猪细小病毒2型分子流行病学调查、结构蛋白免疫原性分析和抗体检测ELISA方法的建立

发布时间：2018-09-14 09:37

【摘要】：猪细小病毒(PPV)是细小病毒科中以猪为宿主的一类能导致猪群流产、腹泻以及呼吸道等症状的病毒的统称。近年来国内外陆续报道出PPV2等多种新型的猪细小病毒,且以PPV2为代表的新型猪细小病毒在猪群中的阳性率居高不下,给世界养猪业带来重大的威胁。本研究利用PCR方法调查了 PPV2等新型细小病毒在我国不同地区的流行情况,并对PPV2 ORF2基因进行了测序分析;利用原核的大肠杆菌经典表达系统和真核的昆虫细胞/杆状病毒表达系统同时表达了 PPV2VP蛋白主要抗原表位区,并对其免疫原性进行了研究;同时还利用原核表达的PPV2VPb蛋白建立间接ELISA检测方法,为PPV2的血清学检测提供了支持。具体研究内容如下:1.新型猪细小病毒的检测及猪细小病毒2型ORF2基因的序列分析本研究对2013-2014年本实验室采集保存的253份组织样品进行了 PCR检测;并对PPV2进行ORF2基因的扩增与测序。结果显示PPV2、PPV3、PPV4和PBoV 的检出率分别为 54.5%、11.1%、8.7%和 9.9%,且 PPV2、PPV3、PPV4 存在着一定的季节性流行;对PPV2基因测序显示,不同地区来源的PPV2 ORF2基因之间的同源性为92.6%-100%,对其进行进化分析,结果显示,PPV2和PPV3以及人细小病毒4、5型一同归入PARV4-like virus。本研究为进一步调查新型猪细小病毒流行病学以及PPV2的遗传进化分析奠定了基础。2.猪细小病毒2型(PPV2)VP蛋白主要抗原表位区的原核表达及其免疫特性研究本实验利用原核表达系统分别表达了 PPV2 ORF2 VP蛋白的第172-412(VPa基因514-1236)位和第713-924(VPb基因,2137-2772)位氨基酸的两段截短蛋白;纯化的蛋白制备成亚单位疫苗免疫小鼠,通过测定抗体效价判定两组蛋白的免疫原性。结果显示,两组蛋白在大肠杆菌中均成功获得表达;小鼠免疫实验表明,两组蛋白均能诱导小鼠产生特异性抗体,但VPb蛋白免疫小鼠产生的抗体效价明显高于VPa,表明VPb蛋白的免疫原性优于VPa蛋白。本实验初步探讨了两种蛋白的免疫原性,为建立PPV2血清学检测方法奠定了基础。3.猪细小病毒2型(PPV2)VP蛋白主要抗原表位区的真核表达及其免疫特性研究本实验将 PPV2 ORF2 的 VPa 基因(514-1236)和 VPb 基因(2137-2772)克隆入转移载体pFastBacTMDual,转化DH10Bac后进行重组,形成重组Bacmid,提取重组Bacmid转染Sf9细胞获得重组杆状病毒,经Western-blot方法鉴定重组蛋白VPa和VPb在Sf9中的表达。表达的蛋白制备成亚单位疫苗免疫小鼠,通过测定抗体效价判定两组蛋白的免疫原性。结果显示,两组蛋白均成功获得表达;小鼠免疫实验表明,经三次免疫后VPb蛋白免疫小鼠产生了较高的抗体效价,而VPa蛋白免疫小鼠产生的抗体效价很低,表明VPb蛋白的免疫原性优于VPa蛋白。本研究为进一步研究VPa蛋白和VPb蛋白的免疫特性以及PPV2VP亚单位疫苗的研发奠定了基础。4.猪细小病毒2型间接ELISA抗体检测方法的建立和应用本研究选择了原核表达的VPb蛋白为包被抗原建立了检测PPV2抗体的间接ELISA方法,优化后反应条件为:抗原包被浓度为0.8μg/mL,血清的最佳稀释度为1:100,酶标二抗的工作浓度为1:20 000,检测的临界值为0.4005(OD≥ 0.4005判定为阳性)。特异性试验证明,与猪细小病毒1型(PPV1)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)、猪口蹄疫病毒(FMDV)血清抗体无交叉反应,批内、批间重复性实验变异系数均小于10%。应用此方法对江苏地区的480份临床血清样本进行了检测,PPV2抗体阳性率为31%。该结果表明,以此pET-32a-VPb蛋白作为包被抗原建立的间接ELISA方法具有较高的特异性和敏感性,可以用于临床血清PPV2抗体的检测。本研究为临床新型细小病毒的流行病学研究奠定了理论基础,同时为PPV2的遗传进化分析提供了支持。建立的间接ELISA检测方法,为PPV2血清学检测方法的研究奠定了基础。
[Abstract]:* * porcine parvovirus (PPV) is a general name of a group of viruses that cause pig abortion, diarrhea and respiratory symptoms in the parvovirus family. In recent years, many new types of porcine parvovirus * such as PPV2 have been reported at home and abroad, and the positive rate of new swine small disease virus represented by PPV2 is still high in pigs. This study investigated the epidemic situation of PPV2 and other new parvovirus in different areas of China by PCR and sequenced the PPV2 ORF2 gene. The main anti-PPV2VP proteins were expressed simultaneously in the prokaryotic E.coli classical expression system and eukaryotic insect cell/baculovirus expression system. The original epitope region and its immunogenicity were studied. At the same time, the indirect ELISA detection method was established by using prokaryotic expression of PPV2VPb protein, which provided support for serological detection of PPV2. The * * contents are as follows: 1. detection of new swine parvovirus and sequence analysis of ORF2 gene of porcine parvovirus type 2, this study is 2013-2014 years old. The results showed that the detection rates of PPV2, PPV3, PPV4 and PPV4 were 54.5%, 11.1%, 8.7% and 9.9% respectively, and there were seasonal epidemics of PPV2, PPV3 and PPV4. The sequencing of PPV2 gene showed that PPV2 ORF2 was from different regions. Because of the homology between 92.6%-100% and evolution analysis, the results showed that PPV2 and PPV3 together with human