伪狂犬病病毒通用转移载体的构建及应用

Construction and Application of a Universal Vector of Pseudoriabies Virus

将来自质粒pFSV40的 3 0 0bpBamHI/PstI片段〔其中含有SV40poly(A)和部分多克隆位点〕插入到质粒pUSK相应的酶切位点中 ,获得重组质粒pUSKSV40 ,该重组质粒中gG基因 5’端编码区缺失了 42 8bp。将来自质粒pcDNA3 .1( +)的 946bpBglIIEcoRI片段 (其中含CMV启动子及部分多克隆位点 )插入到质粒pUSKSV40的BamHIEcoRI位点 ,构建了通用载体pPRVCMV_uni,其中含有CMV启动子、SV40poly(A)以及NheI、Pmel、BamHI、BstXI、EcoRI、StuI、XbaI等 7个单一克隆位点。将eGFP基因插入到该通用载体的BamHI和EcoRI之间 ,用所获得的转移载体与TK_/gG_/LacZ+ PRV基因组共转染PK_1 5细胞 ,经检测eGFP基因在重组伪狂犬病病毒中获得表达 ,从而证实该通用载体的构建是可行的。本研究研制以伪狂犬病病毒为载体的二价或多价基因工程疫苗奠定了物质基础。

A 300bp fragment containing SV40 poly(A) signal and part of nulitiple cloning sites(MCS),was inserted into Bam HI Pst I of pUSK,resulting in a recombinant pUSKSV40,which gave rise to a deleton of 428bp in the 5'-end of PRV gE gene.A 946bp Bgl II Eco RI fragment from pcDNA3.1(+) inchuding human cytomegalovirus(hCMV) promoter and partial multiple cloning sites was cloned into the Ban HI Eco RI sites of the pUSKSV40,resulting a universal transfer vector,which contained the hCMV promoter,SV40 poly(A) signal and 7 nultiple cloning sites including Nhe I, Pme l, Bam HI, Bst XI, Eco RI, Stu I and Xba I.To test the applicability of the universal vactor,e GFP was selected as a reporter gene and inserted into the Bam HI Eco RI Sites of the pPPVCMV-uni to obtain a recombinant plasmid pPRVCMV-eGFP.The PK-15 cell cultures were cotransfect with pPRVCMV-eGFP and the genome of a TK -/gG -/LacZ + PRV,generating a recombinant PRV,which was confirmed to express eGFP.The results indicated that the constructed pPRVCMV-uni was feasible.It will be useful for developing the recombinant PRV exzpressing foreign gene(s).