本文關(guān)鍵詞： PRV新流行毒株 血清流行病學(xué)調(diào)查和分子進化分析 鑒別診斷ELISA 免疫抗體水平評價ELISA 病毒抗原檢測的雙抗夾心 ELISA和膠體金試紙　出處：《吉林大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：偽狂犬�。≒R）是多種動物共患的傳染�。黄洳≡w偽狂犬病病毒（PRV）是一種豬的皰疹病毒，分類學(xué)命名為豬皰疹病毒I型（Suid herpesvirus1）。豬是PRV的傳染源和自然貯存宿主，感染后表現(xiàn)為發(fā)熱、仔豬神經(jīng)癥狀和高死亡率、大中豬出現(xiàn)呼吸道癥狀等臨床特征。消化道、呼吸道、精液、胎盤及空氣等是PRV的主要傳播途徑。PRV對于口鼻部呼吸道的上皮細胞具有非常強的嗜性；其基因組是大小約為150kb的線性雙鏈DNA，編碼70-100余種蛋白。 2011年底，在河北、河南、山東、山西等多個省份許多使用Bartha-K61疫苗免疫豬場爆發(fā)了疑似PR的疫病，發(fā)病豬出現(xiàn)高溫(40-42°C）、食欲不振、咳嗽、腹瀉并伴隨有系統(tǒng)性神經(jīng)癥狀，新生仔豬死亡率高；病毒分離等實驗證實引起這場疫病爆發(fā)的源頭是變異的PRV病毒，這對PR的防控提出了嚴(yán)峻挑戰(zhàn)。因此本研究對河南省及周邊部分省份PRV的流行進行了血清學(xué)調(diào)查和分子進化分析；表達了PRV新流行毒株的gE蛋白和gB蛋白并制備了單抗，以期建立的PRV抗原抗體ELISA檢測方法和制備的膠體金快速檢測試紙能在PRV鑒別診斷和免疫抗體水平評價等方面發(fā)揮作用。 血清流行病學(xué)分析結(jié)果表明河南省PRV gE抗體陽性率在2012年升高至38.0%，2013年仍居高不下（31.3%）。種豬群陽性率為36.3%，育肥后期（19-25周齡）陽性率高達59.3%，后備種豬陽性率為23.8%。在2013年檢測的347個豬場中，豬場陽性率為83.6%，其中豫北豬場陽性率最高為93.8%。這些數(shù)據(jù)表明PR在河南省呈嚴(yán)重流行態(tài)勢�；诜蛛x的12株P(guān)RV新流行株的gE、gB和gC基因開展的分子進化分析顯示新流行株處于獨立的進化分支，，與國內(nèi)早期PRV分離株和其它新流行株進化關(guān)系較近，與Bartha疫苗株進化關(guān)系較遠。 為建立針對PRV新流行毒株抗體檢測的ELISA方法，我們表達了PRV新流行株（湯陰株）的gE蛋白和gB蛋白。其中g(shù)E蛋白為可溶表達，gB蛋白為包涵體表達；利用Ni-NTA親和層析和離子交換純化的gE蛋白和gB蛋白分別建立了gE抗體檢測的間接ELISA（gE-ELISA）和gB抗體檢測的間接ELISA（gB-ELISA）方法。gE-ELISA與商品化試劑盒g(shù)pI-ELISA（IDEXX）的符合率為88.76%；特異性（DSP）為79.15%；敏感性（DSN）為94.03%。gB-ELISA與商品化試劑盒g(shù)B-ELISA（IDEXX）的符合率為93.14%；DSP為77.78%；DSN為93.64%。在檢測的66個陽性場中，陽性活躍場占95.5%，陽性穩(wěn)定場占4.5%。在PRV gE抗體陰性樣品中，gB抗體陽性率占到了94.9%。 制備了針對gE蛋白和gB蛋白上具有免疫優(yōu)勢的表位的單抗并建立了單抗阻斷ELISA檢測方法。gE單抗阻斷ELISA（blocking gE-ELISA）與商品化試劑盒g(shù)pI-ELISA的符合率為94.10%；DSP為89.90%；DSN為96.18%。gB單抗阻斷ELISA（blocking gB-ELISA）與商品化試劑盒g(shù)B-ELISA的符合率為94.07%；DSP為85.37%；DSN為95.90%；兩種單抗阻斷ELISA較間接ELISA在符合率、特異性和敏感性三方面性能均有所提高。在檢測的72個陽性場中，陽性活躍場占95.8%，陽性穩(wěn)定場占4.2%。在PRV gE抗體陰性樣品中，gB抗體陽性率占到了98.0%，與間接ELISA結(jié)果相符合；說明各個豬場采用的常規(guī)疫苗免疫誘導(dǎo)產(chǎn)生了gB抗體，并能夠在一定程度上預(yù)防PRV的感染或流行。 同時我們利用gE蛋白或gB蛋白高免豬血清IgG及特異性單抗酶標(biāo)物，以NP-40處理的PRV病毒抗原為檢測靶標(biāo)建立了雙抗體夾心ELISA并制備了膠體金快速檢測試紙。以gE高免豬血清IgG建立的夾心ELISA和試紙均只與PRV野毒抗原反應(yīng)，不與gE缺失疫苗株和正常BHK-21細胞對照反應(yīng)；可檢測到的病毒樣品的最低檢測限約為105.0TCID50/0.1mL左右。