猪血清中PPV的分离与鉴定及培养条件的优化

发布时间：2019-05-24 03:41

【摘要】：猪细小病毒是一种主要引起母猪繁殖障碍的病原,怀孕母猪感染猪细小病毒可能会出现返情、流产、产死胎、产木乃伊胎、产弱仔等临床症状。除了能引起母猪繁殖障碍外,猪细小病毒还能引起哺乳仔猪非化脓性心肌炎、仔猪渗出性皮炎、育肥猪间质性肾炎等疾病,另外与猪圆环病毒共同感染能够加重仔猪多系统衰竭综合征。本实验室从一育肥猪早、中期生产水平较差的猪场收集不同年龄段的育肥猪血清214份,用DNA提取试剂盒提取血清中的DNA,设计猪细小病毒特异性引物对214份血清DNA进行核酸检测,并选取其中7份经PCR检测为猪细小病毒阳性的血清用ST细胞进行病毒分离,盲传3代后,将病毒液冻融三次,用猪细小病毒特异性引物对7份血清接种的细胞液进行PCR鉴定,其中一份血清检测到了目的条带。确定分离到一株猪细小病毒,命名为PPV-16WS。通过观察细胞病变、测定血凝效价、分析部分全基因序列等方法研究病毒特性。结果显示,该病毒传至第5代能够出现较稳定的细胞病变,血凝效价能达到28,对其部分全基因组进行测序发现与GenBanK收录的传统的PPV序列同源性较高,其中与2012年时乐分离的YL株同源性最高,达到了99.0%,表明16WS是PPV。本研究建立检测PPV实时荧光定量PCR方法,为PPV病毒的初步定量检测奠定了基础。并对实验室分离株16WS进行传代对其培养条件进行摸索,主要包括：ST细胞生长状态、病毒孵育时间、细胞密度、病毒接种量、病毒收毒时间、培养体积、有无二氧化碳等条件,希望通过优化病毒培养条件来提高病毒滴度。结果显示：将生长48h的ST细胞传代12h后待细胞密度为60%(6.0-7.5×106cells/ml)时,进行分步接毒,孵育1h,接种量为107copies/ml,六孔板培养体积为2ml,放置5%二氧化碳培养箱培养72h,病毒滴度最高,能够稳定在3.59×109copies/ml左右。
[Abstract]:Pig parvovirus is a kind of pathogen which mainly causes reproductive disorder of sows. Pregnant sows infected with pig parvovirus may have clinical symptoms such as return, abortion, stillbirth, mummification, weak birth and so on. In addition to causing reproductive disorders in sows, swine parvovirus can also cause non-suppurative myocarditis, exudative dermatitis in piglets, interstitial nephritis in fattening pigs and other diseases. In addition, co-infection with pig circovirus can aggravate multisystem failure syndrome in piglets. In our laboratory, 214 serum samples of fattening pigs of different ages were collected from a pig farm with poor production level in the early and middle stages of fattening pigs, and DNA, in serum was extracted by DNA extraction kit. The nucleic acid of 214 serum DNA samples was detected by designing specific primers of pig parvovirus, and 7 of them were detected by PCR and isolated with ST cells. After blind transmission for 3 generations, the virus solution was frozen and thawed three times. The cell fluid inoculated with 7 serum samples was identified by PCR with Porcine parvovirus specific primers, and the target band was detected in one of the seven serum samples. A strain of Porcine Parvovirus named PPV-16WS. was identified. The characteristics of the virus were studied by observing the cytopathic effect, measuring the hemagglutination titer and analyzing the whole gene sequence. The results showed that the virus could produce stable cytopathic effect and the hemagglutination titer could reach 28. Some of the whole genome was sequenced and found to have high homology with the traditional PPV sequence included in GenBanK. Among them, the homology with the YL strain isolated in 2012 was the highest, reaching 99.0%, indicating that 16WS was PPV.. In this study, a real-time fluorescence quantitative PCR method for the detection of PPV was established, which laid a foundation for the preliminary quantitative detection of PPV virus. The culture conditions of laboratory isolate 16WS were explored, including ST cell growth state, virus incubation time, cell density, virus inoculum size, virus collection time, culture volume, carbon dioxide and so on. It is hoped that the virus titer can be improved by optimizing the culture conditions of the virus. The results showed that after passage of ST cells for 48 h for 12 h, when the cell density was 60% (6.0 脳 7.5 脳 106cells/ml), the cells were incubated step by step for 1 h, the inoculum size was 107 copies / ml, and the culture volume of six-well plate was 2 ml. When cultured in 5% carbon dioxide incubator for 72 hours, the virus titer was the highest and could be stabilized at about 3.59 脳 109copies/ml.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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