【作者】 罗晓松；

【导师】 龙良启；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 博士

【摘要】 嗜水气单胞菌（Aeromonas hydrophila）是一种人畜共患的病原菌,也是一种食源性病原菌。嗜水气单胞菌引起的水生动物细菌性败血症,是我国水产养殖史上危害水生动物的种类最多且年龄范围最大、流行养殖水域类别最多且地区最广、流行季节最长且发病率和死亡率均高、造成的经济损失最严重的一种急性传染病。在现阶段水产养殖中预防和控制该病的主要措施是使用抗菌药物和利用细菌灭活疫苗免疫接种。但现有的商品化嗜水气单胞菌灭活疫苗和亚单位疫苗能够降低同源血清型菌引起的水生动物死亡率,不能降低发病率,并且对异源血清型菌的感染也不能提供较好的交叉保护。因而迫切需要更安全、高效的新型疫苗来预防和控制该传染病在水生动物中的发生与流行。与细菌灭活疫苗和亚单位疫苗不同,弱毒活疫苗能够重复提呈抗原和诱导机体产生持续性免疫应答和保护,因此,通过缺失毒力因子构建弱毒活疫苗已成为国内外疫苗研究的热点。本研究旨在以本地分离的Ah2056菌株为亲本菌,缺失其Ⅲ型蛋白分泌系统中编码嗜水气单胞菌外膜蛋白的aopB/aopD基因构建aopB^-/aopD^-缺失株,进一步缺失aopB^-/aopD^-株编码5-磷酸烯醇式丙酮酸莽草酸-3-磷酸合酶的aroA基因构建毒力丧失和营养缺陷型的aopB^-/aopD^-/aroA^-缺失株。并对aopB^-/aopD^-与aopB^-/aopD^-/aroA^-缺失株生物学特性和免疫保护进行研究。从而使aopB^-/aopD^-/aroA^-缺失株有可能成为一种安全、可靠并能够提供有效地交叉保护的嗜水气单胞菌弱毒活疫苗。本研究主要的工作如下:1.aopB^-/aopD^-缺失株的构建参照GenBank中嗜水气单胞菌aopB/aopD序列,从Ah2056基因组中扩增aopB/aopD上下游片断,连接到自杀性质粒pRE112上,构建了缺失369bp的aopB/aopD基因并含有蔗糖敏感基因（sacB）的重组自杀性质粒pREaopB/aopD。重组自杀性质粒pREaopB/aopD转化大肠杆菌X7213,以大肠杆菌X7213阳性克隆为供体菌,与受体菌Ah2056进行接合转移,经涂布氯霉素（Cm）抗性平板并转印到含10%蔗糖的平皿,筛选出Cm抗性（Cm^R）蔗糖敏感（Suc^S）的接合子,并用PCR鉴定重组自杀性质粒已经整合到染色体中。阳性接合子在不含NaCl的培养基中培养促使第二次同源重组。再涂布含10%蔗糖的平皿,并转印到Cm平板上,筛选出Cm敏感（Cm^S）蔗糖抗性（Suc^R）的克隆,并用PCR鉴定第二次交换,从而构建aopB^-/aopD^-缺失株。2.aopB^-/aopD^-/aroA^-缺失株的构建参照GenBank中嗜水气单胞菌aroA序列,从Ah2056基因组中扩增aroA上下游片断,连接到自杀性质粒pSUP202上,构建缺失1290bp的aroA基因并含有庆大霉素抗性基因（aacC1）的重组自杀性质粒pSUParoA。重组自杀性质粒pSUParoA转化大肠杆菌S17-1,以大肠杆菌S17-1阳性克隆为供体菌,与受体菌aopB^-/aopD^-缺失株进行接合转移,经涂布庆大霉素（Gm）和氯霉素（Cm）抗性平板,筛选Gm抗性（Gm^R）Cm抗性（cm^R）的接合子,阳性接合子在不含NaCl的培养基中培养促使第二次同源重组。再经涂布Gm的平板,并转印Cm到平板上进行筛选,筛出Cm敏感（Cm^S）Gm抗性（Gm^R）的克隆用PCR进行鉴定,确定发生第二次交换,从而构建aopB^-/aopD^-/aroA^-缺失株。3.aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株生物学特性经培养生化检定,aopB^-/aopD^-缺失株与亲本株的生化特性相似,aopB^-/aopD^-/aroA^-缺失株不能利用麦芽糖、甘露醇和蔗糖以及不能产尿酶、精氨酸脱羧酶和赖氨酸脱羧酶。aopB^-/aopD^-、aopB^-/aopD^-/aroA^-缺失株产胞外蛋白酶（酪蛋白酶、弹性蛋白酶）、淀粉酶、DNA酶和溶血素的能力都降低,说明同时降低了多个胞外毒力因子的毒力。aopB^-/aopD^-、aopB^-/aopD^-/aroA^-缺失株对EPC细胞无毒性作用,亲本株有很强的EPC细胞毒性作用,加入10^-3-10^-4μL的培养上清滤液即可抑制50%的细胞生长。在LB液体培养基中培养,aopB^-/aopD^-/aroA^-缺失株生长速度明显慢于亲本株,培养8-9h后停止生长,aopB^-/aopD^-缺失株生长速度与亲本株一致;在M9液体培养基中培养,aopB^-/aopD^-/aroA^-缺失株不能生长,aopB^-/aopD^-缺失株生长速度与亲本株一致;添加5种氨基酸培养试验说明aopB^-/aopD^-/aroA^-缺失株是依赖芳香族氨基酸生长的营养缺陷株。对银鲫的毒力试验,亲本株Ah2056 LD₅₀为1.0×10⁵CFU;aopB^-/aopD^-缺失株LD₅₀为9.7×10⁷CFU,其毒力比亲本株Ah2056降低了近1000倍;aopB^-/aopD^-/aroA^-缺失株LD₅₀大于10¹⁰CFU,其毒力丧失。4.注射aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株疫苗对银鲫的免疫和保护试验aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株组与Ah2056灭活疫苗组以1.0×10⁸CFU/尾,注射免疫100-150g银鲫。以超声波处理的Ah2056抗原包被,进行ELISA检测免疫后不同时间银鲫血清中的抗体效价。结果表明,各免疫组注射免疫7d后可以检测到血清抗体,以后抗体效价逐渐上升,21d后Ah2056灭活疫苗达到最大值,随后逐渐下降;而aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株在免疫7d后可检测到较高的血清抗体,随后抗体效价一直上升,并且能够维持较高的血清抗体水平,aopB^-/aopD^-缺失株组到第35d,仍可达到2¹²。将注射免疫35d后的银鲫,每组再分成2组,以同源株Ah2056剂量为100×LD₅₀和异源血清株AhJ-1剂量为100×LD₅₀进行注射攻毒。结果表明,灭活疫苗组鱼体内血清抗体效价35d后下降迅速,但对同源菌株的免疫保护力达到63.3%,而对异源血清菌株的免疫保护力明显下降仅为30.0%;aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株相对具有较高的免疫保护力,分别达到66.7%和63.3%,两者都显现出对异源血清菌株的免疫保护力,达到60.0%。