【作者】 陈杨；

【导师】 郭万柱；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2011， 博士

【摘要】 猪细小病毒(Porcine Parvovims, PPV)是引起猪繁殖障碍的主要病原之一,猪圆环病毒2型(porcine circovirus type2, PCV2)引起以断奶仔猪多系统衰竭综合征(PMWS)为代表的系列疾病。该两种疾病在世界范围内广泛流行,造成了巨大的经济损失,目前,对这两种疾病的防制主要靠预防接种。然而,目前广泛使用的猪细小病毒灭活疫苗存在灭活不完全的风险,而且由于PPV体外培养较困难,病毒增殖滴度较低,这给灭活疫苗的生产带来一定困难；同时PCV2在细胞上增殖滴度很低,发展传统疫苗都存在一定障碍,因此有必要发展新型疫苗来防制这两种疾病。这两种疾病常发生混合感染,并具有协同性,如果能研发一种可同时预防这两种疾病的疫苗,将具有重要意义。据此,本研究展开了对四川省PPV和PCV2的抗原流行病学调查,以掌握其流行现状；对PPV VP2基因和PCV2 ORF2基因及其串联基因设计核酸疫苗；并以猪伪狂犬病毒(Pseudorabies Virus, PRV)为活病毒载体,构建了PRV-PPV-PCV2三价基因工程疫苗,为进一步预防和控制PRV、PPV和PCV2及其混合感染打下坚实的基础。具体如下：1.四川省猪细小病毒、猪圆环病毒2型流行病学调查从四川省21个规模化猪场共采集273份样品,通过PCR诊断技术进行了PPV、PCV2病原流行病学调查,结果检出PPV抗原阳性样品47份(17.22%)、PPV阳性猪场8个(38.1%),具有种猪的感染率较高,而在仔猪感染率相对较低的特点：检出PCV2抗原阳性样品143份(52.38%)、PCV2阳性猪场18个(85.7%),具有PCV2感染随猪年龄的增长而升高的趋势；检出PPV和PCV2混合感染样品29份(10.62%)、同时混合感染PPV和PCV2的猪场6个(28.7%),混合感染主要集中在母猪和保育仔猪阶段。2.猪细小病毒和猪圆环病毒2型核酸疫苗的构建通过PCR方法扩增PPV VP2基因和PCV2 ORF2基因,并分别插入真核表达载体pCI-neo中,成功构建了真核表达质粒pCI-VP2和pCI-ORF2,同时,将PPV VP2基因和PCV2 ORF2基因进行串联,两基因间以高亲水性氨基酸(Gly-Gly-Gly-Ser)编码核苷酸连接,插入到PCI-neo中构建真核表达质粒pCI-VP2-ORF2。将pCI-VP2. pCI-ORF2和pCI-VP2-ORF2转染Vero细胞后,通过RT-PCR法检测目的基因转录、SDS-PAGE电泳、Western blotting分析和间接免疫荧光分析蛋白表达,结果表明,3种真核表达质粒转染哺乳动物细胞后,既可扩增到目的基因,也可检测到特异性蛋白的表达,成功地表达了外源基因。3.猪细小病毒和猪圆环病毒2型核酸疫苗对小鼠免疫效果研究将84核酸疫苗pCI-VP2、pCI-ORF2和pCI-VP2-ORF2以100μg/只剂量免疫健康昆明小白鼠,2周后相同剂量加强免疫一次,于首免后O d、7 d、14 d、21 d、28 d、42 d、56 d断尾采血并分离血清进行抗体检测,发现首免后14d,可检测到PPV和PCV2特异性抗体的出现,加强免疫后7d,其抗体转化为阳性,但其诱导抗体水平均低于疫苗组所诱导的抗体水平：于首免后0 d、21 d、56 d分离血清进行细胞因子检测,发现其均可诱导较高水平的IL-2、IL-4、IFN-y;于首免后56d,采集抗凝血一份,进行T淋巴细胞亚群检测,发现CD4+/CD8+比例明显下降；首免后56d在每组随机挑选一只小鼠颈椎脱臼处死,取脾脏制备单个脾细胞悬液,用ConA进行刺激并测定刺激指数,发现能够诱导较强的脾细胞增殖；首免后56d采集小鼠的实质器官,进行安全性检测,均未检测到3种核酸疫苗的外源基因与小鼠基因组DNA整合的情况,表明3种核酸疫苗是安全的。这些结果说明,3种核酸疫苗均能够诱导小鼠产生较强的细胞免疫应答和体液免疫应答,并具有良好的安全性。4. PPV-PCV2-PRV三价基因工程疫苗PRV SA215(D1)的构建将VP2-ORF2融合基因从真核表达载体pCI-VP2-ORF2切下,连接到PRV通用转移载体pPI-2.EGFP中,成功构建了转移载体pPI-2.EGFP-VP2-ORF2。将PRV SA215基因组DNA与转移质粒pPI-2.EGFP-VP2-ORF2共转染Vero细胞,通过同源重组,构建重组病毒。挑斑筛选后,通过绿色荧光检测和PCR检测表明成功构建了PRV-PPV-PCV2三价基因工程疫苗株PRV SA215 (Dl);通过SDS-PAGE电泳、Western blotting分析和间接免疫荧光分析,结果表明,VP2基因和ORF2基因在重组PRV SA215 (D1)获得了成功表达,融合蛋白具有良好的免疫反应原性。5. PRV SA215(D1)部分生物学特性研究将PRV SA215(D1)接种Vero细胞,观察其致病变效应、重组病毒的形态,测定一步法生长曲线,结果表明,外源PPV VP2和PCV2 ORF2基因的插入不影响重组病毒的增殖和病毒粒子的包装。将PRVSA215(D1)在Vero细胞上连续传6代,未发现PPV VP2和PCV2 ORF2基因丢失,表明PRV SA215(D1)具有遗传稳定性。将PRVSA215(D1)以104PFU剂量免疫健康昆明小鼠,发现其对小鼠有良好的安全性,以104PFU免疫小白鼠可对106 PFU PRV Fa株强毒攻击产生完全保护。将PRVSA215(D1)以105PFU剂量免疫仔猪,可引起仔猪外周血CD4+T淋巴细胞阳性细胞数明显提高,CD8+T淋巴细胞阳性细胞数相对减少；可诱导产生较高水平的细胞因子IL-2、IL-4、IFN-γ,表明能够显著的增强Thl和Th2型免疫应答；可诱导仔猪产生较高水平的PRV、PPV和PCV2的抗体,其中PRV水平抗体与其亲本株PRV SA215相当,PPV和PCV2抗体水平略低于灭活疫苗组;这些结果表明,PRV SA215(D1)可望作为预防控制PRV、PPV和PCV2的三价基因工程疫苗候选株。更多还原

【Abstract】 Porcine parvovirus (PPV) is the major causative agent in a syndrome of reproductive failure in sows. Porcine circovirus type 2(PCV2) causes Porcine Circovirus type 2-associated diseases (PCVD), and particularly postweaning multisystemic wasting syndrome(PMWS). These two diseases which have caused huge economic losses, are worldwide distribution; At present, vaccination is the major method to prevent them. However, porcine parvovirus inactivated vaccine which has been widely used, has the risk of not incompletely inactivated, as well as porcine parvovirus is difficultly cultivation in vitro which causes low titer proliferation. These above short comings bring many difficulties to product porcine parvovirus inactivated