猪伪狂犬病毒TK缺失株PRV/TK^-的构建及其生物学特性  

Construction and biological characteristics of TK-null recombinant pseudorabies virus

作　　者：陈瑞爱[1,2] 邵定勇[2] 韩静芳[2] 

机构地区：[1]中国农业大学生物学院,北京100083 [2]广东大华农动物保健品股份有限公司,广东新兴527400

出　　处：《中国兽医学报》2010年第5期577-582,共6页Chinese Journal of Veterinary Science

基　　金：广东省高新技术产业基金资助项目(2006B16102019)

摘　　要：为了构建伪狂犬病毒容A株(PRV-Ra)TK基因缺失重组病毒,本研究利用PCR从PRV-Ra株基因组分别扩增TK基因两侧的序列LTK和RTK,将LTK和RTK克隆到pUC19载体,构建转移质粒pUC19-TK/HEK。进一步将增强型绿色荧光蛋白基因(EGFP)表达盒克隆到pUC19-TK/HEK质粒中,构建转移质粒pUC19-TK/EGFP。用pUC19-TK/EGFP与PRV-Ra株基因组共转染BHK21细胞,通过噬斑纯化获得重组病毒PRV/TK-/EGFP;用pUC19-TK/HEK与PRV/TK-/EGFP病毒基因组共转染BHK21细胞,噬斑纯化获得不含EGFP基因的重组病毒PRV/TK-。该重组病毒在体外传30代后仍其有良好的遗传稳定性;PRV/TK-体外增殖速度、对家兔的毒力以及对仔猪的安全性和抗体反应性研究表明,重组毒株与原始株(PRV-Ra)在BHK21上具有相似的增殖规律;PRV/TK-对家兔的毒力比PRV-Ra下降了10000倍;以105.0TCID50PRV/TK-免疫小猪后4周,体内产生的平均中和抗体水平达101.9,略高于对照疫苗产生的抗体水平(101.8)。本试验为PRV基因功能的研究及PRV基因缺失苗的研发提供了技术平台。To construct TK-null recombinant pseudorabies virus （PRV）Ra strain as a techonolgial support of the research of genetic engineering vaccine and gene function of pseudorabies virus.Transfer vector pUC19-TK/HEK was constructed using both ends of PRV TK gene,which were successfully amplified by PCR from PRV-Ra genome.Subsequently enhanced green fluorescent protein gene （EGFP） expression box was cloned into pUC19-TK/HEK,resulting a universal transfer vector pUC19-TK/EGFP.Co-transfecting pUC19-TK/EGFP and genome of PRV-Ra into BHK21 cells,recombinant PRV/TK-/EGFP was obtained following plaque screening.pUC19-TK/HEK and genome of PRV/TK-/EGFP were co-transfected into BHK21 cells to obtain recombinant PRV/TK-.PCR confirmed its genetical stability after 30 passages in BHK21 cells.There was no significant difference of multiplication capacity between the recombinant PRV/TK-and PRV Ra strain.Comparing to the parent viurs,the virulence of PRV/TK-was significantly reduced to 1：10 000.The recombinant PRV/TK-was safe to weaning piglets at 107.0 TCID50,two weeks post-boosting,101.9 neutralizing antibody against PRV were detected in PRV/TK-vaccinated group with the dose of 105.0 TCID50,which is a little higher than that of the commerical vaccine group（101.8） This research is a bas-is of further study of both PRV gene funcitons and PRV gene deletion vaccines.

关 键 词：伪狂犬病毒容A株 TK缺失 生物学特性 

分 类 号：S852.65[农业科学—基础兽医学]