结果表明注射aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株活疫苗,对同源（O:9）和异源（O:5）血清型菌株的攻毒都能使银鲫产生良好的保护效力,优于传统注射细菌灭活疫苗。5.口服aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株疫苗对银鲫的免疫和保护试验aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株组与Ah2056灭活疫苗组以1.0×10¹⁰CFU/尾,口服免疫100-150g银鲫。以超声波处理的Ah2056抗原包被,进行ELISA检测免疫后不同时间银鲫血清中的抗体效价。结果表明,各免疫组口服免疫7d后可以检测到血清抗体,以后抗体效价逐渐上升,14d后口服Ah2056灭活疫苗组达到最大值,随后逐渐下降;aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株在免疫7d后可检测到相对较高的血清抗体,随后抗体一直上升,并且能够维持一定的血清抗体水平,aopB^-/aopD^-缺失株到第35d,仍可达到2⁷。将口服免疫35d后的银鲫,每组再分成2组,以同源株Ah2056剂量为100×LD₅₀和异源血清株AhJ-1剂量为100×LD₅₀进行注射攻毒。结果表明,口服疫苗组比注射免疫组免疫保护力明显降低;口服疫苗组内,aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株对同源和异源血清菌株都具有相似的免疫保护力,在46.7%-50%之间;口服灭活疫苗组对同源和异源血清菌株的免疫保护力分别为23.3%和10%,口服aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株免疫保护力明显高于口服灭活疫苗组。结果说明口服aopB^-/aopD^-,aopB^-/aopD^-/aroA^-缺失株活疫苗,对同源（O:9）和异源（O:5）血清型菌株的攻毒都能使银鲫产生较低的保护效力,但其免疫保护力高于口服灭活疫苗。6.嗜水气单胞菌外膜蛋白在枯草芽孢杆菌中的表达用PCR方法从枯草芽孢杆菌、鱼源嗜水气单胞菌的基因组DNA中,分别扩增P43启动子和不含信号肽的外膜蛋白基因ompTS。各PCR产物经测序、酶切、连接到大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N相应位点构建穿梭表达载体pNWP430mp。电转枯草芽孢杆菌构建菌株BS01（pNWP430mp）,表达的外膜蛋白经SDS-PAGE和western blot检测。结果表明,穿梭表达载体pNWP430mp构建成功,构建的枯草芽孢杆菌在P43启动子的调控下,实现了外膜蛋白基因ompTS的表达。为实现枯草芽孢杆菌表达系统在水产养殖中的应用打下基础。更多还原

【Abstract】 Aeromonas hydrophila is a zoonotic pathogens,is also a food-bome pathogens.Aquatic bacterial septicemia is caused by Aeromonas hydrophila which induces high incidence of a disease and mortality in aquatic animals each year.It endangers the most aquatic species and scopes and different stage of fish and breed aquatic area.As an acute infectious disease,aquatic bacterial septicemia brings huge economic loss.At present,the extensive use of antibiotics and immunization are main methods to prevent and control it in acquculture.The current dominant commercial vaccines are still killed whole cell bacterins and subunit vaccines,which generally reduce the mortality of A.hydrophila infection.However those vaccines frequently failed to prevent severer morbidity and economic losses.Above all,it’s very urgent to develop a safe,effective,inexpensive and convenient vaccine to control A.hydrophila.In contrast,the attenuated live vaccines could re-present protective antigens and stimulate a lasting immune response that may be more efficacious in preventing the disease.Therefore,studies of A.hydrophila vaccines tend to focus on live attenuated vaccines constructed by inactivating virulence-associated genes.Based on above considerations,this study was aimed to develop a safer,effective vaccine strain and exploit A.hydrophila as a live vaccine.Ah2056 strain was used as the parent strain.The aopB/aopD（encoding A.hydrophila out membrane protein B,D） gene was deleted in the Ah2056 genome to decrease virulence and remove the risk of virulence return.Then the aroA