小鼠SRS白血病病毒前病毒DNA片段的分子克隆

MOLECULAR CLONING OF MOUSE SRSV PROVIRAL DNA FRAGMENT

从重组质粒pSF_(11)中提取SRS白血病病毒(SRSV)5kb DNA片段,用双脱氧法对其两末端进行测序,每端测得约150bp,从中筛选出PCR引物。SRSV新鲜感染NIH/3T3细胞,从感染细胞的胞质内提取前病毒DNA。用PCR法对SRSV前病毒DNA中的约3.4kb片段进行扩增,扩增产物用BamHⅠ酶切后与碱性磷酸单酯酶处理的pUC_(19)载体连接,并转化大肠杆菌JM_(103),得到含约3.4kb前病毒DNA片段插入子的重组质粒,并经Southern Blot证实,定名为pSF_(12),该克隆的SRSV DNA片段含有部分的env和pol基因。pSF_(12)和pSF_(11)构成SRSV的完整基因克隆。

SRSV 5 kb DNA fragment, extracted from the recombinant pSPu, was sequenced about 150 bp at each terminal by the dideoxymediated method. Two primers for polymerase chain reaction (POE) were designed from the each terminal. SRSV proviral DNA was extracted from the cytoplasm of NIH/3T3 cells freshly infected by SRSV. 3.4kb DNA fragment was amplified from the SRSV proviral DNA by POR. The POR product was digested with BamH I and ligated to pUC19 vector which was treated with calf intestinal alkaline phosphatage. Following the ligation reaction, transformed the plaemid DNA into competent cells of E. coli JM103. A recombinant, containing 3.4kb DNA insert, was constructed and identified by southern blot. It was named pSFia. A part of env gene and pol gene is involved in, this recombinant. The whole gene clone of SRSV is formed by pSF12 and pSF11.