CONSTRUCTION OF A RECOMBINANT ADENOVIRUS VECTOR OF HUMAN PAPILLOMAVIRUS TYPE 16 LI_E7C
Abstract
Objective: Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these properties of HPV have seriously hampered the development of HPV vaccine. HPV type 16 L1 proteins can assembled into virus-like particles (VLP), which are morphologically identical to the nature virion. In order to develop the recombinant adenovirus vectors of HPV, we constructed a recombinant adenovirus shuttle plasmid pCA14 L1-E7C.
Methods: Haman papillomavirus type 16 L1 open reading frame without terminator codon (TAA) (5559-7152) and E7C (682-855) were amplified using PCR. The L1 and E7C fragments were inserted into pGEM-T easy vectors by T-A strategy, named pTAL1 and pTAE7C, pTAL1 was cut with Hind III and BglII, the pTAE7C with BamHI and ClaI. The L1 DNA fragment, E7C and pBluesscript SK were ligated together using T4 DNA ligase, pBSL1-E7C and pBSL1-E7C was digested with Hind III and Xhol. The L1-E7C fragment was inserted into adenovirus shuttle plasmids pCAI4, named pCAI4L1-E7C. DNA sequence results indicated that The L1-E7C DNA fragment can encode the HPV16L1-E7 fusion protein correctly.
Results: The L1 and E7C DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7C, pBSL1-E7C and pCA14L1-E7C were constructed correctly. The pCAi4L1-E7C can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10.
Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCALI-E7C for the further research.
Methods: Haman papillomavirus type 16 L1 open reading frame without terminator codon (TAA) (5559-7152) and E7C (682-855) were amplified using PCR. The L1 and E7C fragments were inserted into pGEM-T easy vectors by T-A strategy, named pTAL1 and pTAE7C, pTAL1 was cut with Hind III and BglII, the pTAE7C with BamHI and ClaI. The L1 DNA fragment, E7C and pBluesscript SK were ligated together using T4 DNA ligase, pBSL1-E7C and pBSL1-E7C was digested with Hind III and Xhol. The L1-E7C fragment was inserted into adenovirus shuttle plasmids pCAI4, named pCAI4L1-E7C. DNA sequence results indicated that The L1-E7C DNA fragment can encode the HPV16L1-E7 fusion protein correctly.
Results: The L1 and E7C DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7C, pBSL1-E7C and pCA14L1-E7C were constructed correctly. The pCAi4L1-E7C can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10.
Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCALI-E7C for the further research.