以gB高免IgG建立的夾心ELISA和試紙可同時與PRV野毒抗原和gE缺失的疫苗株反應(yīng)，不和正常BHK-21細胞對照反應(yīng)。 本研究中的血清和分子流行病學(xué)調(diào)查將為河南省PRV的流行和進化關(guān)系提供參考；建立的間接和單抗阻斷ELISA可用于區(qū)分疫苗免疫抗體與野毒感染抗體，并為PRV疫苗免疫抗體水平評價提供手段；建立的檢測PRV病毒抗原的雙抗體夾心ELISA和制備的膠體金快速檢測試紙能夠直觀簡便地進行PRV野毒感染的檢測和疫苗質(zhì)量評估。因此，本研究為我國PR的凈化提供了抗原、抗體快速檢測的方法。
[Abstract]:Pseudorabies virus (PR) is a variety of animal zoonotic infectious disease; the pathogen of pseudorabies virus (PRV) is a swine herpesvirus taxonomy named pig herpes simplex virus type I (Suid herpesvirus1). The pig is PRV source of infection and the natural reservoir, after infection characterized by fever, neurological symptoms and high mortality in piglets the clinical features of respiratory symptoms, and other large and medium-sized pig. The digestive tract, respiratory tract, semen, placenta and air is.PRV PRV main transmission way has a very strong tropism for the nose and mouth of respiratory epithelial cells; its genome is about the size of 150kb linear double stranded DNA, encoding 70-100 kinds of protein.
By the end of 2011, in Hebei, Henan, Shandong, Shanxi and other provinces to use many Bartha-K61 vaccine of swine outbreak of suspected PR disease, swine high temperature (40-42 degrees C), loss of appetite, cough, diarrhea and accompanied by systemic neurological symptoms, high rate of death of newborn piglets; virus isolation experiments confirmed by the source the outbreak is a variant of the PRV virus, which has posed a severe challenge to prevent and control PR. This study analyzed the serological investigation and molecular evolution of Henan province and surrounding provinces part of the popularity of PRV; expression of PRV monoclonal antibody preparation of new strains of gE protein and gB protein and preparation, in order to establish the PRV ELISA antigen antibody detection method and preparation of colloidal gold strip for rapid detection can play a role in the differential diagnosis of PRV and antibody level of evaluation.