gene（encoding 5-enolpyruvyl shikimate 3-phosphate synthase） was deleted in the aopB^-/aopD^-to construct the aopB^-/aopD^-/aroA^- strain for removing the risk of virulence return again.This aopB^-/aopD^-/aroA^-strain was lower virulence and aromatic amino acids dependent for removing the risk of virulence diffusion.The main research was described as follows:1.Construction of aopB^-/aopD^- mutantAccording to the GenBank sequences of A.hydrophila,the upstream and downstream of aopB/aopD gene were respectively amplified from Ah2056 genome,subcloned into suicide plasmid pRE112.The recombination suicide plasmids were designated as pREaopB /aopD which contained 369bp-deleted aopB/aopD and sucrose sensitive（sacB） gene. The aopB^-/aopD^-mutant,from which aopB/aopD gene was deleted,was constructed first. The E.coil donor strain X7213 transformed with the suicide plasmid pREaopB/aopD was conjugated with the recipient strain,the wide-type Ah2056 strain.After transconjugation,Chloramphenicol-resistant（Cm^R） and sucrose-sensitivity（Suc^S） transconjugants in which the whole plasmid had been incorporated into the recipient chromosome were analyzed by PCR.Colonies with the correct PCR profile were incubated in LB.Aliquots were then plated on to NA plates containing 10%（w/v） sucrose. Sucrose-resistant（Suc^R） colonies were tested for the chloramphenicol-sensitivity（Cm^S） phenotype,which was indicative of loss of plasmid vector sequences.Following this, Sue^R and Cm^S colonies were identified using PCR to determine the presence of the second crossover.2.Construction of aopB^-/aopD^-/aroA^- mutantThe upstream and downstream of aroA gene were respectively amplified from Ah2056 genome according to the GenBank sequences of A.hydrophila,subcloned into suicide plasmid pSUP202.The recombination suicide plasmids were designated as pSUParoA which contained 1290bp-deleted aroA and gentamycin resistance（aacCl） gene.The aopB^-/aopD^-/aroA^- mutant,from which aroA gene was deleted,was constructed first.The E.coil donor strain S17-1 transformed with the suicide plasmid pSUParoA was conjugated with the recipient,the aopB^-/aopD^- mutant.After transconjugation,Chloramphenicolresistant （Cm^R） and gentamycin-resistance（Gm^R） transconjugants were analyzed for the presence of a first crossover event.Colonies were then plated on to NA plates containing gentamycin.Gentamycin-resistance（Gm^R） colonies were tested for the chloramphenicol-sensitivity（Cm^S） phenotype,which was indicative of loss of plasmid vector sequences.Following this,Gm^R and Cm^S colonies were identified using PCR to determine the presence of the second crossover.3.Biological characteristic of aopB^-/aopD^-,aopB^-/aopD^-/aroA^- mutantBiochemical assays of aopB^-/aopD^- mutant was the same as those of Ah2056,while AhaopB^-/aopD^-/aroA^- mutant failed to ferment maltose,sucrose,xylose and produce urea, arginnine