The Study Of Constructing The Recombinant Adenovirus Expressing The Soluble Extracellular Domain Of T βRⅡ-RANTES Fusion Gene And Using In Anti-Tumor Experiments

Section one: Construction and Indentify recombinant adenovirus expressing the secretive extracellular domain of T β Râ…¡-RANTESfusion GeneProjective: To construct recombinant adenovirus expressing the secretive extracellular domain of T P R II -RANTES fusion gene and indentiyy its expression. Methods: (1) Mouse origin extracellular domain of TβRâ…¡ and RANTES gene were amplified by RT-PCR method; (2) extracellular domain of T βRâ…¡ -RANTES fusion gene was amplified by overlapping PCR method, (3) extracellular domain of TP RII-RANTES fusion gene was cloned into pDC316 vector, recombinant adenovirus expressing the fusion Gene was constructed by the AdMax Adenovirus Vector Creation System; (4) recombinant adenovirus expressing the fusion Gene was transfected into LA795 cells, the expression of recombinant adenovirus was checked by Westen blot. Results: (1) extracellular domain of TβRâ…¡, RANTES gene amplified by RT-PCR were proved by DNA sequence analysis; (2) extracellular domain of TβRâ…¡ -RANTES fusion gene amplified by overlapping PCR was proved by DNA sequence analysis; (3)restrictive enzyme digestion analysis showed that and recombinant vector was constructed correctively, recombinant adenovirus expressing the fusion gene was constructed successfully by the AdMax Adenovirus Vector Creation System, its titer was 8×10¹⁰ pfu/ml; (4) recombinant adenovirus expressing the secretive extracellular domain of TβRâ…¡ -RANTES fusion gene was transfected into LA795 cells, and had specific protein fragment by Western Blot. Conclusion: (1) recombinant adenovirus expressing the secretive extracellular domain of TβRâ…¡ -RANTES fusion gene was constructed successfully; (2) recombinant adenoviruscan express the fusion protein effectively in transfected LA795 cells.Section two : The Study of Recombinant Adenovirus Expressing the Soluble extracellular domain of T P RII-RANTESFusion Gene Using in Anti-Tumor ExperimentsProjective: To analyze anti-tumor effects of recombinant adenovirus expressing the secretive extracellular domain of T 0 RII -RANTES fusion Gene. Methods: (1) Ad- extracellular domain of T P RII-RANTES, Ad-RANTES> Ad- extracellular domain of T P RII -RANTES fusion Gene were transfected into LA795 cells, the levelof TGF- P i > RANTES in supernatant were checked by ELISA; (2)transfected cells(2000/well) were plated in 96 well plates, trysinized and counted using a hemocytometer every day for seven days; (3) apoptosis of transfected cells was detected by Annexin V FITC method; (4) supernatant of transfected cells chemotacte splenic lymphocytes; (5) transfected cells(lX 105) were inoculated into the T739 mice, observe tumor growth and survival time; (6) Ad- extracellular domain ofTPRH-RANTES, Ad-RANTES. Ad- extracellular domain of TP RII -RANTES fusion Gene(l X 10 °pfu) were injected into the tumor of T739 mice, observe tumor site, tumor weight and count tumor growth inhibition rate; (7) peripheral blood was obtained to detect the level of cytokine IL-2, INF-r of injected mice; (8) tumor was obtained to detect apoptosis. Results: (1) the concentration of TGF- P \ was decreased and RANTES was increased in supernatant of transfected cells; (2)growth curve showed that recombinant adenovirus expressing the fusion gene could delay tumor development; (3)recombinant adenovirus expressing the fusion gene could induce apoptosis, apoptosis rate in vitro was 16.9%; (4)supernant of cells transfected with...