伪狂犬病病毒gG基因缺失通用转移载体的构建

对克隆有伪狂犬病病毒Ea株基因组DNA SphI 15kb片段的质粒pBSA用SphI和KpnI消化，将含gG全基因及其上游PK基因，下游gD基因部分编码区的约2．6kb的片段亚克隆到消除了EcoRI位点的载体pUC19中，获得重组质粒pUSK(E)。以pEGFP-N1为模板，通过PCR扩增约0．3kb的SV40Poly(A)片段，并将其克隆到杆状病毒转座载体pFastBac1的NotI和PstI位点，利用pFastBac1的BamHI和PstI将含有9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区约9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区的400bp的缺失，构建了由gG启动子驱动gG部分编码区缺失的通用转移载体pgG-Uni。序列分析进一步证实：有7个单一酶切位点可供外源基因直接插入。上述结果为构建以伪狂犬病病毒作载体的多价基因工程疫苗以及利用标记蛋白探讨伪狂犬病病毒在体内的增殖与分布奠定了基础。

Construction of a Universal Transfer Vector Deleting gG of Pseudorabies Virus

The plasmid pBSA containing the SphI 15kb fragment of genomic DNA of pseudorabies virus (PRV) Ea strain was digested with Sph I and Kpn I. About 2.6kb DNA fragment containing the complete gG gene PK and gD partial gene were recovered and cloned into the same sites of modified vector pUC19 eliminating the Eco RI site, which resulted in the recombinant plasmid pUSK(E). 0.3kb SV 40 Poly(A) DNA fragments were amplified with pEGFP N1 by PCR and were cloned into pFastBac1.Tha same fragments with 9 restriction sites were sub cloned into the plasmid pUSK(E), and replaced in gG the 5' partial coding region. As a result, in a universal transfer vector pgG Uni was constructed.. The results of sequences analysis showed that there were 7 single restriction sites in M.C.S. which enable the foreign gene to be inserted directly.