Construct Recombination Of Fibroblast Growth Factor Receptor2Promoter Luciferase Report Gene

Alveolar bone defect is one of the most common bone defects in dental clinic, and alsoone of the issues affecting denture repair. The reasons causing Alveolar bone defect containboth pathological and physiological factors, such as congenital developmental abnormalities(such as cleft lip and palate, etc.), periodontal disease, trauma, tumor and hormonal changes,etc. Alveolar bone loss will affect the stability and function of jaw, the appearance, and evenseriously impact on body health. Complete denture repair is unable to get good retentionbecause of alveolar bone defection for the patients lost of denture. Fixed denture repair cannot be beautiful as a result of alveolar bone absorption severe for the patients defected ofdenture. Implant denture patients alveolar bone must be health with a certain height andwidth before surgery. The teeth of periodontal disease patients will be lost eventually alsobecause of alveolar bone defect. Therefore, alveolar bone repair and regeneration has asignificant theoretical and clinical application value in the dental field.Basic fibroblast growth factor（bFGF） is a cytokine with wild functions. Studiesdemonstrated that it could promote the bone regeneration through the following ways: suchas promoting angiogenesis, stimulating bone cell and cartilage cell proliferation anddifferentiation, regulating the function of other growth factors and so on. Fibroblast growthfactor receptors（FGFRs）, as the receptor of bFGF, are the base of bFGF activatingbiological function. Among them, fibroblast growth factor receptor-2（FGFR2）is one of thebFGF high affinity receptors, which plays an important role in promote osteoblastproliferation and differentiation.Purpose: In this research, the key gene, fibroblast growth factor receptor-2(FGFR2)that can regulate the regeneration of alveolar bone, was selected as a target direction, andFGFR2promoter as a target point, and then FGFR2promoter luciferase reporter gene wasconstructed. The research provides foundation of the drug screening for alveolar bone repairand regeneration. It has a profound theoretical significance and extensive clinical applicationvalue.Method: FGFR2promoter sequence was searched in NCBI database, and primerpremier5.0(primer design software) was used to design FGFR2promoter primer, and then the human genome was used as the template to PCR. The amplified fragments were ligatedwith pGEM-T Easy vector and the ligation products were transformed into E.coli DH5α, forscreening positive plasmids, culturing, extraction, enzyme digestion and sequencing. Then,ligation of pGEMT-FGFR2p recombinant plasmid and pGL2vector after double enzymedigestion was processed with following steps, those were transformation, culturing,extraction, restriction enzyme digestion. Then the recombinant plasmid pGL2-FGFR2p wasconstructed successfully.Result: Human FGFR2promoter sequence was cloned by PCR, sequence comparisonrevealed that there is only one point mutation, which could be explained that the pointmutation occurred during PCR or the template itself had had mutation already. Theconstruction of pGL2-FGFR2p recombinant plasmid was successfully constructed.Conclusion:1Human FGFR2promoter sequence was successfully cloned.2Recombinant plasmid of pGL2-FGFR2promoter luciferase reporter gene was successfullyconstructed. The research has a profound theoretical significance and extensive clinicalapplication value. It lays the foundation for the subsequent drug screening stuty.