本文選題：犬細(xì)小病毒 + 分離鑒定�。� 參考：《延邊大學(xué)》2015年碩士論文

【摘要】：犬細(xì)小病毒病(canine parvovirus infection, CP)是由犬細(xì)小病毒(canine parvovirus, CPV)感染而引起犬科及其肉食性經(jīng)濟(jì)型動(dòng)物患病的一種急性接觸性傳染病。CP臨床患病犬主要有兩種表現(xiàn)型,即出血性腸炎型和非化膿性心肌炎型,且發(fā)病率及致死率均較高,是危害我國(guó)犬科類動(dòng)物健康的主要疫病之一,對(duì)犬科類動(dòng)物的生存構(gòu)成極大威脅,同時(shí)也嚴(yán)重影響我國(guó)經(jīng)濟(jì)動(dòng)物養(yǎng)殖業(yè)的健康發(fā)展。我國(guó)專家學(xué)者在CP流行病學(xué)、CP病原學(xué)、CP診斷及CP預(yù)防等多方面進(jìn)行了大量研究,且已經(jīng)取得階段性研究進(jìn)展。為豐富我國(guó)CP流行病學(xué)調(diào)查資料,了解延邊地區(qū)CPV的遺傳進(jìn)化趨勢(shì),進(jìn)一步深入研究CPV的生物學(xué)特性,制備更有針對(duì)性的地區(qū)性疫苗,本研究采集延邊地區(qū)疑似感染CPV犬組織病料,將病料預(yù)處理后接種F81細(xì)胞進(jìn)行病毒分離試驗(yàn),并對(duì)分離株病毒進(jìn)行形態(tài)學(xué)及血清學(xué)鑒定，，克隆CPV-VP2基因,分析其遺傳進(jìn)化規(guī)律,并構(gòu)建pGEX-CPV-VP2重組表達(dá)質(zhì)粒,對(duì)重組融合表達(dá)蛋白進(jìn)行優(yōu)化誘導(dǎo)表達(dá)及純化,且經(jīng)Western-blot分析純化蛋白反應(yīng)原性后,將純化的重組CPV-VP2蛋白免疫BALB/c小鼠,制備鼠抗CPV-VP2蛋白多克隆抗體,采用ELISA方法監(jiān)測(cè)其抗體效價(jià)。結(jié)果顯示,病毒接種F81細(xì)胞后,可致細(xì)胞變圓、腫脹、脫落等明顯細(xì)胞病變(CPE)；經(jīng)IFA鑒定,可觀察特異性綠色熒光；回歸動(dòng)物試驗(yàn)可致試驗(yàn)幼犬出現(xiàn)典型CP臨床特征,且剖檢可見與自然感染相似的病理變化；CPV-VP2基因測(cè)序結(jié)果經(jīng)分析后進(jìn)一步證明,本試驗(yàn)已成功分離到延邊地區(qū)本地株犬細(xì)小病毒,命名為YBYJ株CPV,YBYJ株CPV-VP2基因全長(zhǎng)約為1755bp,編碼584氨基酸；該分離株病毒與GenBank中收錄的國(guó)內(nèi)外47株CPV-VP2基因核苷酸同源性為98.7～99.9%,氨基酸同源性為96.9～99.8%,且與參考株比對(duì)后有氨基酸位點(diǎn)變異；經(jīng)鑒定已成功構(gòu)建原核表達(dá)重組質(zhì)粒pGEX-CPV-VP2,經(jīng)IPTG誘導(dǎo)表達(dá),SDS-PAGE分析鑒定表達(dá)蛋白約為118 Ku,且以包涵體形式存在；純化蛋白經(jīng)Western-blot分析鑒定,具有較好的反應(yīng)原性；免疫BALB/c小鼠,能夠誘導(dǎo)其產(chǎn)生抗CPV-VP2抗體,表明該重組表達(dá)蛋白同時(shí)具有較好的免疫原性。本研究成果將為下一步CPV基因工程疫苗研制、單克隆抗體制備,以及后期開展CPV相關(guān)研究奠定基礎(chǔ)。
[Abstract]:Canine parvovirus infection (CPV) is an acute contact infectious disease caused by canine parvovirus (CPV) infection in canine canine parvovirus.CP has two main phenotypes.Hemorrhagic enteritis type and non-suppurative myocarditis type, with high morbidity and mortality, are one of the major diseases that endanger the health of canines in China, and pose a great threat to the survival of canines.At the same time, it also seriously affects the healthy development of China's economic animal husbandry industry.Chinese experts and scholars have made a great deal of research on CP epidemiology, CP etiology, CP diagnosis and CP prevention, and have made progress in stages.In order to enrich the epidemiological investigation data of CP in China, to understand the genetic and evolutionary trend of CPV in Yanbian area, to further study the biological characteristics of CPV, and to prepare a more targeted regional vaccine.In this study, samples of suspected infected CPV canine tissue in Yanbian area were collected and inoculated with F81 cells for virus isolation test after pretreatment. The virus was identified by morphology and serology, CPV-VP2 gene was cloned and its genetic evolution was analyzed.The recombinant pGEX-CPV-VP2 expression plasmid was constructed, and the recombinant fusion protein was optimized for induction and purification. After Western-blot analysis, the purified recombinant CPV-VP2 protein was immunized with BALB/c mice to prepare anti- polyclonal antibody.The antibody titers were detected by ELISA method.The results showed that after inoculation with F81 cells, the cells became round, swelling, exfoliation and other obvious cytopathic changes. The specific green fluorescence could be observed by IFA, and the typical clinical features of CP could be observed in the experimental puppies.The results of CPV-VP2 gene sequencing showed that the local canine parvovirus strains in Yanbian area had been isolated successfully.The total length of CPV-VP2 gene of YBYJ strain was about 1755 BP, encoding 584 amino acids, and the nucleotide homology of CPV-VP2 gene was 98.799. 9, amino acid homology was 96. 9%, and there was amino acid mutation after comparison with reference strain.The prokaryotic expression plasmid pGEX-CPV-VP2was successfully constructed, and the expressed protein was identified to be about 118Kuand existed as inclusion body by IPTG induced SDS-PAGE analysis. The purified protein was identified by Western-blot analysis and had good reactivity. BALB/c mice were immunized with pGEX-CPV-VP2.It can induce the production of anti CPV-VP2 antibody, which indicates that the recombinant protein has good immunogenicity at the same time.The results of this study will lay a foundation for the further development of CPV gene engineering vaccine, the preparation of monoclonal antibody, and the later research on CPV.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.655

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