decarboxylase,lysine decarboxylase.The exoenzyme activities analysis of revealed that aopB^-/aopD^- and aopB^-/aopD^-/aroA^-mutant could not produce the detectable exoproteases,haemolysin,amylase and Dnase, while Ah2056 had a high level of exoenzyme activities.The cultural supematant of aopB^-/aopD^-,aopB^-/aopD^-/aroA^- were inoculated in EPC cell culture,and the result indicating that lost the toxigenicity to EPC cell.The cultural supematant of Ah2056 had highly the toxigenicity to EPC cell,which 10^-3-10^-4μl cultural supernatant could restrain the growth of 50%EPC cell.The growth curve in LB of aopB^-/aopD^-/aroA^- showed that the mutant grew significantly more slowly than aopB^-/aopD^- and Ah2056 did,and didn’t grow after incubation for 8-9h.The growth curve in M9 of aopB^-/aopD^-/aroA^- showed that the mutant didn’t grow,while aopB^-/aopD^- and Ah2056 did.The results suggest aopB^-/aopD^-/aroA^- was lower virulence and aromatic amino acids dependent. The i.p LD₅₀ value of Ah2506 and aopB^-/aopD^- mutant were determined to be approximately 1.0×10⁵ and 9.7×10⁷ CFU,respectively.The virulence of aopB^-/aopD^-mutant in Carassius auratus gibelio reduced about 1000 times than parent strain. The i.p LD₅₀ value of AhaopB^-/aopD^-/aroA^- mutant was higher than 1.0×10¹⁰,which lose its virulence.4.The immune and protective assay of the intraperitoneally immunized with aopB^-/aopD^-,aopB^-/aopD^-/aroA^- mutants in Carassius auratus gibelio100-150g Carassius auratus gibelio were intraperitoneally immunized with the aopB^-/aopD^-,the aopB^-/aopD^-/aroA^- mutant and the killed cell vaccine at the same dose of 1.0×10⁸CFU.ELISA was used to assay serum antibodies tirtre to A.hydrophila by coating with A.hydrophila sonicated antigen.The results showed that the serum antibodies tirtre of all groups were detect at week after intraperitoneally immunization. The antibody level for killed Ah2056 group was the highest at week 3 after intraperitoneally immunization,and then to descent gradually.The antibody level for the aopB^-/aopD^-,aopB^-/aopD^-/aroA^- mutant groups were both higher than those of the killed cell vaccine group at week after oral immunization,and then to increase gradually,which were keeping a highly serum antibodies tirtre.Thirty-five days later,the serum antibodies tirtre of aopB^-/aopD^-mutant group was 2¹².Carassius auratus gibelio were intraperitoneally immunized with the aopB^-/aopD^-, aopB^-/aopD^-/aroA^- mutant and the killed cell vaccine at the same dose of 1.0×10⁸CFU. Thirty-five days later,Carassius auratus gibelio were challenged i.p.with 100×LD₅₀ of Ah2056（O:9） and AhJ-1（O:5）.Relative percent survivals（RPS） of intraperitoneally immunized mutants were higher than those of the killed cell vaccine,and were 66.7%, 63.3%respectively.With the hige dose challeng,still offered Carassius auratus gibelio 63.3%-66.7%protection against