the OMPpreparation. SDS-PAGE profiles appeared 5-6 main bands with molecular weights(MW) about 21kDa to 54kDa, four common bands with MW of about 21kDa, 28 kDa,and 35 kDa were present among the strains. Western blotting results showed that theOMP preparations were differently recognized by various antiserum. Both of the twomost abundant bands with MW of about 28kDa and 35kDa were recognized bY rabbitantiserum against OMP, which indicated that the main outer membrane proteins ofV.parahaemolyticus are immunogenic. The other main band with MW of about 21kDawas the dominantly one that reacted with rabbit antiserum against FKC; and mainly fourbands with MW of about 21kDa, 28kDa, 35kDa and 44kDa were recognized by the fishconvalescent sera, it is suggested such OMPs should have been exposed to the host’simmune system during vaccination and in vivo infection and may be of some significancefor vaccine development. The protein band with MW of about 21 kDa reacted with all theantiserum, which well displayed the strong immunogenicity of this protein.The immunogenic OMP of Vibrio parahaemolyticus zj2003 with MW of about21kDa was preliminary confirmed as ompW by method of N terminal amino acidresidues sequencing. Primers were designed to amplify the mature peptide codingsequence of the protein, and the sequence was sent to GenBank under the accessionnumber DQ425109. The sequence was ligated into prokaryotic expression vector pET30a,then the recombinant plasmid pET30a-ompW was transferred to Escherichia coli BL21,the strain was induced to express the recombinant protein by addition of IPTG.SDS-PAGE showed the recombinant protein appeared the expected MW of 27kDa. The recombinant protein was expressed in fusion bodies and purified with Ni-IDA affinitychromatography. Rabbit antiserum prepared against the purified protein well recognizedthe protein, which indicated that the protein was appropriately expressed. And therecombinant protein also reacted with rabbit antiserum against FKC of V.parahaemolyticus and large yellow croaker convalescent sera. The results suggested thatomp W was strong immunogenic and may act as target gene for species specific rapididentification of V.parahaemolyticus and may be considered a powerful vaccine Candidateagainst the bacteria.Four OMPs and two iron-regulated outer membrane proteins (IROMPs) ofV.parahaemolyticus zj2003, ompV, ompK, ompU, TolC, psuA and pvuA were cloned andthe genes were registered in GenBank (the accession number DQ016303, DQ016304,DQ006287, DQ141606, DQ141607 and DQ141608, respectively). Then the maturepeptide coding sequences of the proteins were ligated into prokaryotic expression vectorpET30a, the recombinant plasmids pET30a-OMP were transferred to E.coli BL21 and thestrains were induced to express the recombinant proteins by addition of IPTG. Therecombinant proteins were expressed in fusion bodies and purified with Ni-IDA affinitychromatography. Except ompU, the other five recombinant proteins showed expectedMW in SDS-PAGE, 32kDa, 33 kDa, 48 kDa, 80kDa and 78 kDa, respectively.Recombinant ompU moved more slowly in SDS-PAGE, appeared a MW of 47kDa, somehigher than the calculated value (39.6kDa). Western blotting results showed that threerecombinant proteins, ompK, ompU and TolC reacted with rabbit antiserum against OMPof V. parahaemolyticus, while the other three proteins not. It is suggested that the formerthree proteins were expressed under routinely culture condition and they remained thereactivity of native proteins. Combined with results of SDS-PAGE, it is indicated thatompV, psuA and pvuA were not expressed under the present culture condition or notenough expressed to elicit antibody response. Further study must carry out to solve theproblem for expression of these proteins. And rabbit antiserum against ompK of V.harveyicross-reacted with recombinant ompK of V.parahaemolyticus, which indicated thehomology of the protein between the two vibrio species. That’s may be of somesignificance for vaccine development.The purified seven OMPs of V. parahaemolyticus were classified into four groupsconsidering of their biological function: (1) ompW&ompV; (2) ompK; (3) ompU&TolC;(4) psuA & pvuA. The protein or proteins combination was administrated to large yellowcroaker by method of intraperitoneall injection (i. p.) with the dose of 100μg per fish. Specific antibody level was detected by indirect ELISA during 4-8 weeks postvaccination and challenge experiment was undertaken for determination of relativepercent survival (RPS) of the immunized fish. Western blotting was carried out with largeyellow croaker convalescent sera. The results showed that antibody titers to theimmunized recombinant proteins increased 4-8 weeks post vaccination. The recordedRPS of vaccinated groups reached above 80%. And the fish convalescent sera reactedwith all recombinant proteins except TolC. It is suggested that six OMPs ofV.parahaemolyticus ompV, ompW, ompK, ompU, psuA and pvuA, are immunogenicduring in vivo infection, and may be of great significance for vaccine development.Especially, fish group immunized with recombinant ompK achieved a high protection of90%. For the wide expression of this conserved protein among vibrio species, ompKmust be paid enough attention for vaccine development against vibriosis.Partial sequence of pvuA of Vibrio parahaemolyticus zj2003, was ligated intoeukaryotic expression vector pCI together with EGFP, the gene encoding enhanced greenfluorescent protein, resulted the recombinant plasmid pCI-pvuAl-EGFP with a targetORF of 1551bp, encoding a fusion protein with the expected MW of 57.16kDa. EGFPexpression by Vero cells transferred with the recombinant vector was observed withfluorescent microscope. The DNA vaccine prepared with the purified plasmids wasadministrated to large yellow croaker by intramuscular injection (i.m., 20μg per fish).Challenge experiment with live bacteria resulted with a RPS of 50% 4 weeks postvaccination. And antibody against the recombinant pvuA was detected by Westernblotting with antiserum of the immunized fish. It is indicated that the target Protein wasexpressed in vivo and elicited antibody reponse.更多还原