本文選題：豬瘟　切入點(diǎn)：病毒　出處：《河南科技學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬瘟(Classical Swine Fever)，早期稱豬霍亂(Hog Cholera, HC)，是由豬瘟病毒(Classical Swine Fever Virus, CSFV)引起豬的一種急性、熱性和高度接觸性傳染病，其特征為高熱稽留和全身廣泛性出血。豬瘟病毒(CSFV)屬于黃病毒科（Flaviviridae）瘟病毒屬（Pestivirus），是RNA病毒，具有感染性。CSFV在豬體內(nèi)復(fù)制增殖，破壞機(jī)體多種細(xì)胞，使豬迅速發(fā)病并死亡。而在體外細(xì)胞培養(yǎng)中，CSFV并不能使細(xì)胞產(chǎn)生病理變化。CSFV在體外增殖的病毒滴度較低，而提高病毒滴度對(duì)研究CSFV的分子生物學(xué)特性和免疫學(xué)特性，以及制備CSFV單抗和疫苗都具有非常重要的作用。 豬瘟病毒是單股正鏈線性RNA病毒，ORF編碼一個(gè)由3898個(gè)氨基酸組成的多聚蛋白，在病毒和宿主細(xì)胞酶的作用下，形成至少12種成熟的病毒蛋白。用于CSFV分子流行病學(xué)分析的核苷酸片段主要有NS5B、E2和5’UTR等，其中NS5B和E2可用來精確區(qū)分毒株。根據(jù)CSFV的NS5B、E2和E0基因分群的結(jié)果一致，初步認(rèn)為這3個(gè)基因在系統(tǒng)發(fā)生分析中具有高度的一致性。本研究通過設(shè)計(jì)E0、E2和NS5B基因特異性引物，擴(kuò)增目的片段，然后通過測(cè)序及序列分析，確定使用的CSFV與shimen株同源性最高，為后續(xù)試驗(yàn)奠定背景基礎(chǔ)。 PK15細(xì)胞廣泛應(yīng)用于CSFV的分離與培養(yǎng)，但是其不均一性導(dǎo)致了病毒培養(yǎng)滴度不高。研究表明豬瘟病毒在體外培養(yǎng)中釋放的病毒的量與細(xì)胞感染數(shù)量（陽性細(xì)胞）有關(guān)，所以提高細(xì)胞感染數(shù)量同時(shí)提高感染速度對(duì)提高病毒滴度有至關(guān)重要的作用。研究顯示感染PCV2的PK15細(xì)胞培養(yǎng)物，約有20％PK-15細(xì)胞是PCV2易感的細(xì)胞群，該研究表明PK-15細(xì)胞的異質(zhì)性與PCV2滴度的高低有密切關(guān)系。本實(shí)驗(yàn)通過IPMA檢測(cè)發(fā)現(xiàn)CSFV對(duì)PK-15的感染也僅僅只有20%左右，所以獲得均一的PK-15細(xì)胞有助于提高CSFV滴度。CSFV病毒滴度，即50％組織細(xì)胞感染劑量（TCID50），得到每毫升PK15細(xì)胞培養(yǎng)通常從104到105不等。為了獲得均一的、針對(duì)培養(yǎng)CSFV高病毒滴度的PK15細(xì)胞，本研究通過有限稀釋法對(duì)PK15親代細(xì)胞進(jìn)行克隆，獲得兩株P(guān)K15細(xì)胞克隆株，分別為PK15-1A6和PK15-3B1，，與PK15親代細(xì)胞相比，PK15-1A6和PK15-3B1TCID50分別能達(dá)到106.8TCID50/mL和103.61TCID50/mL，而PK15親代細(xì)胞的TCID50則為104.88TCID50/mL。PK15-1A6單克隆細(xì)胞株感染CSFV的強(qiáng)度遠(yuǎn)遠(yuǎn)高于PK15母細(xì)胞，為以后CSFV疫苗評(píng)價(jià)及診斷研究奠定了基礎(chǔ)。
[Abstract]:Hog Cholera, early known as hog Cholera, is an acute, feverish and highly contact infectious disease in pigs caused by classical Swine Fever virus (CSFV). Swine fever virus (CSFV) belongs to the family Flaviviridae (Flaviviridaeae), it is a RNA virus, and it is infectious. CSFV replicates and proliferates in pigs and destroys many kinds of cells. In vitro cell culture could not make the cells produce pathological changes. The virus titer of the proliferation of CSFV in vitro was lower, but the increase of virus titer was helpful to study the molecular biological and immunological characteristics of CSFV. The preparation of CSFV monoclonal antibodies and vaccines are very important. CSFV is a single-stranded linear RNA virus that encodes a polyprotein consisting of 3898 amino acids, acting on the virus and host cell enzymes. At least 12 mature viral proteins were formed. The nucleotide fragments used in CSFV molecular epidemiology analysis mainly included NS5BnE2 and 5UUTR, among which NS5B and E2 could be used to distinguish the virus strains accurately. According to the results of CSFV NS5BUE 2 and E0 gene subgroups, It is considered that these three genes are highly consistent in phylogenetic analysis. In this study, the target fragments were amplified by designing E0E _ 2 and NS5B gene specific primers, and then sequenced and sequenced. The highest homology between CSFV and shimen strain was determined, which laid a background for further test. PK15 cells are widely used in the isolation and culture of CSFV, but their heterogeneity leads to the low titer of the virus. Studies have shown that the amount of virus released by CSFV in vitro is related to the number of infected cells (positive cells). So increasing the number of infected cells and increasing the rate of infection are crucial to increasing the titer of the virus. Studies have shown that about 20 PK-15 cells infected with PCV2 are susceptible to PCV2. This study shows that the heterogeneity of PK-15 cells is closely related to the titer of PCV2. In this study, IPMA detection showed that the infection of CSFV to PK-15 was only about 20%, so obtaining homogeneous PK-15 cells was helpful to increase the titer of CSFV. That is to say, the infected dose of 50% tissue cells was TCID50, and the culture of PK15 cells usually ranged from 104 to 105.In order to obtain a uniform culture of PK15 cells with high virus titer of CSFV, the parent cells of PK15 were cloned by limited dilution method. Two PK15 cell clones, PK15-1A6 and PK15-3B1, were obtained. Compared with PK15 parent cells, PK15-1A6 and PK15-3B1TCID50 could reach 106.8 TCID50 / mL and 103.61 TCID50 / mL, respectively, while the TCID50 of PK15 parental cells was 104.88 TCID50 / mL.PK15-1A6 and the intensity of CSFV infection was much higher than that of PK15 mother cells. It lays a foundation for the evaluation and diagnosis of CSFV vaccine.
【學(xué)位授予單位】：河南科技學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

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