人乳头瘤病毒HPV16L1-E7C重组腺病毒载体的构建

CONSTRUCTION OF A RECOMBINANT ADENOVIRUS VECTOR OF HUMANPAPILLOMAVIRUS HPV16L1 E7C

采用ＰＣＲ方法，从ＨＰＶ１６型野毒株质粒中分离出ＨＰＶ１６Ｌ１（５５５９７１５１ｂｐ）、ＨＰＶ１６Ｅ７Ｃ（６８２８５５ｂｐ）基因片段，采用ＰＣＲ产物的ＴＡ克隆法，将Ｌ１、Ｅ７Ｃ分别插入ｐＧＥＭＴＥａｓｙ载体，构建克隆载体ｐＴＡＬ１、ｐＴＡＥ７Ｃ。ＢａｍＨⅠ、ＨｉｎｄⅢ酶切ｐＴＡＥ７Ｃ，Ｂｇ１Ⅱ、ＣｌａⅠ酶切ｐＴＡＬ１，回收Ｌ１、Ｅ７Ｃ片段，利用ＢａｍＨⅠ、Ｂｇ１Ⅱ酶切产生相同粘末端的特点，产生Ｌ１Ｅ７Ｃ重组基因片段，将其克隆入质粒ｐＢｌｕｅＳｃｒｉｐｔｓｋ，构建了ｐＢＳＬ１Ｅ７Ｃ重组克隆质粒，ＨｉｎｄⅢ、ＸｈｏⅠ酶切ｐＢＳＬ１Ｅ７Ｃ，释放Ｌ１Ｅ７Ｃ基因片段，定向重组入ｐＣＡ１４腺病毒载体，成功构建真核表达载体ＨＰＶ１６Ｌ１Ｅ７Ｃ重组腺病毒载体：ｐＣＡ１４Ｌ１Ｅ７Ｃ。

The entire HPV16L1 gene and C terminal HPV16E7 gene were amplified from wild type HPV16 plasmid using PCR method.After using T A cloning of PCR products,E7C gene and L1 gene are inserted into the pGEM T easy vector.After digested pTAL1 with restriction endonuclease Bgl Ⅱ,Hind Ⅲ and digested pTAE7C with restriction endonuclease BamH Ⅰ,Cla Ⅰ,E7C and L1 gene are inserted into the polycloning site of clone vector pBlueScriptks in which E7C and L1 gene are fused .Then using Hind Ⅲ and Xho Ⅰ in polycloning site of recombinant vector pBlueScriptsk ,L1 E7C gene is released and inserted into adenovirus vector pCA14,a recombinant adenovirus expressing vector is generated.