【作者】 韩国全；

【导师】 郭万柱；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2009， 博士

【摘要】 为适应以减毒胞内寄生菌为运载体的兽用基因疫苗研究发展趋势,研究构建含有减毒胞内寄生菌自身内源性诱导启动子的新型双功能基因疫苗表达载体,以增强基因疫苗诱导有效保护性免疫。基于以上考虑,本课题研究旨在研制含猪霍乱沙门菌内源性诱导启动子的新型双功能基因疫苗通用表达载体,既能够在运载体原核细胞中可调控表达携带抗原基因,又能够在真核细胞中启动表达携带抗原基因,以期与S.C500合用开发适于粘膜免疫的猪群重组活菌疫苗。为此,本研究首先扩增出猪霍乱沙门菌nirB、pagC两基因启动子区域片段,然后分析其启动活性,进一步构建新型双功能表达载体pCB-neo、pCC-neo,系统研究表达载体的启动活性特点,检验S.C500运送以pCB-neo、pCC-neo基础构建的基因疫苗的可行性。本课题研主要研究内容包括:Ⅰ.猪霍乱沙门菌nirB、pagC基因启动子区域分子克隆及其生物信息学分析为获得沙门菌nirB、pagC两基因启动子区域片段,利用PCR技术从猪霍乱沙门菌疫苗株S.C500基因组DNA中扩增出启动子PnirB、PpagC基因区域片段,经TA克隆筛选及测序鉴定,成功构建克隆重组质粒pUCX-PnirB、pUCX-PpagC。序列分析结果显示启动子PnirB、PpagC与其它沙门菌相关序列核苷酸同源性较高,分别为96%～100%、93%～99%,其中与霍乱沙门菌SC-B67核苷酸同源性分别高达100%、99%,说明两启动子基因区域高度保守。生物信息学网络软件NNPP分析结果显示,PnirB启动子基因区域序列有3个较强的启动子Promotor序列,PpagC启动子基因序列有12个较强的启动子Promotor序列,说明试验所扩增的两个基因启动子区域片段具有启动基因表达的基本元件。Ⅱ.沙门菌nirB、pagC基因启动子启动活性研究为考察沙门菌启动子的启动表达能力及特点,运用基因工程技术构建携带有沙门菌内源性启动子PnirB、Ppagc的单一启动子表达载体pPB、pPC,进一步构建其报告基因载体pPB-EGFP、pPC-EGFP,并转化到S.C500中进行相应的诱导性表达。通过表达菌液荧光观察、菌细胞表达荧光检测、SDS-PAGE检测,确证EGFP的获得成功表达,证实克隆获得的启动子PnirB、PpagC区域基因片段在S.C500中具有启动活性。Ⅲ.新型双功能基因疫苗通用表达载体的构建及表达活性研究为研制含沙门菌内源启动子的新型双功能基因疫苗表达载体,利用PCR技术亚克隆启动子必需区域基因片段PnirB（CB）（190bp）、PpagC（CC）（490bp）,并设计添加SD-Kozak杂合序列,替换pCI-neo启动子Pcmv下游的Chimeric intron基因区域,构建双功能疫苗表达载体pCB-neo、pCC-neo。进一步构建报告基因载体pCB-EGFP、pCC-EGFP。实现EGFP在S.C500中表达,证实PnirB（CB）、PpagC（CC）可以在S.C500中驱动外源基因的表达;实现EGFP在Vero细胞中表达,证实原载体中启动子Pcmv可以在Vero中驱动外源基因的表达,说明下游新插入的启动子不影响其启动活性:实现EGFP在小鼠体外培养巨噬细胞中表达,进一步证实S.C500运送以pCB-neo、pCC-neo为表达载体构建的新型基因疫苗的可行性。Ⅳ.内江猪白细胞介素15基因的分子克隆、表达及免疫佐剂作用研究为获得内江猪白细胞介素15基因序列片段,利用RT-PCR技术从经由Con A刺激的内江猪PMBC扩增了pIL-15基因,经TA克隆筛选及测序鉴定,成功构建克隆重组克隆质粒pUCX-njpIL-15。构建原核表达载体pMAL-pmIL-15,实现pIL-15在大肠杆菌中高效表达。MTT法试验显示融合产物MBP-pmIL-15经初步纯化、透析复性后,可明显增强淋巴细胞增殖。构建真核表达载体pCI-pIL-15,与pCI-gD（PRV）联合肌注免疫BALB/c雌性小鼠试验显示,pCI-pIL15能够促进小鼠脾脏CD4+T、CD8+T细胞数量增殖,加强基因疫苗pCI-gD（PRV）特异性中和抗体的产生:试验证实内江猪IL-15具有免疫佐剂作用。Ⅴ.猪瘟病毒E2蛋白主要抗原区编码基因的分子克隆与原核表达研究为获CSFV-E2基因片段,利用RT-PCR技术从PK-15细胞增殖的CSFV四川分离株中成功分子克隆E2基因,含有编码E2蛋白完整基因序列,共1119 bp,编码373 aa。构建原核表达载体pET-mE2（pe）、pET-E2（pe）,转化到宿主菌E.coli Rosetta-gami-TM（DE3）plysS进行表达研究,SDS-PAGE电泳检测及Western blot分析结果显示,试验成功表达的mE2（pe）、E2（pe）均具有一定的生物学免疫反应活性。CSFV-E2主要抗原位点区域（mE2）的成功原核表达,为进一步研究新型猪瘟病毒基因疫苗奠定理论基础。Ⅵ.猪IL-15与CSFV-E2双基因融合表达载体的构建及表达研究运用分子生物学实验技术将内江猪IL-15成熟肽基因与CSFV-E2主要抗原位点区域基因有机连接,构建融合双基因的克隆重组质粒pUCX-ME2-IL-15。构建真核表达载体pEGFP-ME2-IL-15,转染到Vero细胞中,荧光检测证实表达出增强型绿色荧光蛋白融合蛋白,证实试验构建的目的融合双基因ME2-IL-15可以在真核Vero细胞中获得表达,为进一步研究融合双基因ME2-IL-15奠定理论基础。构建融合双功能重组表达载体pCB-ME2-IL-15、pCC-ME2-IL-15及对照真核重组表达载体pCI-ME2-IL-15,分别将pCB-ME2-IL-15、pCC-ME2-IL-15转化到S.C500,SDS-PAGE电泳及Western-blott检测显示融合双基因ME2-IL-15在S.C500中成功表达,证实PnirB（CB）、PpagC（CC）可以在S.C500中驱动融合双基因ME2-IL-15的表达:将pCB-ME2-IL-15、pCC-ME2-IL-15、pCI-ME2-IL-15转染到哺乳动物Vero细胞中,mRNA转录、间接免疫荧光及夹心ELISA检测结果证实Pcmv可以在Vero中驱动外源基因ME2-IL-15的表达。为进一步研究S.C500运载双基因融合表达基因疫苗奠定试验基础。Ⅶ.融合双基因疫苗在S.C500中的稳定性及其对运载菌侵袭力影响的研究为了考察融合双基因疫苗表达载体在运载菌S.C500中的稳定性及其对运载菌侵袭力的影响,试验对构建的重组工程活菌苗进行系统研究。结果显示,双功能表达载体pCB-ME2-IL-15、pCC-ME2-IL-15及对照pCI-ME2-IL-15存在与否不影响运载菌S.C500的生物学特性,对其生化鉴定特性亦不影响,除Amp抗性外,重组菌与运载菌的药敏性一致;基因疫苗表达载体所插入的融合双基因ME2-IL-15增加其在运载菌S.C500体内的稳定性;基因疫苗的存在使运载菌S.C500对ST细胞的粘附能力、侵袭能力及其在ST细胞体内的增殖能力均有一定程度的下降,对运载菌本身的免疫效应有一定影响;口服免疫28日龄BALB/c雌性小鼠具有一定的稳定性和免疫安全性,可以有效的将基因疫苗运载至免疫小鼠机体细胞;为研究S.C500运载基因疫苗的免疫应答奠定前提。