PRV特异性糖蛋白gp50基因片段的克隆探针制备

Cloning and Probe Preparation of the Specific Glycoprotein gp50 of PRV

在ＢＨＫ２１细胞中增殖伪狂犬病病毒（ＰＲＶ），提纯ＰＲＶＤＮＡ，选编码特异性糖蛋白ｇｐ５０基因中的２６２ｂｐ片段进行ＰＣＲ扩增。扩增产物经ＫｐｎⅠ和ＳａｌⅠ双酶切后，将２２２ｂｐ片段与质粒ｐＵＣ１９连接构成重组子ｐＵＰ。通过转化大肠杆菌ＪＭ８３、Ａｍｐｒ和α互补效应筛选后，扩增、提纯重组子ｐＵＰ，用ＫｐｎⅠ和ＳａｌⅠ酶切，电泳回收克隆的２２２ｂｐ片段。用光敏生物素标记２２２ｂｐ片段和重组子ｐＵＰ制备的探针，分别检出４６．８ｐｇ和９３．６ｐｇ的ＰＲＶＤＮＡ，且ｐＵＰ探针只与ＰＲＶ呈杂交阳性反应，与ＨＳＶ－１、ＨＳＶ－２及ＣＭＶ呈阴性反应。

Pseudorabies viruses (PRV) were replicated in cells of hamster origin (BHK 21 ) and then the PRV DNA extracted. The segment selected for polymerase chain reaction DNA amplification was consisted of 262 base pairs within the gene coding the specific glycoprotein gp50. The production of amplification was digested with KpnⅠ and SalⅠ and then inserted into the vector plasmid pUC19 to construct a recombinant pUP. The E coli 83 cells were transformed by the recombinant pUP. The clones with the recombinant pUP which was screened by Amp r, IPTG and X gal amplification, were extracted from E coli JM83 and were digested with KpnⅠ and SalⅠ. The cloned fragment of 222 base pairs was isolated by gel electrophoresis. The cloned 222 bp fragment and the recombinant pUP were labelled with photobiotin and utilized to hybridize with PRV DNA, PRV, HSV 1, HSV 2 and CMV. The two probes were proved to be specific and more sensitive by the hybridization, with the 46.8 pg and 93.6 pg of PRV DNA being detected.