【摘要】 地高辛标记伪狂犬病病毒(PRV)E a株短区段蛋白激酶(PK)基因3′端0.4 kb片段,Sou thern杂交确定短区段PK基因定位在基因组DNA B amHⅠ4.0 kb片段中。将该片段克隆获得重组质粒pSB 304,对pSB 304亚克隆,构建了仅含完整PK基因约1.3 kb片段的重组质粒pSB 305,并进行了序列测定。结果表明,PK基因存在2种可能的同框编码方式,分别编码388或334个氨基酸残基,并具有真核细胞蛋白激酶催化结构域序列。同国外PRV N IA-3、K a株相比,氨基酸同源性分别为98.8%和97.3%,有意义的是E a株、K a株均较N IA-3株在同一位置缺失2个氨基酸(A sp,G ly)。进一步对pSB 305和含gG全基因以及部分gD基因的质粒pU SK进行酶切拼接,将PK基因大部分编码区、gG基因5′端部分编码区进行缺失,构建成两端同源侧翼分别为3.1 kb和1.6 kb的PK、gG双缺失转移载体pLR 001。上述结果为深入研究PK基因功能及研制更安全的TK-/PK-/gG-三缺失基因工程疫苗奠定了基础。更多还原

【Abstract】 A 4.0 kb BamHⅠ fragment which containing the PK gene of unique short region of pseudorabies virus Ea strain was determined and cloned by Southern blot using Dig-labeled 0.4 kb fragment which locating in the 3′ of PK gene,resulting in the recombinant plasmid pSB304.A 1.3 kb fragment through EcoRⅤ to BamHⅠ from the plasmid pSB304 was subcloned into pBluescriptⅡ SK~+ to obtain the recombinant plasmid pSB305.The complete sequence was also determined by Sanger′s technique.The sequence analysis indicated that there existed two kinds of open read frame(ORF) in which the ATG was located in the 97 and 259 respectively.The amino acids of the PK contains most of the conserved motifs of eukaryotic protein kinase.When compared with the PK gene of PRV NIA-3 and Ka strain,the homology of deduced amino acids were 98.8% and 97.3% respectively.It is interesting that the PK of NIA-3 strain have additional two amino acids(Asp,Gly) in the same sites than Ea and Ka strain.A recombinant transfer vector pLR001,in which about 900 bp of encoding region of the PK gene and about 400 bp of the 5′ encoding region of the gG were deleted simultaneously,was further constructed by using DNA recombination technique from the plasmid pSB305 and pUSK containing the complete gG gene and partial gG gene of PRV Ea strain.The above results might be contributive to the study of the construction and function of the PK gene and the development of more safe and effective genetically engineered TK~-PK~-gG~-PRV vaccine.更多还原