【作者】 温建新；

【导师】 王金宝； 牛钟相；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2008， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种以母猪繁殖障碍和仔猪呼吸道疾病为特征的病毒性传染病。细菌DNA具有广谱的免疫刺激作用,实验证明人工合成的CpG-ODN可以模拟细菌DNA诱发机体产生强烈的Th1型免疫应答。CpG-ODN作为一种新型的免疫佐剂已成为分子免疫学研究的热点之一。在本研究中,首先筛选出对猪具有最强免疫刺激活性的CpG-ODN,将PRRSV ORF5基因克隆至哺乳动物真核表达载体pVAX1,构建了pVAX1-ORF5真核重组质粒;将优选的CpG-ODN插入到pVAX1-ORF5中,构建成含有CpG基序的真核重组载体质粒CpG-pVAX1-ORF5。为了进一步检测其免疫原性,进行SPF小鼠动物试验,检测其免疫反应指标。用CpG-pVAX1-ORF5免疫仔猪,检测对本体动物的免疫保护效果。1、不同序列的CpG-ODN对猪体外周血淋巴细胞和脾脏免疫细胞免疫刺激作用的研究:为了筛选出对猪体有免疫刺激活性的CpG寡聚脱氧核苷酸(CpG-ODN),人工设计合成了9种未甲基化CpG-ODN不同序列,分别作用于健康猪外周血淋巴细胞和脾脏免疫细胞,进行体外转化试验。用四甲基偶氮唑盐(MTT)比色法检测外周血淋巴细胞转化效果和脾细胞的增殖能力和代谢能力,通过测得的OD490值,计算出淋巴细胞增殖指数(SI),筛选CpG-ODN的免疫刺激作用。结果显示:九种不同序列的CpG-ODN对猪外周血淋巴细胞和脾脏免疫细胞均具有不同程度的刺激作用,其中CpG-ODN7对淋巴细胞增殖能力的增强效果最为明显(SI=3.5),CpG-ODN6对猪脾脏免疫细胞的刺激指数最大(SI=2.7)。为研制猪DNA疫苗有效的CpG-ODN免疫佐剂奠定了基础。2、含CpG序列PRRSV SD1 ORF5基因真核表达载体的构建与表达:本研究利用已构建的PIRORF5S-ORF5重组质粒,重新设计酶切位点,将ORF5基因插入哺乳动物表达载体pVAX1中,构建出pVAX1-ORF5质粒。再将CpG-ODN6和CpG-ODN7有机组合,人工设计合成CpG-ODN序列,通过在其两端的粘性酶切末端定向插入到经相同酶切处理的真核表达质粒pVAX1-ORF5,成功构建含特定CpG序列的真核重组表达质粒CpG-pVAX1-ORF5。将重组质粒转染COS7细胞,经间接免疫荧光试验(IFA)证实CpG-pVAX1-ORF5可表达PRRSV GP5蛋白。构建成功的表达PRRSV GP5的DNA重组质粒,为研究其所诱导的免疫反应的效果和强度以及对猪体产生的保护性反应奠定基础。3、真核重组表达载体质粒CpG-pVAX1-ORF5免疫原性的研究:通过小鼠试验对CpG-pVAX1-ORF5真核重组表达载体质粒进行了免疫原性检测。免疫体重约22-26克SPF小鼠,检测体液免疫与细胞免疫水平。通过ELISA法检测鼠血清中抗猪繁殖障碍与呼吸道综合征病毒抗体水平,并用流式细胞仪检测外周血淋巴细胞的CD4+、CD8 +数值,用MTT法检测淋巴细胞增殖指数(SI),试验结果表明:CpG-ODN能显著提高鼠的特异性抗体滴度以及淋巴细胞增殖反应。4、真核重组表达载体质粒CpG-pVAX1-ORF5对猪体免疫作用研究:通过猪体试验对含CpG-pVAX1-ORF5 DNA疫苗进行了免疫效力评价和病毒保护性试验。在猪体进行了免疫试验。检测猪PRRSV的特异性抗体滴度、IL-2水平以及淋巴细胞增殖反应(MTT法)水平,并做了攻毒保护试验。试验结果表明本试验构建的含CpG基序真核重组表达质粒CpG-pVAX1- ORF5能诱导产生针对PRRSV的体液免疫与细胞免疫(显著的淋巴细胞增值反应水平)。攻毒结果表明CpG-pVAX1-ORF5组能够有效地抵抗经鼻攻毒,可以保护猪轻微病毒血症,比对照组减少80%,也不表现临床症状,对肺的损害能提供部分保护。CpG的加入可使免疫猪的病变减轻或消失。从而表明含有CpG-ODN能作为PRRSV DNA疫苗的有效佐剂。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease found in swine farms worldwide and it is characterized by reproductive failure such as late-term abortions in sows and respiratory illness and mortality in young pigs.Bacterial DNA has immunostimulatory effects and adaptive immune responses in numerous species.It has been proved that synthetic CpG-ODN motifs could mimic bacterial DNA to induce a potent Th1 immune response. In the study, the CpG-ODN of the strongest immunostimulatory activity on the swine was screened out.PRRSV ORF5 gene of swine were amplified and cloned into the mammalian eukaryotic expression vector pVAX1 and constructed the eukaryotic recombinant expression vector plasmid pVAX1-ORF5. The DNA recombinant plasmids CpG-pVAX1-ORF5 containing ORF5 gene coding region and CpG motifs were constructed, respectively. Furthermore, In order to detect the immunogenicity of CpG-pVAX1-ORF5. SPF mice were immunized with these DNA recombinant plasmids and the immune responses were examined. Piglets were vaccinated with CpG-pVAX1-ORF5. The protective efficacy was also evaluated.1、The Preliminary Study of Different CpG-ODN Sequences with Immunostimulatory in Peripheral Blood Lymph cells and Spleen Cells of Swine.In the study, nine different artificial unmethylated oligodexynucleotides (CpG-ODN) sequences based on the swine peripheral blood lymph cells and spleen cells respectively. The effect of the invitro lymphocyte transformation was detected by MTT assay. According to the tested OD ,calculated the lymph cells SI to study the immune effect of CpG-ODN. It is showed that the nine CpG-ODNs had different extent of immunostimulatory effects on the swine peripheral blood lymph cells. The stimulation index of CpG-ODN was screened out . After elaborate analysis ,the conclusion was drawn that the CpG-ODN7 had the strongest immunostimulatory activity on the swine