| Objective this studying constructed the human mutant p27 gene recombinant Adenovirus vector. It was used to transfect colorectal carcinoma cell line SW480, and the expression of p27 protein and the growth inhibition and apoptosis was observed , and mechanism was explored.Methods hp27mt cDNA was digested with Age I and Nhe I from plasmid of pORF9-hp27mt and subcloned into plasmid of pBluescript II sk(+) with Xma I and Xba I .the same direction recombination was continued, the mid-vector was constructed.lt was named after pBluescript-hp27mtthe plasmid of pShuttle-CMV and pBluescript-p27mt was liberated with Not I and Kpn I .The 7388bp fragment and 699bp fragment were collected respectively at low melting-temprature agarose gel electrophoresis .The same direction recombination was continued. The shuttle plamids of pSuttle-CMV-hp27mt was constructed. The pAdeasy-1 plasmid was transformed into completent coli bacteia BJ5183,and prepared ultra completent BJ5183. The shuttle plasmid pShuttle-CMV-hp27mt was linealized with Pme I and transformed into ultracompletent BJ5183 containing pAdeasy-1,positive clone of homologous recombinant Adenovirus was selected. It was right after the identification performed with Pac I and PCR. The homologous recombinant Adenovirus plasmid of pAdeasy-l-hp27mt was transducted into Ad293 cell. The recombinant Adenovirus Ad-p27mt was packing in it. The identification was performed with PCR. It was approved that Ad-p27mt contained p27mt gene. After the Ad-p27mt transfected into colorectal carcinoma cell line SW480 ,the expression of p27 protein was detected with Wastern blot analysis, the growth inhibition was observed with cell counting , cell cycle and apoptosis was observed with flow cytometry, The apoptosis was also observed with DNA fragment analysis and TUNEL analysis.Results (1) The over 30% positive recombinant plasmid was obtained after 12-20h that linealized pShuttle-CMV-hp27mt was transformed into ultracompletent BJ5183 containing pAdeasy-l.The enzyme cutting appraisal was conducted ,a over 20kb fragment and a special 3.0kb fragment was observed at agarose gel eletrophoresis, it applificated275bp target gene fragment by PCR. It approved that target gene p27mt was successfully inserted Adenovirus plasmid. (2) the 14th day after Ad-p27mt transfected into Ad293 cell, the Ad293 cell underwent pathologic changes and floated as in grape clusters, which suggested that Ad-p27mt was produced through Ad293 package. Ultracentrifugation in CsCl gradient and purification. Spectrophotometer detection showed that the virus titer was 7.95 X 1015 pfu/L. It was right with the identification by PCR.(3) The transduction efficiency was conduced with Ad-LacZ. When multiplicity was more than or equal 50, the transduction efficiency was 100%.(4) There was high expression of p27 protein by Wastern blot analysis at 24h after Ad-p27mt transfected into SW480.(5) p27mt gene can inhibited cell cycle in GO/Gl.The inhibition efficiency was 77.9%,while Ad-LacZ group and blank contrast group was 27.6% and 25.3% respectively, and Ad'p27mt group appeared obviously sub-diploid peak.(6) The Ad-p27mt group showed 180-200bp special ladder band with DNA fragment analysis ,but no ladder was found hi Ad-LacZ group and blank contrasting group. (7) The apoptosis index of Ad-p27mt group was 82.6%, while the blank contrasting group was 5.0%.(8) Ad-p27mt inhibited markedly growth of SW480 in 24h and continued 7d.Conclusion (1) The method of homologous recombinant in bacterium could prepare recombinant adenovirus plasmid containing p27mt.(2) Adenovirus is an efficient vector for mediating transfer and expression of tumor suppressor gene hi human tumor cells. It has advanced applied furture at tumor gene therapy.(3) p27mt gene can obviously bar cell cycle at GO/G1 and inhibit tumor cell growth.(4) p27mt gene has efficiency to induce tumor cell apoptosis. (5) Mutant p27 gene is an efficient gene for gene therapy of carcinoma and has important significance.
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