parvovirus 4,5 type belong to PARV4-like virus. * this study laid the foundation for further investigation of the epidemiology of new swine parvovirus and the genetic evolution of PPV2 * the major antigen epitope of.2. porcine parvovirus type 2 (PPV2) VP protein. Prokaryotic expression and immunological characteristics of PPV2 ORF2 VP protein were studied in this study. Two fragments of amino acids at positions 172-412 (VPa gene 514-1236) and 713-924 (VPb gene 2137-2772) were expressed in the prokaryotic expression system. The purified protein was prepared into subunit vaccine to immunize mice, and the antibody titer was determined. The results showed that the two proteins were successfully expressed in E. coli. The mouse immunoassay showed that both proteins could induce mice to produce specific antibodies, but the antibody titer of mice immunized with VPb protein was significantly higher than that of VPa, indicating that the immunogenicity of VPb protein was superior to that of VPa protein. The immunogenicity of the two proteins has laid the foundation for the establishment of PPV2 serological detection methods. * the eukaryotic expression and immunological characteristics of the major epitope regions of.3. VP (PPV2) VP protein. The VPa gene (514-1236) and VPb gene (2137-2772) of PPV2 ORF2 were cloned into transfer vector pFastBacTMDual to transform DH10Bac. The recombinant baculovirus was obtained from Sf9 cells transfected with recombinant Bacmid. The expression of recombinant protein VPa and VPb in Sf9 was identified by Western blot. The expressed protein was prepared into subunit vaccine and immunized mice. The immunogenicity of the two proteins was determined by antibody titer. The immunogenicity of VPb protein was superior to that of VPa protein. The immunological characteristics of VPa protein and VPb protein and the subunit epidemic of PPV2VP were further studied in this study. * the establishment and application of the indirect ELISA antibody detection method for.4. porcine parvovirus type 2 has been established. This study chose the prokaryotic expression VPb protein as the inclusion antigen and established an indirect ELISA method for detecting PPV2 antibody. The optimized reaction conditions were as follows: the antigen coating concentration was 0.8 g g, the optimal dilution of the serum was 1:100, and the enzyme labeled two The working concentration of the antibody was 000 at 1:20 and the critical value of detection was 0.4005 (OD * 0.4005 was positive). Specificity test showed that there was no cross reaction with serum antibody of swine parvovirus 1 * * (PPV1), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), swine pseudorabies virus (PRV), porcine circovirus 2 (PCV2), Swine Foot and mouth disease virus (FMDV). The results showed that the indirect ELISA method based on pET-32a-VPb protein as coating antigen had high specificity and sensitivity and could be used for clinical serum PP. This study laid a theoretical foundation for the epidemiological study of new clinical parvovirus, and provided support for the genetic evolution analysis of PPV2. The established indirect ELISA detection method laid a foundation for the study of serological detection of PPV2.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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