摘要
以小鼠腺病毒作为无病原性“孤儿病毒”的模型,进行了构建真核细胞基因工程载体的研究。通过对小鼠腺病毒DNA的提取、酶切和克隆,将病毒DNA片段插入pBR322质粒中,从中选出重组质粒pBAD-8,进而在病毒DNA的BglⅡ单一切口处,插入Ⅰ型单纯疱疹病毒TK基因,构成新的重组质粒pBAT。将重组质粒pBAT和野生小鼠腺病毒在TK^-的3T3细胞内进行同源重组,使TK基因插入小鼠腺病毒的DNA序列中。通过选择性培养基培养及α-^(32)P标记的核酸探针检测,证明在经同源重组的小鼠腺病毒DNA中插入了TK基因,并得到了表达。将重组病毒接种小鼠,进行体内存留实验,结果表明所构建的小鼠腺病毒载体在小鼠体内至少可存活40d。
Mouse adenovirus FL(MAD-FL) was used as an experiment-al model for constructing 'orphan virus), vector in eukaryotic geneticengineering. MAD-DNA was extracted, digested and cloned with routinetechnique. Plasmid pAD-8 was selected from tens of recombined plas-mids containing MAD-DNA Bam HI fragments and further characterizedwith digestion by restriction endonucleases BglII, SalI,Hind III and BamHI. TK gene was inserted into BglII site of MAD-DNA fragment in pAD-8, and a new plasmid pBAT formed. Through homogenous recombinationof pBAT with a wild strain of MAD in 3T3 cells (TK^-), TK gene wastransferred into MAD-DNA. The insertion and the expression of TK genehave been identified by growth selection in HAT medium and. detectionwith α-^(32)p labeled nucleic acid probe. The recombined MAD was foundto be able to reside long time in animal host and the insertion of foreigngene did not affect the ability of infection and multiplication of the virus.The carried foreign gene can be transcribed and translated effectively inthe host cells. The recombined MAD is expected to be used as a longterm vector for eukaryotic genes in genetic engineering.