Ⅷ.融合双基因疫苗重组S.C500活菌苗口服接种家兔的免疫应答初步研究为检测家兔血清中S.C500抗体水平,建立猪霍乱沙门菌血清抗体间接ELISA检测方法,确立抗原最佳包被浓度为0.305μg/mL,血清最适稀释度为1:40,试验测定血清样品的D_{492 nm}值,判定标准通过标准阴性血清按照P/N比值公式进行判断,P/N≥2.1为阳性,1.5＜P/N≤2.1为可疑,1.5＜P/N为阴性;可以间接反应抗体水平的高低。家兔血清CSFV抗体检测结果显示对照A组（口服PBS缓冲液对照）无CSFV抗体,B组（口服S.C500/pCB-ME2-IL-15）、C组（口服S.C500/pCC-ME2-IL-15）、D组（口服S.C500/pCI-ME2-IL-15）、E组（口服S.C500/pCI-ME2）首免后14天即已产生CSFV抗体,第二、三次均呈上升趋势,B、C组免疫效果优于D、E组,B、C组之间相比C组免疫效果稍好,D、E组之间相比D组免疫效果稍好,试验研制的双功能疫苗通用表达载体pCB-neo、pCC-neo具有一定优势;S.C500抗体检测结果显示,试验构建的四个重组工程菌组经灌服均产生了较高的抗体水平,对照灌服PBS缓冲液组未能检测到抗S.C500特异性抗体的存在。免疫家兔T淋巴细胞增殖检测分析显示,对照A组家兔三次T淋巴细胞增殖率均低于15%,之间差异亦不大;B、C、D、E四个口服免疫组T淋巴细胞增殖率均高于18%以上,末次免疫后14d,B组增殖率有小幅度下降,C、D、E三组增殖率均有不同程度的增强提高。于末次免疫后14d,用4头份猪瘟兔化弱毒脾淋苗肌肉注射进行攻毒,以热反应为判定标准,保护率依次为0%、40%、60%、40%、25%,说明S.C500为运载体的猪瘟基因疫苗能够诱导家兔产生一定抗CSFV的保护性特异抗体,其中试验研制的基因疫苗通用表达pCC-neo携带的抗原基因保护率较高,具有一定优势。采用4×10¹⁰ CFU/只剂量的猪伤寒沙门菌野毒株口服接种攻毒试验,结果表明试验所构建的四个重组工程菌均能够诱导家兔产生一定水平的抗猪伤寒沙门菌野毒株的保护性特异抗体。更多还原

【Abstract】 Construction of a new dual-function gene vaccine expression vector containing hyperparasite intracellular endogenous inducible-promoter enhances the effective protective immunity induced by the DNA vaccine.This adapts to the research and development trend of veterinary gene vaccine carried by attenuated intracellular parasites. Based on the above considerations,this research aims to develop a new type expression vector for gene vaccine,using vaccine strain of Salmonella choleraesuis（S.C500） as the transporter with the endogenous promoter of its own.Thereby to creat swine live recombinant vaccines that can activate mucosal immunity.Therefore,this study first amplified the nirB,pagC promoter gene fragments of Salmonella choleraesuis,and then to carry out systematic analysis of promoters activity.Based on this,constructing the new type expression vector pCB-neo and pCC-neo,and analysising the characteristics of promoting activity of the vectors;in the end,verify the feasibility of gene vaccines deliveried by S.C500 built on pCB-neo,pCC-neo.The main research contents include:Ⅰ.Molecular cloning and bioinformatics analysis of the nirB,pagC promoter genes of Salmonella choleraesuisThe nirB,pagC promoter gene fragments were amplified from the genome DNA of the vaccine strain Salmonella choleraesuis S.C500.The recombinant plasmid pUCX-PnirB and pUCX-PpagC were successfully constructed,after TA cloning and sequencing.The sequential analysis showed that the homology between the PnirB,PpagC promoter genes of S.C500 and other Salmonellaes related nucleotide sequence are 96-100%,93-99% respectively.The nucleotide homology between the PnirB,PpagC promoter gene of S.C500 and Salmonella cholera SC-B67 are up to 100%,99%respectively.These revealed that the two promoter gene regions were highly conservative.The bioinformatics software NNPP analysis revealed that the PnirB promoter gene includes three strong promotor sequences,and the PpagC promoter gene includes 12 promotor sequences.It indicated that the two Promotor genes have the basic components to activated gene expression.