The Establishment And Identification Of The Adenovirus Vector 5/35 Of Coding MCD20

[Objective]: B-cell lymphoma is one of the most common types of lymphomas,and immunotherapy plays an important role in the treatment of the lymphoma. Dendritic cells (DC) which is infected by adenovirus and encode as the tumor associated antigen (TAA) is a promising neoplasm tumor vaccine, however, the commonly used adenovirus subgenus C serotypes 5 (Ad5) carrier DC infections is of low efficiency. This research aims at comparing the efficiency of adenovirus subgenus C serotypes 5(Ad5) carrier DC infection with adenovirus subgenus B serotypes 5/35 (Ad5/35) carrier DC infection, cloning the CD20 (mCD20) gene of mouse, establishing the adenovirus vector 5/35 of coding mCD20 (Ad5/35mCD20), identifying expression outcomes,providing experimental evidence and initial basis for DC modification of adenovirus vector 5/35 coding CD20 gene being used as the immune gene therapy of B-cell lymphoma.[Method]: To culture DC in Peripheral blood mononuclear cell (PBMC) in the healthy people, infect DC with Ad5EGFP or Ad5/35EGFP of different virus tites, double-dye it with the flow cytometry device,check the expression of EGFP,and then compare the efficiency of the two different adenovirus vector infecting DC. The molecular clonal technology is employed to clone the mouse CD20 from mouse C57BL/6, insert cDNA segment of the gene into adenovirus shuttle plasmid pDC315 and establish pDC315mCD20 after PCR, restriction enzyme digesting, identification and DNA sequencing being confirmed. Ad MaxTM adenovirus vector packing system is employed to transfect pDC315mCD20 and the adenovirus framework plasmid containing adenovirus vector 5/35 fiber to 293 cells through the calcium phosphate precipitation method, and obtain Ad5/35mCD20 by repeated amplification and purification. The plasmid encodging the fusion genemCD20 was transfected into HepG2 cells which will be detected and identified by flow cytometry.[Result]: To infect the human DC with 100,50 or 10 pfu Ad5EGFP and Ad5/35EGFP of each cell, after double-dying CD11 a-PE and EGFP through the flow cytometry device the infection efficiencies are 28.41%,17.36%, 3.22% and 69.12%,49.03%,36.89% respectively. The cloned mCD20cDNA from the mouse C57BL/6 proves through DNA sequencing to have the same sequence (No.M62541) provided by Gene Bank. The plasmid was transfected into HepG2 cells and the stable expression clone was obtained. After dying CD20, the expression of mCD20 in the cells which is 38.72% was detected by flow cvtometry.[Conclusion]: The efficiencies of the two adenovirus vector carrier DC infections are in direct ratio with the tites of virus infection, and the higher tite will have the higher efficiency of DC infection; with the same virus tite, 5/35 adenovirus vector has an obviously higher efficiency of DC infection than adenovirus subgenus C serotypes 5 (Ad5). The Ad5/35mCD20 established by inserting the gene of the mouse CD20 into 5/35 adenovirus vector without section El can effectively express protein of the mouse CD20, thereby providing a useful tool for further research on 5/35 adenovirus vector coding CD20 gene, DC modification could be used as an approach for the immune gene therapy of B-cell lymphoma.