vaccine. Meanwhile, porcine circovirus type 2 also has low titer proliferation in vitro, which lead to hardly develop the traditional vaccines. These two kinds of infectious diseases, which have synergistic effect, often co-infect swine herds. If a vaccine to simultaneously prevent these two diseases could be developed, it will have vital significance. In this work, antigen epidemiology of porcine parvovirus and porcine circovirus type 2 in Sichuan province were investigated; DNA vaccines against porcine parvovirus and porcine circovirus type 2 as well as a recombinant pseudorabies virus that could express PPV VP2 protein and PCV2 ORF2 protein were constructed, and their immunogenicity was further demonstrated. These experiments were aiming to provide a novel vaccine to be used in prevention and control these above diseases in the future. The main research contents are the following:1. Antigen Epidemiology Survey of Porcine Parvovirus and Porcine Circovirus Type 2 in Sichuan ProvinceTwo hundred and seventy-three samples were collected from 21 large pig farms in Sichuan province, and antigen epidemiology of porcine parvovirus and porcine circovirus type 2 were investigated by PCR detecting. The antigen positive rate of porcine parvovirus was 17.22%(47/273), and positive rate of pig farms was 38.1% (8/21), which displaied a feature that infection rate of boar was much higher than that of piglets; The antigen positive rate of porcine circovirus type 2 was 52.38% (143/273), and positive rate of pig farms was 85.7% (18/21), which showed a trend that the infection rate of porcine circovirus type 2 was rising with the piglets growth. The co-infection rate of porcine parvovirus and porcine circovirus type 2 was 10.62% (29/273), and co-infection rate of pig farms was 28.7% (6/21). Co-infection was mainly take place in the sow and nursing piglets.2. Construction of DNA Vaccines of Porcine Parvovirus and Porcine Circovirus Type 2Porcine parvovirus VP2 gene and porcine circovirus type 2 ORF2 gene were amplified by PCR, then were inserted into eukaryotic expression vector pCI-neo to constructed pCI-VP2 and pCI-ORF2 respectively. Meanwhile, these two genes were linked by high hydrophilia amino acid (Gly-Gly-Gly-Ser) encoding nucleotides, and then were also inserted into pCI-neo to constructed pCI-VP2-ORF2. Plasmids pCI-VP2, pCI-ORF2 and pCI-VP2-ORF2 were transfected to Vero cells, and target gene expression analysis was detected by PCR, Western blotting and IFA assay. The results suggested that the three kinds of DNA vaccines were successfully constructed and expressed.3. Immunogenicity of DNA Vaccines in MiceVaccinate healthy Kunming mouse with DNA vaccines pCI-VP2, pCI-ORF2 and pCI-VP2-ORF2 in the dose of 100μg, and booster injection was given with the same dose at 14 days post-vaccination. Collected mouse serum by tail cutting for antibodies detection at 0、7、14、21、28、42 and 56 days post-vaccination. Specific antibodies of PPV and PCV2 could be detected on 14 days post-vaccination. After 7 days of booster immunization, antibodies converted into positive, but the titer of immunized DNA vaccines groups were lower than that of inactivated vaccine groups. Collected mouse serum for cytokine detection at 0,21 and 56 days post-vaccination, and comparative high-level of IL-2, IL-4, IFN-y were induced by these three kinds of DNA vaccines. Collected