peripheral blood lymph cells(SI=3.5). and CpG-ODN6 had the biggest immunostimulatory index on the swine spleen immue cells (SI=2.7) .This would be useful for study of effective CpG-ODN immune adjunct for gene vaccination in future.2、Construction and express of eukaryotic expression vector containing PRRSV SD1 ORF5 Gene and CpG motifs. PRRSV ORF5 gene of swine were amplified by the recombinant expression vector plasmid PIRORF5S-ORF5 and were chosen two new enzyme sites; Then ORF5 gene was cloned into the mammalian eukaryotic expression vector pVAX1 and constructed the eukaryotic recombinant expression vector plasmid pVAX1-ORF5. CpG-ODN of the strongest immunostimulatory activity on the swine was cloned into recombinant expression vector plasmid pVAX1-ORF5; The DNA recombinant plasmids CpG-pVAX1-ORF5 containing ORF5 gene coding region and CpG motifs were constructed, respectively. The DNA recombinant plasmids CpG-pVAX1-ORF5 was identified by enzyme analysis and nucleotide sequencing test .To improve the effect of the gene immunization against porcine reproductive and respiratory syndrome virus(PRRSV)SD1 the recombinant expression vector plasmid CpG-pVAX1-ORF5 was transfected into COS-7 cells by using lipofectamine methods.The ORF5 mRNA of transfected cells were detected by RT-PCR. The transient expression of PRRSV SD1 ORF5 nucleocapsid protein was detected by indirect immunofluorescence assay(IFA). In some transfected COS-7 cells, the green fluorescence was showed, thus we can conclude that the eukaryotic expression vector CpG-pVAX1-ORF5 was successfully constructed and expressed in vitro. This would place the base for study of CpG-ODN immune adjunct effective and intensity of immue protective reaction on piglets for gene vaccination in future.3、Studies of immunogenicity responses of mice immuned with eukaryon recombinant expression plasmid CpG-pVAX1-ORF5.In order to detect its immunogenicity level ,the recombinant expression plasmid was inoculated into SPF female mice(22-26).The PRRSV antibody protein in serum were confirmed by ELISA. The number of CD4+、CD8 + and the lymphocyte proliferation test(MTT) in peripheral blood of mice vaccinated were used to detect. The results showed that the recombinant plasmid CpG-pVAX1-ORF5 could induce higher response of cellular immune response and specific humoral immune responses in mice . CpG-ODN was effective adjuvant which could significantly enhance the immunogenicity of PRRSV SD1 gene vaccine .It will lay a foundation for further studying on pig immunoreaction intensity and the protective reaction induced by PRRSV SD1 ORF5 gene vaccine .4、Study on immunological responses to eukaryotic recombinant expression vector plasmid CpG-pVAX1-ORF5 on pigs. piglets were immunized with DNA recombinant plasmids which expresses PRRSV GP5 for three immunity.The PRRSV antibody in serum, the concentration of IL-2 and the lymphocyte proliferation test(MTT) in peripheral blood of piglets vaccinated were used to detect.The vaccinated pigs were challenged intranasally with PRRSV SD1. The results showed that GP5 DNA immunization with CpG resulted in the production of both PRRSV antibodies and cellular immune(a significant enhancement of a lymphoproliferative response ) . The CpG-pVAX1-ORF5 showed significantly better protection from the PRRSV challenge compared with the control plasmid. This immune response was characterized by a significantly decreased frequency of viraemia with a decrease of 80% compared with the control group.There was little clinic symptom and protection in part to lungs. The results indicated that CpG-ODN could be used as immune adjuvant of PRRSV DNA vaccine.更多还原