猪瘟病毒高感染性PK-15细胞单克隆细胞株的筛选

发布时间：2018-03-21 11:01

本文选题：猪瘟　切入点：病毒　出处：《河南科技学院》2015年硕士论文　论文类型：学位论文

【摘要】：猪瘟(Classical Swine Fever)，早期称猪霍乱(Hog Cholera, HC)，是由猪瘟病毒(Classical Swine Fever Virus, CSFV)引起猪的一种急性、热性和高度接触性传染病，其特征为高热稽留和全身广泛性出血。猪瘟病毒(CSFV)属于黄病毒科（Flaviviridae）瘟病毒属（Pestivirus），是RNA病毒，具有感染性。CSFV在猪体内复制增殖，破坏机体多种细胞，使猪迅速发病并死亡。而在体外细胞培养中，CSFV并不能使细胞产生病理变化。CSFV在体外增殖的病毒滴度较低，而提高病毒滴度对研究CSFV的分子生物学特性和免疫学特性，以及制备CSFV单抗和疫苗都具有非常重要的作用。猪瘟病毒是单股正链线性RNA病毒，ORF编码一个由3898个氨基酸组成的多聚蛋白，在病毒和宿主细胞酶的作用下，形成至少12种成熟的病毒蛋白。用于CSFV分子流行病学分析的核苷酸片段主要有NS5B、E2和5’UTR等，其中NS5B和E2可用来精确区分毒株。根据CSFV的NS5B、E2和E0基因分群的结果一致，初步认为这3个基因在系统发生分析中具有高度的一致性。本研究通过设计E0、E2和NS5B基因特异性引物，扩增目的片段，然后通过测序及序列分析，确定使用的CSFV与shimen株同源性最高，为后续试验奠定背景基础。 PK15细胞广泛应用于CSFV的分离与培养，但是其不均一性导致了病毒培养滴度不高。研究表明猪瘟病毒在体外培养中释放的病毒的量与细胞感染数量（阳性细胞）有关，所以提高细胞感染数量同时提高感染速度对提高病毒滴度有至关重要的作用。研究显示感染PCV2的PK15细胞培养物，约有20％PK-15细胞是PCV2易感的细胞群，该研究表明PK-15细胞的异质性与PCV2滴度的高低有密切关系。本实验通过IPMA检测发现CSFV对PK-15的感染也仅仅只有20%左右，所以获得均一的PK-15细胞有助于提高CSFV滴度。CSFV病毒滴度，即50％组织细胞感染剂量（TCID50），得到每毫升PK15细胞培养通常从104到105不等。为了获得均一的、针对培养CSFV高病毒滴度的PK15细胞，本研究通过有限稀释法对PK15亲代细胞进行克隆，获得两株PK15细胞克隆株，分别为PK15-1A6和PK15-3B1，，与PK15亲代细胞相比，PK15-1A6和PK15-3B1TCID50分别能达到106.8TCID50/mL和103.61TCID50/mL，而PK15亲代细胞的TCID50则为104.88TCID50/mL。PK15-1A6单克隆细胞株感染CSFV的强度远远高于PK15母细胞，为以后CSFV疫苗评价及诊断研究奠定了基础。
[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.
【学位授予单位】：河南科技学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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