摘要
为了以国内地方分离株鄂A株为亲本构建伪狂犬病病毒双基因缺失株 ,采用外切酶Ⅲ和绿豆核酸酶酶切 ,构建了缺失主要毒力基因TK基因部分编码区的重组质粒 pSTK1-4 ,进一步改造成为转移质粒 pUCPB4。用HindⅢ将质粒pUCPB4线性化 ,然后与用EcoRI消化的伪狂犬病病毒鄂A株TK-/LacZ+ 突变株基因组DNA共转染PK- 15细胞 ,待完全病变后 ,收毒作空斑试验 ,PCR筛选TK缺失的重组病毒。重组病毒空斑纯化 3次 ,随机挑取空斑进行PCR扩增 ,证实所获得的病毒为均一的无TK-/LacZ+ 突变株污染的TK缺失的重组病毒。分别以TK缺失株病毒与鄂A野毒株为模板对TK基因进行PCR扩增 ,扩增产物经酶切分析和测序后发现 :TK缺失重组病毒的TK基因缺失了 2 0 5个碱基 ,XhoI和SalI位点消失 ,SmaI酶切片段发生变化。两种病毒在PK - 15细胞上形成空斑的大小和增殖的滴度无明显的差别。进一步提取TK缺失突变株基因组DNA ,与含gG -LacZ的转移质粒pUSKZ通过磷酸钙法共转染PK - 15细胞 ,待完全病变后 ,在X - gal存在下筛选蓝斑 ,将挑取的蓝斑纯化 3次后 ,对纯化的重组病毒进行TK、LacZ扩增 ,结果既能扩增出较以鄂A野毒株为模板扩增的要小的TK基因片段 ,同时又能扩增出LacZ基因 ,证实所得到的重组病毒为TK-/ gG-/LacZ+ 突变株。此双缺失突变株的构?
In order to obtain an ideal TK-/gG-/LacZ+ deleted vaccine strain of pseudorabies virus(PRV), several TK-deleted plasmids were constructed by Exonuclease III and Mung Bean nuclease cleavage. One of them, pSTK 1-4, was further constructed as the transferring plasmid pUCPB4. Linerized pUCPB4 was cotransfected into PK-15 cells with EcoRI-digested TK-/LacZ+ mutant DNA. After CPE, TK-deleted recombinants were selected by PCR and plaque-purified three times. The partial sequence of TK gene of the resultant TK-deleted mutant virus was determined and compared with that of parent PRV strain Ea. The results showed that the TK gene of the TK-deleted mutant was shorter by 205bp than that of PRV strain Ea and the deletion region involved the conservation region of TK. Comparing the size of plaques and the titers in PK-15 cells of the TK- mutant with that of the PRV strain Ea, there were no obvious differences between them. Then a plasmid pUSKZ containing gG-LacZ expression cassette was cotransfected into PK-15 cells with DNA of TK- mutant of PRV Ea using the calcium phosphate coprecipitation technique. After CPE was observed, the transfection progenies were plated onto PK-15 cells in the presence of X-gal, the blue plaques were picked up and purified three times. Recombinants, named TK-/gG-/LacZ+, were obtained. They were further confirmed by polymerase chain reaction(PCR).
出处
《病毒学报》
CAS
CSCD
北大核心
2001年第1期69-74,共6页
Chinese Journal of Virology
基金
"九五"国家科技攻关计划生物技术项目! (96-C0 1-0 4-0 3 )
