The Study Of Treating RCC On The Recombinant AAV Mediated Secretive Expression Of NT4-TAT-6×His-VHLβ Fusion Peptide

Backgroud and AimsSporadic clear cell renal carcinoma is a main clinical type of RCC(renal cell carcinoma).Bioresearch of renal tumor cell revealed that the morbility of RCC was associated with a biallelic loss of anti-oncogene VHL.This tumor suppressor gene is located on chromosome 3p25-26 with a total size of 15 kb and has been reported to be a potent down-regulator of several hypoxia-inducible genes including VPF/VEGF and hypoxia inducible factor-1α(HIF-1α).The crystal structure of the pVHL published recently revealed aβ-sheet structure referred to asβ-domain in the NH₂ terminus of pVHL and anα-domain consisting ofα-helices in the COOH-terminus of pVHL.The pVHL is found to bind with elongin B and C and CUL-2,a member of the cullin family of proteins,and is thought to form a complex that closely resembles the yeast E3 type ubiquitin ligase complex.The complex is confirmed being involved in the degradation of a broad variety of cellular proteins,then realizing cytokine inhibition and inhibiting cell proliferation by regulating cell cycle.Previous research had shown that one of the major functions of pVHL is its role in the ubiquitination machinery.Apart from its role in the proteasomal degradation,the pVHL could also inhibit IGF-IR-mediated signaling pivotal for the growth and development of RCC.This inhibition is accomplished by the ability of the 104-123 amino acid region of theβ-domain of the VHL molecule.Theβ-domain of the VHL molecule is sufficient to block the interaction between the cytoplasmic domain of the IGF-IR and its downstream signaling molecule PKCδ,thereby blocking PKCδkinase activity,which may ultimately affect cell proliferation and induce apoptosis of renal carcinoma cells.However,like other peptide the VHLβdomain peptide used for therapeutic applications now arise from artificial synthesis in vitro and the cost is expensive.The therapeutic use of VHLβdomain peptide has also remained limited due to problems of specificity,bio-availability,proteolytic degradation and poor membrane permeability.Gene therapy represents an attractive alternative to recombinant protein administration for several reasons.We hypothesize that an alternative approach is to design a method to increase synthesis and release of endogenous VHLβdomain peptide during a given time period.Recent progress in the recombinant DNA and the protein expression technology,particularly a variety of mammalian and microbial systems render the attractive alternatives to produce therapeutic peptides possible. The overall costs of peptide development and manufacture may also be lower than for chemical entities,particularly if simple unmodified peptides are involved,since these could potentially be synthesized using microbial or cellular expression systems.The aims of present study are:(1)cloning and sequencing of VHLβcDNA.(2) The cDNA of HIV-1 Tat PTD(protein transduction domains)and tag peptide 6×His was adopted to Construct recombinant plasmid pGEM-T-TAT-6×His-VHLβcDNA.(3)VHLβcDNA was ligated to the signal and leader peptides of neurotrophin 4(NT4),the fusion gene was named NT4-TAT-6×His-VHLβcDNA.(3)to construct a novel adeno-associated virus expression plasmid and obtain a higher-titer secretory human VHLβdomain peptide rAAV by a new production system.(4)to evaluate the effects of the rAAV on Hela cells.(5)to determine whether recombinant adeno-associated virus-mediated overexpression of VHLβdomain peptide could inhibited the renal cell growth.Methods and Results(1)According to sequence of VHLβcDNA supplied by GenBank,the fragments encodingβ-domain of pVHL(the VHL gene product)and TAT-6×His were gained by means of asymmetrical primer/template.Then the fragments were cloned into vector pGEM-T easy.The positive clone was identified by restriction enzymes.After digestion with the restriction enzyme of EcoR I,the two fragments(with length of 69bp,75bp)and the fragment of plasmid pGEM-T Easy vector(with length of 3015bp)was achieved,which demonstrated that the two interest cDNA fragments were inserted into pGEM-T easy vector respectively.