Ⅱ.The activity study of nirB,pagC promoter genes of Salmonella choleraesuisIn order to study the activity of PnirB,PpagC promoter,we builted single-promoter expression vectors pPB,pPC with endogenous promoter of Salmonella by using genetic engineering techniques,and further constructed reporter gene vectors pPB-EGFP, pPC-EGFP,and then Transformed them into S.C500 for inducible expression.The results of observing the fluorescence of expression bacilli,detection the fluorescence of bacteria cells,and SDS-PAGE tests confirmed the successful expression of EGFP.The results showed that have promoter activity of the cloned PnirB,PpagC promoter gene fragments of S.CS00.Ⅲ.Construction and Expression of activity Research of New type dual-functional gene vaccine expression vectors To develop the new type dual-functional gene vaccine expression vector containing endogenous promoter of Salmonella,we subcloned necessary promoter gene core-region fragments of PnirB（CB） 190bp),PpagC（CC）（490bp）,and also added SD/Kozak Hybrid sequences to replace the Pcmv promoter downstream Chimeric intron genes of pCI-neo, and then built dual-functional gene vaccine expression vectors pCB-neo and pCC-neo. Further Constructed reporter gene vectors pCB-EGFP and pCC-EGFP.The successful expression of EGFP in S.C500 confirmed that PnirB（CB）,PpagC（CC） could drive foreign genes expression.The realization of EGFP expression in Vero cells,confirmed the original promoter vector Pcmv could also drive the expression of foreign genes,and the new inserted promoters don’t affect its promoter activity;the realization of EGFP expression in mice,confirmed the feasibility of delivery the gene vaccine based on pCB-neo,pCC-neo by S.C500.Ⅳ.Molecular cloning,expression and the effects of immune adjuvant of the Neijiang porcine interleukin-15 genePorcine interleukin-15 gene fragment was amplified from Neijiang porcine PMBC stimulated by Con A by RT-PCR technology.After the TA cloning to identify the selection and sequencing,the cloned recombinant plasmid pUCX-njpIL-15 was constructed successfully.Prokaryotic expression vector pMAL-pmIL-15 was constructed,and expressed at a high level in E.coli.MTT test showed that after initial purification, renaturation and dialysis,the fusion-protein MBP-pmIL-15 could significantly enhance the proliferation of lymphocyte.Construction of eukaryotic expression vector pCI-pIL-15, combining with pCI-gD（PRV）,and to immune BALB/c female mice by intramuscular injection,the result demonstrated that pCI-pIL15 contributed to proliferation of spleen CD4+T,CD8+T cells,strengthened the production of PRV-gD gene vaccine specific neutralizing antibody.The experiment confirmed that Neijiang porcine IL-15 has the immunity adjuvant function.