Construction Of Recombinant Plasmid Prins1GFP And Exploration On Expression Of Prins1GFP Transfected Into Pancreatic B-Cells

Objective To construct recombinant plasmid prIns1GFP and observe the expression after transfection of INS-1cells with prIns1GFP.Methods The first part The recombinant plasmid prIns1GFP was constructed by Nanjing GenStrip Corporation. Preliminary verification of returned recombinant plasmid by using of enzyme digestion method. Transfection experiments were divided into two groups:recombinant plasmid transfection group:INS-1cells were transfected with prIns1GFP; no-load plasmid control group:INS-1cella were ransfected with pEGFP-C1. The second part As a result of green fluorescence was not observed after recombinant plasmid transfection, Western-Blot and immunohistochemical assay were used to detect the expression of green fluorescent protein and gene sequence analysis.Results The first part1. Double enzyme digestion of Eukaryotic recombinant plasmid prIns1GFP was consistent with the expected size of DNA fragments.2. Transfection results showed:fluorescence expression was not observed until72hours after transfection with recombinant plasmid prIns1GFP, weak green fluorescence could be observed12hours after transfection with pEGFP-C1, and was significantly enhanced after24～36hours. The second part1. Results of Western-Blot showed that expression of green fluorescent protein was negative in the recombinant plasmid group, in no-load plasmid transfection group expression green fluorescent protein was positive.2. Immunohistochemical analysis of transfected cells showed:expression of green fluorescent protein in the cells transfected with pEGFP-C1was positive, however, negative expression in the cells transfected with prIns1GFP.3. sequence alignment analysis found: five site mutations on the sequence of the gene encoding green fluorescent protein, including three sites with two base pairs was simultaneous mutation in, respectively,633～634bp position of AC mutated into GG,873bp location of A mutated to T,894～895bp position of the TG mutated into CC,924～925bp location of the TG mutated into CC,1463bp location of C mutated to G.Conclusion In this experiment, expression of fluorescent protein has not yet been detected after transfecion with the recombinant plasmid prInslGFP in INS-1cells, which may be related to mutations of genes encoding fluorescent protein.