(2)We construct the recombinant plasmid of pGEM-T-TAT-6×His-VHLβwith the two segments after digestion of recombinant plasmids including pGEM-T-TAT-6×His and pGEM-T-VHL with the restriction enzymes BamH I and Eco 721.The products were used to transform the competent E Coli.DH5αprepared by CaCl₂.Select the positive bacterial colonies and reclaim the DNA of the recombinant plasmid by the method of alkali lysis.After digestion with the restriction enzymes EcoR I,we got a fragment of plasmid pGEM-T Easy vector(with length of 3015bp) and another one with length of 132bp,including the fragment of VHLβcDNA(114bp),two recognition sequences of restriction enzymes(15 bp)and a stop codon(3bp),which showed that TAT-6×His-VHLβcDNA fragment was inserted into pGEM-T easy vector.The resulting DNA fragment was sequenced by Shanghai Shenggong biotechnology Ltd.Co.The sequencing result of positive clone was identical to sequence supplied by GeneBank.(3)The gene of TAT-6×His-VHLβcDNA was ligased to the signal peptide and prepeptide of human neurotropin-4 and precisely fused,and a secretory vector pBV220/NT4-TAT-6×His-VHLβwas constructed.The pBV220/NT4-TAT-6×His-VHLβwas confirmed by endonuclease analysis.The recombinant gene of NT4-TAT-6×His-VHLβcould encode the just amino acids as we projected.(4)The aforementioned pBV220/NT4-TAT-6×His-VHLβwas digested with EcoR I/BamH I to release the fragment of NT4-TAT-6×His-VHLβcDNA(363bp),and it was inserted into the AAV vector pSSHGCMV,resulting in the construction of pSSHGCMV/NT4-TAT-6×His-VHLβ.Recombinat virus rAAV/NT4-TAT-6×His-VHLβwas produced by a 3-plasmid(including vector plasmid pSSHGCMV/ NT4-TAT-6×His-VHLβ,packaging plasmid pAAV/Ad and helper adenovirus plasmic pFG140)one-step co-transfection method.To purify rAAV particles,its stocks was precipitated by addition of saturated(NH₄)₂SO₄ and dialyzed.Recombinat virus vector titer was determined by quantitative dot blot hybridization.The secretory recombinant AAV expressing VHLβdomain peptide was packaged and produced,which contained the signal peptide and prepeptide of NT4.The titer of the rAAV/NT4-TAT-6×His-VHLβwas about 3.4×10¹⁰/ml.(5)Cultured Hela cells were infected with rAAV/NT4-TAT-6×His-VHLβ. Immunocytochemistry staining showed that fusion peptide Expression significantly increase comparing with the control group(P＜0.01).(6)Cultured human renal carcinoma cells,GRC-â… cells,were infected with rAAV/NT4- NT4-TAT-6×His-VHLβ.The MTT assay was used as described to assess proliferation of GRC-â… .The result displayed rAAV/NT4-TAT-6×His-VHLβhad significant effect on renal carcinoma cells proliferation,as assessed using the MTT assay and flow cytometry.It exhibited cytotoxicity effects and induced cells to die as apoptosis manner in VHLβfusion peptide cellular model.Conclusion(1)Cloning the TAT-6×His-VHLβcDNA gene,the sequencing result of positive clone was identical to that of what sequence and supplied by GenBank.It has paved the way for further study on gene therapy of tumor.(2)The recombinant plasmid expressing TAT cell penetrating peptides and theβ-domain of pVHL was constructed.Then prokaryotic expression vector pBV220-NT4-TAT-6×His-VHLβwas constructed by routine molecular biological methods.These results lay the foundation for further research of using fusion gene to treat RCC.(3)We prepared the viral stock of rAAV/NT4-TAT-6×His-VHLβThe rAAV constructed can efficiently mediate expression of exogenous gene in Hela cells in immunocytochemistry staining and be used to transfect eukaryotic cell and gene therapy.(4)Cultured human renal carcinoma cells were infected with rAAV/NT4-NT4-TAT-6×His-VHLβ.It exhibited cytotoxicity effects and induced cells to die as apoptosis manner in VHLβfusion peptide cellular model.