噬菌体T7基因6.5和6.7的克隆及其缺失变种的构建

 用适当的限制性内切酶,将噬菌体T7基因6.5和6.7从整个噬菌体T7基因组中分离出来,插入到质粒pBR322中去,转化E.Coli HMS174,筛选出这两个基因的成功克隆。运用同样手段,从整个噬菌体T7基因组中分离出含有部分基因6编码序列,而不含基因6.5和6.7编码序列的T7DNA片段,插入到pBR322的衍生质粒中去,转化Ecoli C1757,再用含有基因6和基因7的双突变噬菌体T7去感染这一转化菌,通过同源交叉而得到缺失基因6.5和6.7的噬菌体T7缺失变种。这种噬菌体只能在载有噬菌体T7基因6.5和6.7,或者只载有基因6.7质粒的寄主中增殖。通过噬菌体结构蛋白电泳分析证明,这种噬菌体丢失了野生型菌体T7所具有的两条结构蛋白带。

CLONING 6.5 AND 6.7 OF BACTERIOPHAGE T7 AND CONSTRUCTION OF ITS DELETION MUTANT

Gene 6.5 and 6.7 of bacteriophage T7 were isolated from intact genome of bacteriophage T 7 by appropriate restriction endonucleases. These genes were inserted into plasmid pBR322 to transfect E. coli HMS 174 (rk12- mkl2+ recAl rifR nonsuppressor). The successful clones harboring T7 gene 6.5 and 6.7 were screened. Using the same strategy, a fragment that contained the partial gene 6 without coding sequence of gene 6.5 and 6.7 from T7 DNA were inserted into the derivative of pRR322, pAR767 to transfect E. coli C1757 (suppressor).The cultures of E.coli C1757 carrying the indicated plasmid were infected by a phage T7 double mutant in gene 6 and gene 7. During the growth in culture, a deletion mutant phage lacking gene 6.5 and 6.7 was obtaind due to the lysate homologous cross between the plasmid and the double mutant.This deletion mutant could only grow on hosts that would supply gene 6,5 and 6.7 or gene 6.7 complementing plasmid. Electrophoresis of the structural protein from the deletion mutant showed that two bands were missing from the protein pattern of wild-type T7.