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[Construction of recombinant adenovirus vector expressing extracellular domain of TbetaR-II-RANTES fusion gene and its anti-tumor effects].

OBJECTIVE
To construct a recombinant adenovirus vector expressing TbetaR-II extracellular domain-RANTES fusion gene and evaluate its anti-tumor effects.


METHODS
Mouse origin TbetaR-II extracellular domain and RANTES gene were amplified by RT-PCR. The TbetaR-II extracellular domain-RANTES fusion gene was amplified by overlapping PCR method. TbetaR-II extracellular domain-RANTES fusion gene was cloned into pDC316 vector. The recombinant adenovirus vector expressing the fusion gene was constructed by adMax adenovirus vector creation system. Recombinant adenovirus vector expressing the fusion gene was transfected into LA795 cells. The expression of recombinant adenovirus was checked by Westen blot. The levels of TGF-beta1, RANTES in supernatant were checked by ELISA. The transfected cells were counted and growth curve was obtained. Apoptosis of transfected cells was detected by Annexin V FITC method. The chemotactic activity of supernatant of transfected cells to splenic lymphocytes was assayed. Transfected cells (1 x 10(5)) were inoculated into T739 mice and to observe the tumor growth and survival time. Ad-TbetaR-II extracellular domain, Ad-RANTES and Ad-TbetaR-II extracellular domain-RANTES fusion gene(1 x 10(10) pfu) were injected into the tumor in T739 mice. The tumor size and tumor weight were recorded and tumor growth inhibition rate was counted and statistically analyzed.


RESULTS
TbetaR-II extracellular domain and RANTES gene were amplified by RT-PCR and TbetaR-II extracellular domain-RANTES fusion gene amplified by overlapping PCR, were identified by DNA sequence analysis. Restriction enzyme digestion analysis showed that the recombinant vector was constructed correctly. The recombinant adenovirus vector expressing the fusion gene was constructed successfully using the AdMax Adenovirus Vector Creation System. Its titer was 8 x 10(10) pfu/ml. Ad-TbetaR-II extracellular domain-RANTES fusion gene was transfected into LA795 cells and had specific protein fragment proved by Western Blot. The concentration of TGF-beta1 was decreased and RANTES was increased in supernatant of transfected cells. The growth curve showed that recombinant adenovirus vector expressing the fusion gene could delay tumor development and induce apoptosis, with an apoptosis rate in vitro of 16.9%. The supernant of infected cells showed chemotactic activity to splenic lymphocytes. Tumor growth and survival time were prolonged significantly in group tranfected with recombinant adenovirus vector expressing the fusion gene, and tumor growth was effectively inhibited after injecting recombinant adenovirus vector expressing the fusion gene, with a tumor growth inhibition rate of 37.6%.


CONCLUSION
A recombinant adenovirus vector expressing TbetaR-II extracellular domain-RANTES fusion gene has been constructed successfully. The recombinant adenovirus vector can bind TGF-beta1 effectively, counteract immune suppression mediated by TGF-beta, enhance immune function, induce significant antitumor immune respone, inhibit tumor growth, and prolong the survival time of tumor-bearing mice.