anti-coagulated blood for T-lymphocyte subsets detection at 56 days post-vaccination, and CD4+/CD8+ was declined evidently. At 56 days post-vaccination, killed a mice by cervical dislocation in a random way to prepare splenic cell suspension which were then stimulated with ConA, and examined the stimulation index. The result showed that ConA had an marked effect on promoting spleen cell proliferation. Collected the murine parenchymal organs for the potential integration detection of DNA between the exogenous gene of the 3 kinds of DNA vaccines and the mice genome at 56 days post-vaccination, and the result indicated that the 3 kinds of DNA vaccines were security for the experimental animals. All the above results demonstrated that three kinds of DNA vaccines had high security, and could induce strong cell mediated immune response as well as humoral immune response. 4. Construction of the Triple Genetic Engineering Vaccine Strain PRV SA215 (D1)Cut off the VP2-ORF2 fusion gene from pCI-VP2-ORF2, and connected into a universal transfer vector pPI-2.EGFP of pseudorabies virus, then eukaryotic expression transfer vector pPI-2.EGFP-VP2-ORF2 was successfully constructed. PRV SA215 genomic DNA and the transferred plasmid pPI-2.EGFP-VP2-ORF2 were co-transfected into Vero cells to construct recombinant virus via homologous recombination. Then the recombinant virus PRV SA215 (D1) was purified by plaque purification using green fluorescent assay and PCR identification, and confirmed by detecting the presence of VP2 gene by PCR, Western blotting analysis and IFA assay. The results suggested the recombinant PRV SA215 (D1) strain could steadily express exogenous protein, and the fusion protein had good Immunogenicity.5. Study on the Partial Biological Characteristics of PRV SA215 (D1)Vero cells were infected with the recombinant PRV SA215 (D1), then cytopathic effect(CPE) and the morphology of virus were observed, and one step growth curve was determined. The results showed that the insertion of exogenous PPV VP2 and PCV2 ORF2 gene do not affected the proliferation and packaging of recombinant virus. Vero cells inoculated PRV SA215(D1), and then subcultured 6 rounds, which found that PPV VP2 and the PCV2 ORF2 gene did not loss. These results suggested that PRV SA215 (D1) was genetic stability. Healthy Kunming mouse were immunized with 104 PFU PRV SA215 (D1), found that all immunized mouse were alive which suggesting good security. Mouse immunized with the recombinant developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal dose of 106 PFU PRV challenge. Piglets immunized with 105 PFU PRV SA215 (D1) induced significant cell mediated immune response and high levels of PRV, PPV and PCV2 antibodies. CD4+ T lymphocytes in peripheral blood was significantly increased, and CD8+T lymphocytes in peripheral blood was relatively reduced. High levels of cytokines IL-2, IL-4 and IFN-γshowed that PRV SA215 (D1) can significantly enhance Thl and Th2 immune response. The PRV antibody level induced by PRVSA215 gourp was equal to that of their parents strain PRV SA215, as well as PPV and PCV antibody levels were slightly lower than that of inactivated vaccine group, respectively; All above-mentioned results suggested that PRV SA215 (D1) is expected as the candidate strain of trivalent genetic engineering live vaccine to prevent and control PRV, PPV and PCV2.更多还原