湖南省4株伪狂犬病毒的分离鉴定与培养高滴度伪狂犬病毒的研究

发布时间：2018-08-11 17:59

【摘要】：2012-2014年从湖南平江、泪罗、浏阳、长沙四地的四个规模猪场送检的病料(脑组织)中检测到伪狂犬病毒(PRV)野毒。将脑组织接种PK-15细胞,经PCR和动物接种鉴定为伪狂犬病毒。4个毒株分别命名为PRV-XiangA、PRV-GA、PRV-YY和PRV-LY。本研究通过对这4株PRV的免疫(gB、gG、gH、gI、gL、gM)与毒力相关基因(gE、PK、TK)进行序列分析,结果显示这4株PRV的免疫与毒力相关基因核苷酸同源性在99.8%-100%,说明分离得到的4株PRV变异极小；将这4株PRV与国内外的主要毒株进行序列比对,结果表明这4株PRV与国内的分离毒株同源性很高,与BJ-YT株和TJ株的亲缘关系最近,为99.8%-100%,说明当前国内流行的PRV毒株变异较小。对从湖南省分离得到的4株伪狂犬病毒进行毒价测定后,筛选出一株培养滴度较高的伪狂犬病毒,即PRV-YY株,该株伪狂犬病毒在MDBK细胞上的病毒滴度能够达到107.41 TCID50/0.1mL。通过建立MDBK单克隆细胞库,筛选出1株单克隆细胞MDBK-1培养PRV-YY株的病毒滴度能够达到107.71 TCID50/0.1mL,通过终点稀释法将PRV-YY株在MDBK-1上进行传代,PRV-YY株在该株单克隆细胞MDBK-1上的病毒滴度能够稳定在107.5TCID50/0.1mL以上,PRV-YY株的平均病毒滴度达到107.692TCID50/0.1mL, PRV-YY株的病毒滴度在原有的基础上提高了约2倍。这为制备伪狂犬病毒野外流行毒株灭活疫苗打下了良好的基础。
[Abstract]:Pseudorabies virus (PRV) was detected from four pig farms in Pingjiang, Yiluo, Liuyang and Changsha from 2012 to 2014. The brain tissue was inoculated with PK-15 cells and identified as pseudorabies virus by PCR and animal inoculation. The four strains were named PRV-XiangAv GAPRV-YY and PRV-LY. In this study, the nucleotide homology of immune and virulence related genes (PRV) of the four strains of PRV were analyzed. The results showed that the nucleotide homology of the four strains of PRV was 99.8-100, which indicated that the PRV mutation of the four isolated strains was very small. The four PRV strains were sequenced with the main strains at home and abroad. The results showed that the four PRV strains had high homology with the domestic isolates, and had the closest relationship with BJ-YT and TJ strains (99.8-100), indicating that there was little variation in the current prevalent PRV strains in China. Four strains of pseudorabies virus isolated from Hunan Province were tested for virus titer. A pseudorabies virus strain, PRV-YY strain, was selected. The titer of pseudorabies virus on MDBK cells was 107.41 TCID50 / 0.1 mL. By establishing a MDBK monoclonal cell library, The virus titer of PRV-YY strain cultured with a monoclonal cell line MDBK-1 could reach 107.71 TCID 50 / 0.1 mL, and the virus titer of PRV-YY strain on MDBK-1 could be stabilized above 107.5TCID50/0.1mL by terminal dilution method. The virus titer of PRV-YY strain on MDBK-1 was stable at the end point dilution method. The average titer of PRV-YY strain was 107.692TCID 50 / 0.1mL, and the virus titer of PRV-YY strain increased by about 2 times. This lays a good foundation for the preparation of pseudorabies virus inactivated vaccine.
【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】：S852.65

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