Serum epidemiological analysis results show that the PRV of Henan province gE antibody positive rate in 2012 increased to 38%, 2013 is still high (31.3%). Among the positive rate was 36.3%, the latter fattening period (19-25 weeks) positive rate was as high as 59.3%, the positive rate for 23.8%. reserve pigs on 347 farms in 2013 detection, positive rate pig is 83.6%, the positive rate of 93.8%. was the highest in swine these data indicate that PR is a serious epidemic situation in Henan province. 12 strains of gE isolated PRV strains based on the molecular evolution of gB gene and gC gene in the analysis show the new strains in the evolution of branch independence, and domestic PRV isolates and other early the new epidemic strain evolution relationship is near, far from the relationship between Bartha vaccine strains evolution.
In order to establish the ELISA method for detection of PRV new strains of antibodies, we expressed PRV new strains (Tang Yinzhu) of the gE protein and gB protein. The expression of gE protein was soluble, gB protein expressed as inclusion bodies; indirect ELISA gE antibody detection was established using Ni-NTA affinity chromatography and ion exchange of gE protein and gB protein white purified respectively (gE-ELISA) and gB antibody detection by indirect ELISA (gB-ELISA).GE-ELISA method and commercial gpI-ELISA Kit (IDEXX) and the coincidence rate was 88.76%; the specificity was 79.15% (DSP); sensitivity (DSN) for 94.03%.gB-ELISA with a commercial gB-ELISA Kit (IDEXX) with the rate of 93.14%; DSP 77.78%; DSN is 93.64%. in the detection of 66 positive field, positive active field accounted for 95.5%, accounted for 4.5%. of PRV in the stability field of positive gE antibody negative samples, the positive rate of gB antibody accounted for 94.9%.
The preparation of the gE protein and gB protein with immunodominant epitope McAbs and established the monoclonal antibody blocking ELISA detection method of.GE monoclonal antibody blocking ELISA (blocking gE-ELISA) with a commercial gpI-ELISA kit with the rate of 94.10%; DSP 89.90%; DSN 96.18%.gB ELISA (blocking gB-ELISA) monoclonal antibody blocking and commercial reagent box of gB-ELISA with the rate of 94.07%; DSP 85.37%; DSN 95.90%; two kinds of monoclonal antibody blocking ELISA than indirect ELISA in coincidence rate, sensitivity and specificity of the three aspects of performance are improved. In the detection of 72 positive field, positive active field accounted for 95.8%, accounted for 4.2%. of PRV in the stability field of positive gE antibody negative in the sample, gB antibody positive rate accounted for 98%, consistent with the results of indirect ELISA; the conventional vaccine induced by pig each gB antibody, and to prevent PRV in the extent of infection or Popular.
At the same time, we use the gE protein or gB protein from porcine serum IgG and specific monoclonal antibody ELISA, the double antibody sandwich ELISA and preparation of colloidal gold strip for rapid detection of PRV virus antigen based on NP-40 processing for target detection. With gE high immune serum IgG to establish sandwich ELISA and test paper are only with PRV wild virus antigen, and the deletion of gE vaccine strain and normal BHK-21 cells in the control reaction; the minimum detection limit of the virus can be detected in the sample is about 105.0TCID50/0.1mL. The sandwich ELISA and test gB high free IgG can be established at the same time with PRV wild virus antigen and gE deletion vaccine strain response, with normal BHK-21 cells the control reaction.
Serum and molecular epidemiological survey in this study will provide a reference for the epidemic and evolutionary relationship of PRV in Henan province; the establishment of indirect and blocking ELISA can be used to distinguish between antibody vaccine and wild virus infection and PRV antibody, vaccine antibody levels provide a means of evaluation; quality inspection and evaluation of PRV vaccine virus antigen set the double antibody sandwich ELISA and preparation of colloidal gold strip for rapid detection of PRV can directly and simply wild virus infection. Therefore, this study provides for our PR antigen purification method, rapid detection of antibodies.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S852.65

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