【摘要】：豬細小病毒(PPV)是細小病毒科中以豬為宿主的一類能導致豬群流產(chǎn)、腹瀉以及呼吸道等癥狀的病毒的統(tǒng)稱。近年來國內(nèi)外陸續(xù)報道出PPV2等多種新型的豬細小病毒,且以PPV2為代表的新型豬細小病毒在豬群中的陽性率居高不下,給世界養(yǎng)豬業(yè)帶來重大的威脅。本研究利用PCR方法調(diào)查了 PPV2等新型細小病毒在我國不同地區(qū)的流行情況,并對PPV2 ORF2基因進行了測序分析;利用原核的大腸桿菌經(jīng)典表達系統(tǒng)和真核的昆蟲細胞/桿狀病毒表達系統(tǒng)同時表達了 PPV2VP蛋白主要抗原表位區(qū),并對其免疫原性進行了研究;同時還利用原核表達的PPV2VPb蛋白建立間接ELISA檢測方法,為PPV2的血清學檢測提供了支持。具體研究內(nèi)容如下:1.新型豬細小病毒的檢測及豬細小病毒2型ORF2基因的序列分析本研究對2013-2014年本實驗室采集保存的253份組織樣品進行了 PCR檢測;并對PPV2進行ORF2基因的擴增與測序。結(jié)果顯示PPV2、PPV3、PPV4和PBoV 的檢出率分別為 54.5%、11.1%、8.7%和 9.9%,且 PPV2、PPV3、PPV4 存在著一定的季節(jié)性流行;對PPV2基因測序顯示,不同地區(qū)來源的PPV2 ORF2基因之間的同源性為92.6%-100%,對其進行進化分析,結(jié)果顯示,PPV2和PPV3以及人細小病毒4、5型一同歸入PARV4-like virus。本研究為進一步調(diào)查新型豬細小病毒流行病學以及PPV2的遺傳進化分析奠定了基礎(chǔ)。2.豬細小病毒2型(PPV2)VP蛋白主要抗原表位區(qū)的原核表達及其免疫特性研究本實驗利用原核表達系統(tǒng)分別表達了 PPV2 ORF2 VP蛋白的第172-412(VPa基因514-1236)位和第713-924(VPb基因,2137-2772)位氨基酸的兩段截短蛋白;純化的蛋白制備成亞單位疫苗免疫小鼠,通過測定抗體效價判定兩組蛋白的免疫原性。結(jié)果顯示,兩組蛋白在大腸桿菌中均成功獲得表達;小鼠免疫實驗表明,兩組蛋白均能誘導小鼠產(chǎn)生特異性抗體,但VPb蛋白免疫小鼠產(chǎn)生的抗體效價明顯高于VPa,表明VPb蛋白的免疫原性優(yōu)于VPa蛋白。本實驗初步探討了兩種蛋白的免疫原性,為建立PPV2血清學檢測方法奠定了基礎(chǔ)。3.豬細小病毒2型(PPV2)VP蛋白主要抗原表位區(qū)的真核表達及其免疫特性研究本實驗將 PPV2 ORF2 的 VPa 基因(514-1236)和 VPb 基因(2137-2772)克隆入轉(zhuǎn)移載體pFastBacTMDual,轉(zhuǎn)化DH10Bac后進行重組,形成重組Bacmid,提取重組Bacmid轉(zhuǎn)染Sf9細胞獲得重組桿狀病毒,經(jīng)Western-blot方法鑒定重組蛋白VPa和VPb在Sf9中的表達。表達的蛋白制備成亞單位疫苗免疫小鼠,通過測定抗體效價判定兩組蛋白的免疫原性。結(jié)果顯示,兩組蛋白均成功獲得表達;小鼠免疫實驗表明,經(jīng)三次免疫后VPb蛋白免疫小鼠產(chǎn)生了較高的抗體效價,而VPa蛋白免疫小鼠產(chǎn)生的抗體效價很低,表明VPb蛋白的免疫原性優(yōu)于VPa蛋白。本研究為進一步研究VPa蛋白和VPb蛋白的免疫特性以及PPV2VP亞單位疫苗的研發(fā)奠定了基礎(chǔ)。4.豬細小病毒2型間接ELISA抗體檢測方法的建立和應用本研究選擇了原核表達的VPb蛋白為包被抗原建立了檢測PPV2抗體的間接ELISA方法,優(yōu)化后反應條件為:抗原包被濃度為0.8μg/mL,血清的最佳稀釋度為1:100,酶標二抗的工作濃度為1:20 000,檢測的臨界值為0.4005(OD≥ 0.4005判定為陽性)。特異性試驗證明,與豬細小病毒1型(PPV1)、豬瘟病毒(CSFV)、豬繁殖與呼吸綜合征病毒(PRRSV)、豬偽狂犬病毒(PRV)、豬圓環(huán)病毒2型(PCV2)、豬口蹄疫病毒(FMDV)血清抗體無交叉反應,批內(nèi)、批間重復性實驗變異系數(shù)均小于10%。應用此方法對江蘇地區(qū)的480份臨床血清樣本進行了檢測,PPV2抗體陽性率為31%。該結(jié)果表明,以此pET-32a-VPb蛋白作為包被抗原建立的間接ELISA方法具有較高的特異性和敏感性,可以用于臨床血清PPV2抗體的檢測。本研究為臨床新型細小病毒的流行病學研究奠定了理論基礎(chǔ),同時為PPV2的遺傳進化分析提供了支持。建立的間接ELISA檢測方法,為PPV2血清學檢測方法的研究奠定了基礎(chǔ)。
[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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.28

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