Construction And Identification Of The Recombinant Adeno-associated Virus Vector Harboring Fusion Peptide NT₄-SAC-HA₂-TAT

Backgroud and AimsProstate cancer is a common cancer of male genitourinary system.Its morbility has been increasing quickly in recent years.Anti-androgen therapy is the most important means of treatment in all stages of prostate cancer.However,the majority of androgen-dependent prostate cancer can change to androgen-independent prostate cancer at an average of 12～18months after the Anti-androgen therapy.The androgen-independent prostate cancer has a poor prognosis.Prostate apoptosis response gene-4 is a recently discovered pro-apoptotic gene.Its core area of apoptosis peptide can induce both androgen-dependent and androgen-independent prostate cancer to apoptosis.We construct expression vector of SAC,which includes a signal peptide Neurotrophin-4(neurotrohphin-4,NT4) and penetrating peptide TAT.We further build the recombinant adeno-associated virus which secrets NT4-SAC-HA2-TAT fusion peptide.These experiment lay the foundation for the therapeutic effect of the Par-4 core domain SAC as a target gene for prostate cancer.Methods1.The forward and reverse primers of SAC were designed and synthesized.By means of asymmetrical primer/template,the fragment encoding SAC was gained, which included NaeI and KpnI restriction enzyme sites.Then the synthesized fragment was cloned into vector pGEM-T easy.The positive clone was identified by restriction enzymes,and then the cloned fragment was sequenced by dideoxy-mediated chain-termination method.2.After pGEM-T-SAC was digested by NaeI and KpnI,the SAC/NaeI,KpnI and HA₂-TAT/KpnI,XhoI were cloned into recombinant vector pBV220-NT₄/NaeI,SalI and the recombinant plasmid pBV220-NT₄-SAC-HA₂-TAT was obtained.When the recombinant plasmid was digested with EcoRâ… and Hindâ…¢,the fragment NT₄-SAC-HA₂-TAT we got was amplified by PCR.Then the fragment was cloned into vector pGEM-T easy and the recombinant plasmid pGEM-T-NT₄-SAC-HA₂-TAT was obtained.The positive clone was identified by restriction enzymes,and then the cloned fragment was sequenced by dideoxy-mediated chain-termination method.3.The fusion gene NT₄-SAC-HA₂-TAT we got was inserted into the vector plasmid pSSCMV and the vecctor of NT₄-SAC-HA₂-TAT recombinant AAV was constructed.4.The recombinant AAV viral sock was packaged.Renal embryo 293 cells were co-transfected with the rAAV vector of plasmid pSSCMV/ NT₄-SAC-HA₂-TAT,packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in renal embryo 293 cells and its titer was measured by quantitative dot blot hybridization.Results1.By DNA sequencing,we found that we obtained the sequence of SAC which included NaeI and KpnI restriction enzyme sites by pcr.Its sequence was consistent with that of gene bank.2.After analyzing the ORF of the cloned NT₄-SAC-HA₂-TAT cDNA by the DNASIS,we found that amino acids encoded by the cloned NT₄-SAC-HA₂-TAT cDNA were identical to the published results.3.The vecctor of NT₄-SAC-HA₂-TAT recombinant AAV was succssfully constructed.High titer of recombinant adeno-associated virus was obtained by homologous recombination in renal embryo 293 cells.Conclusion 1.The sequence of SAC which included NaeI and KpnI restriction enzyme sites was successfully obtained by pcr.2.The NT4-SAC-HA2-TAT fusion gene was successfully constructed.3.The recombinant adeno-associated virus vector encoding gene NT₄-SAC-HA₂-TAT was successfully constructed and high titer of recombinant adeno-associated virus was obtained.