论文部分内容阅读
采用ＰＣＲ方法，从ＨＰＶ１６型野毒株质粒中分离出ＨＰＶ１６Ｌ１（５５５９７１５１ｂｐ）、ＨＰＶ１６Ｅ７Ｃ（６８２８５５ｂｐ）基因片段，采用ＰＣＲ产物的ＴＡ克隆法，将Ｌ１、Ｅ７Ｃ分别插入ｐＧＥＭＴＥａｓｙ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Ｌ１Ｅ７Ｃ重组基因片段，将其克隆入质粒ｐＢｌｕｅＳｃｒｉｐｔｓｋ，构建了ｐＢＳＬ１Ｅ７Ｃ重组克隆质粒，ＨｉｎｄⅢ、ＸｈｏⅠ酶切ｐＢＳＬ１Ｅ７Ｃ，释放Ｌ１Ｅ７Ｃ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ＨＰＶ１６Ｌ１Ｅ７Ｃ重组腺病毒载体：ｐＣＡ１４Ｌ１Ｅ７Ｃ，为研制ＨＰＶ１６Ｌ１Ｅ７Ｃ重组Ａｄ５载体疫苗和ＨＰＶ１６Ｌ１Ｅ７Ｃ嵌合蛋白疫苗打下基础
The HPV16L1 (5559-7151bp) and HPV16E7C (682-855bp) gene fragments were isolated from HPV16 wild-type plasmids using PCR method. The T-A cloning method of PCR products was used to insert L1 and E7C into pGEM-T Easy vector The cloning vectors pTAL1 and pTAE7C were constructed. BamHI, HindIII digestion of pTAE7C, Bg1II, ClaI digested pTAL1, recovered L1, E7C fragment, the use of BamH Ⅰ, Bg1 Ⅱ digestion produced the same sticky end characteristics L1 gene recombinant E7C gene was cloned into the plasmid pBlueScriptsk, constructed pBSL1 E7C recombinant cloning plasmid, Hind Ⅲ, Xho Ⅰ digestion pBSL1 E7C, release L1 E7C gene fragment, directed recombination into pCA14 adenovirus vector, the successful construction of eukaryotic expression vector HPV16L1 E7C recombinant adenovirus vector: pCA14L1 E7C, Development of HPV16L1 E7C recombinant Ad5 vector vaccine and HPV16L1 E7C chimeric protein vaccine laid the foundation