Construction And Identification Of Adenoviral Vector In BJ5183 By Homologous Recombination Expressing Rhesus Fusion Protein Of PD-L1ECD And IgG₁Fc

[Object] To construct fusion gene of PD-L1ECD and IgG1Fc in recombinant adenoviral vector in BJ5183 which can express Rhesus fusion protein of PD-L1ECD and IgG1Fc.[Methods] (1) To discover the Rhesus's sequences of PD-L1ECD and IgG1Fc in Genebank.To design some 59bp primers which overlap 21bp on the basis of the sequences of PD-L1ECD and IgG1Fc according to the Genebank's gaidline to this two genes. To synthesize the gene of PD-L1ECD and IgG1Fc through overlap extension PCR.(2) The synthesized genes of PD-L1ECD and IgG1Fc are ligased to pGEM-T in vitro and transformed to E.coliTOP10 to clone the aimed genes.(3) Linearized pGEM-T/IgG1Fc and PD-L1 ECD gene which are doubly digested by salⅠand EcoⅤrespectively are ligased and transformed to E.coliTOP10 to clone the fusion gene pGEM-T/PD-L1Ig.(4) Linearized pGEM-T/IgG1Fc which is doubly digested by salⅠand EcoⅤand PD-L1 ECD gene are ligased and transformed to E.coliTOP10 to clone the pShuttle-CMV/PD-L1Ig.(5) Linearized pShuttle-CMV/PD-L1Ig and pAdEasy-1 are co-transformed to BJ5183 by electroporation through homologous recombination to clone the pAdEasy-1/PD-L1Ig.(6) Linearized pAdEasy-1/PD-L1Ig is transformed by Lipofectamin2000 to AD293 which is cultured to be original adenoviral fruid.The original adenoviral fruid are infected to the new AD293 that are amplified and purified repeatedly.(7)RT-PCR and Western Bloting testify what the recombinant adenovirus can express.[Result] PD-L1ECD and IgG1Fc were cloned out whose length are 660bp and 280bp respectively in correspondence with the length and sequence in Genebank. PD-L1Ig fusion gene whose length sequence are testified by restriction digestion,PCR and sequencing was constructed in pGEM-T. Recombinant Ad plasmid was testified accurate by restriction digestion and PCR.The titer of Recombinant Ad was measured as 6.5×109pfu/ml after it was transfected to 293 cells and amplified and purified repeatedly.RT-PCR display the band of 940bp matched with the length of PD-L1Ig cDNA.Western bloting display 35.1KDa of band matched with PD-L1Ig protein size.[Conclusion] Constructed the high titer recombinant adenovirus successfully which can express costimulatory molecule PD-L1Ig fusion protein.[Prospect] Recombinant adenovirus which can express PD-L1Ig of costimulatory molecule will be transfected to iDC which could activiate PD-1/PD-L1 pathway that would be predicted to induce immunological tolerance of Rhesus liver transplantation effectively in the continued experimental research.