homologous serovar and 60%protection against heterogenous serovar.The results suggest that intraperitoneally immunized with aopB^-/aopD^- or aopB^-/aopD^-/aroA^- is better than the killed cell vaccine.5.The immune and protective assay of the orally immunized with aopB^-/aopD^-, aopB^-/aopD^-/aroA^- mutants in Carassius auratus gibelio100-150g Carassius auratus gibelio were orally immunized with the aopB^-/aopD^-,the aopB^-/aopD^-/aroA^- mutant and the killed cell vaccine at the same dose of 1.0×10¹⁰CFU. ELISA was used to assay serum antibodies tirtre to A.hydrophila by coating with A. hydrophila sonicated antigen.The results showed that the serum antibodies tirtre of all groups were detect at week after oral immunization.The antibody level for killed Ah2056 group was the highest at week 2 after oral immunization,and then to descent gradually. The antibody level for the aopB^-/aopD^-,aopB^-/aopD^-/aroA^- mutant groups were both higher than those of the killed cell vaccine group at week after oral immunization,and then to increase gradually,which were keeping a highly serum antibodies tirtre. Thirty-five days later,the serum antibodies tirtre of aopB^-/aopD^- mutant group was 2⁷.Carassius auratus gibelio were orally immunized with the aopB^-/aopD^-,the aopB^-/aopD^-/aroA^- mutant and the killed cell vaccine at the same dose of 1.0×10¹⁰CFU. Thirty-five days later,Carassius auratus gibelio were challenged i.p.with 100×LD₅₀ of Ah2056（O:9） and AhJ-1（O:5）.Relative percent survivals（RPS） of orally immunized groups were lower than those of intraperitoneally immunized groups respectively.Both aopB^-/aopD^- and aopB^-/aopD^-/aroA^- group had the same of RPS of orally immunized groups.With the hige dose challeng,offered Carassius auratus gibelio only 46.7% protections against homologous serovar and 50%protection against heterogenous serovar. The results suggest that orally immunized with aopB^-/aopD^- or aopB^-/aopD^-/aroA^- is inferior to the killed cell vaccine.6.Expression of A.hydrophila out membrane protein in Bacillus subtilisWith the technology of PCR,the promoter P43 was amplified from B.subtilis total DNA,the outer membrane protein gene ompTS was amplified from A.hydrophila total DNA without its signal peptide-encoding sequence.The PCR productions were sequenced, digested and cloned into the corresponding site of an E.coli-B.subtilis shuttle vector pNW33N to generate the shuttle expression vector pNWP43omp.The recombinant vector was transformed into B.subtilis BS01.BS01 cells harboring pNWP43omp expressed the outer membrane protein,which were confirmed by SDS-PAGE and western blot.In conclusion,the shuttle expression vector pNWP43omp were successful constructed. Under the control of the promoter P43,the ompTS gene were expressed in B. subtilis.There is a good prospect for the application of B.subtilis expression system in aquaculture.更多还原