Ⅴ.Molecule cloning of Classical Swine Fever Virus E2 major-antigen protein coding gene and Prokaryotic expression reserchThe CSFV-E2 complete gene was successfully cloned from CSFV proliferated in PK-15 cells by using RT-PCR technology.The CSFV-E2 gene contains a complete sequence coding E2 protein,includes 1119bp in total,and encodes 373aa.Construction of prokaryotic expression vector pET-mE2（pe）,pET-E2（pe）.Transformed them into host bacteria E.coli Rosetta-gami-TM（DE3） plysS,and then studied the expression of recombinant bacteria R/pET-mE2（pe） and R/pET-E2（pe）.The results of SDS-PAGE electrophoresis and Western blot test show that mE2（pe）,E2（pe） were expressed successfully have a certain degree of biological activity of the immune response.The successful prokaryotic expression of the main antigen sites（mE2） of CSFV-E2 lays theoretical basis for the further study of Classical Swine Fever virus gene vaccine.Ⅵ.Construction and expression of dual-genes fusion expression vectors of Neijiang porcine IL-15 and CSFV-E2 genes Combining Neijiang porcine IL-15 gene with the CSFV-E2 major-antigen protein encding gene by molecular biology technology to build a dual-genes fusion recombinant cloning plasmid pUCX-ME2-IL-15 and plasmid pUCX-dME2-IL-15.Construction of eukaryotic expression vector pEGFP-dME2-IL-15,transfected into Vero cells,The result of fluorescence detection confirmed the expression of enhanced green fluorescent fusion protein dME2-IL-15-EGFP,and the possibility of expression of the double-genes ME2-IL-15 in eukaryotic Vero cells.This provided theoretical basis for further study of the fusion dual-genes ME2-IL-15.Construction of recombinant eukaryotic expression vectors pCB-ME2-IL-15,pCC-ME2-IL-15,pCI-ME2-IL-15,transformed them into S.C500.The results of SDS-PAGE electrophoresis and Western-blot test showed the successful expression of the fusion dual-genes ME2-IL-15 in S.C500.The experiments confirmed that PnirB（CB）,PpagC（CC） promoter in S.C500 could drive the ME2-IL-15 expression. Transfected pCB-ME2-IL-15,pCC-ME2-IL-15,pCI-ME2-IL-15 into Vero cells,the results of immunofiuorescence and ELISA confirmed that the Pcmv could drive expression of exogenous gene ME2-IL-15 in Vero cells.This provided a experimental basis for further study of the expression of dual-genes vaccines delivering by S.C500.Ⅶ.Stability and immunizing safety of fusion dual-genes vaccines using Salmonella choleraesuis S.C500 as transporterIn order to investigate the stability of the integration of dual-genes vaccine expression vectors in S.C500 and its impact on invasiveness of the carrying bacteria,we built a systematic research of live recombinant vaccine.The results demonstrated that the vaccine expression vectors pCB-ME2-IL-15,pCC-ME2-IL-15,pCI-ME2-IL-15 didn’t affect the biological characteristics and their characteristics of biochemical identification of the carrying bacteria S.C500.The drug-sensitivity of the recombinant bacteria were same to the carrying bacteria except the Amp resistance.The fusion gene ME2-IL-15 that was inserted into the gene vaccine expression vectors increased the stability of the expression vectors in the carrying bacteria S.C500.The gene vaccine declined the adhesion and invasion of the carrying bacteria to ST cell,cut down the proliferative ability of S.C500 in ST cells;and also influenced the immune response of the carrying bacteria.It’s stable and safe to immune the 28-day-old BALB/c female mice at a certain degree.It could deliver the gene vaccines effectively to the immune cells of the mice.These studies provided a experimental basis for further study of the immune response of gene vaccine carrying by S.C500.