Construction And Eukaryoticexpression Of Recombinant Retroviral Vectors Expressing B7-1 Gene Of The Mouse

Objective: To construct recombinant retroviral vectors expressingB7-1 gene and detect its protein expression which is prepared for theapplication of B7-1 gene in immunogene therapy.Method: The total RNA was isolated from mouse,OD260 andOD260/OD280 of total RNA are determined by ultravioletspectrophotometer.PCR was performed with special primers of B7-1 to getB7-1 gene. The cDNA fragment of B7-1 was cut by EcoRⅠand BamHⅠand purified by gel.Then it was ligated into retroviral vectors. then it wasconnected with retroviral vectors which was samely treated by T4 ligase at16℃to stay overnight, product was electrophoresed? cut the band andpurify it ,transfer the competent cell of colibacillus, cultivated at 37℃,sieve the monoclonal colony,analyzed by EcoRⅠand BamHⅠ,sequencingthe DNA. The pancreatic carcinoma cell line of mouse transfeced by vivalthat was transfeced by package cell line, transient expression was analysedby Western blottingResults The amplified fragment of B7-1 gene was 93Obp with RT-PCR.In addition, the sequence was coincidence exactly to what we need. Thetotal length coding region of B7-1 gene was inserted accurately into theenzyme digestion site between HindⅠand BamHⅠand not found mutationor displace of bases.The protein expression was detected in eukaryoticcells.Conclusion: Recombinant and analysis retroviral vectors expressingB7-1 gene of the mouse should provide a foundation for the application ofB7-1 gene in further study.