Ⅷ.Preliminary study on immune response of the fusion dual-genes recombinant live vaccines delivered by S.C500 in rabbitsA method of indirect ELISA of Salmonella choleraesuis was established to detect the level of antibodies against the S.C500 in rabbits serum.The experiment Established the optimal antigen concentration was 0.305μg/mL,and the optimal serum dilution was 1:40. Testing the value D_492nm of the serum sample,determined through a standard negative serum in accordance with the P/N ratio,P/N≥2.1 positive,1.5＜P/N≤2.1 as suspicious,1.5 ＜P/N for negative.This indirectly reflected the level of antibody response.The results of testing the rabbits serum CSFV antibody showed that A group（oral PBS buffer） non-CSFV antibodies,but B group（oral S.C500/pCB-ME2-IL-15）,C group（oral S.C500/pCC-ME2-IL-15）,D group（oral S.C500/pCI-ME2-IL-15）,E group（oral S.C500/pCI-ME2） CSFV antibodies emerged after the first 14 days.The level of antibodies were higher after the second and third immunization.The immune effect of B,C group is superior to that of D,E group.But the immune effect of C Group was better than B group, D groups better than E group.The development of general dual-functional gene vaccine expression vectors pCB-neo and pCC-neo have a certain advantage.The results of S.C500 antibody detection showed that the four recombinant strains groups by gavage produced a higher antibody level,while the control buffer PBS gavage group failed to detect specific antibodies against the existence of S.C500.The analysis of T lymphocyte proliferation of the immuned rabbits showed that the control A group of T lymphocyte proliferation in rabbits were lower than 15%after immuning three times,and there were little differences among each time.The T lymphocytes proliferation rate of B,C,D,E oral immunization group were higher than 18%.After 14 days from the last immunization,the proliferation rate of B group declined a little,while the proliferation rate of C,D,E groups enhanced in different levels.After 14d from the last immunization,to infect the rabbits with four copies of Classical Swine Fever Virus of rabbits spleen vaccine by intramuscular injection,to determine the standards for the thermal reaction,the protection rates were 0%,40%,60%,40%,25%.The result demonstrated that the classical swine fever gene vaccine carrying by S.C500 in rabbits could induce some protective anti-CSFV-specific antibodies,the protection rates of the antigen gene carried by the general dual-functional gene vaccine expression vector pCC-neo was higher.So the pCC-neo has certain advantages.The rabbits infected with 4×10¹⁰ CFU/dose of Salmonella paratyphi wild strain by oral inoculation.The results showed that the four recombinant strains were able to induce rabbits to produce a certain level of anti-pig wild strain of Salmonella protective specific antibodies.更多还原