ABSTRACT
OBJECTIVE@#To detect VHL gene mutation in a pedigree affected with von Hippel-Lindau syndrome (VHL).@*METHODS@#Clinical data of the pedigree was reviewed. Patients were subjected to Sanger sequencing to detect mutation of the VHL gene. Structure of pVHL was predicted by 3D modeling using the swiss-model.@*RESULTS@#A novel c.426delT(p.V142fs) [NM_000551] mutation was found in exon 2 of the VHL gene. 3D modeling suggested that the alpha-structure of pVHL is completely absent.@*CONCLUSION@#The novel c.426delT(p.V142fs) mutation probably underlies the VHL in this pedigree.
Subject(s)
Humans , DNA Mutational Analysis , Exons , Mutation , Pedigree , Von Hippel-Lindau Tumor Suppressor Protein , Genetics , von Hippel-Lindau Disease , GeneticsABSTRACT
Objective To establish a calculation software to determine paternity index(PI) in parentage testing and likelihood ratio (LR) in individual recognition.Methods Based on relevant industry standards and literature, using Visual Basic 6.0 to write the program.Results We successfully developed the calculation software for paternity index (PI) in parentage testing and likelihood ratio (LR) in individual recognition.Conclusion The calculation software can help staff to improve the calculation efifciency, and serve the forensic evidence.
ABSTRACT
Objective To express the recombinant antigen coded by Pagumogonimus skrjabini adult cysteine protease gene fragment and to investigate the immunoreactivity of the fusion protein for further usage on serodiagnosis of the fluke disease. Methods The target gene fragment was amplified by PCR. After purification with gel purification recoverykit, the target gene fragment was ligated with PinPointTM Xa 1 T vector and transduced into E.coli JM109 strain. The expressed fusion protein sample was prepared with alkaline lysis solution, and then analyzed by SDS PAGE. The expression level was determined by Coomassie blue staining and the streptavidin alkaline phosphatase staining. The immunoreactivity of fusion protein was examined by Western blotting. Results A total of 8 positive clones were harvested, and only one had the proper orientation verified by sequencing. The recombinant antigen was obtained after being induced with IPTG (Isopropltio ? D galactoside), and a positive band of 32?10 3 was found by streptavidin alkaline phosphatase staining. In the same position, the fusion protein was also detected by Western blotting. Conclusion The expression vector of adult cysteine protease gene PinPointTM Xa 1 T vector was successfully constructed. The recombinant antigen obtained after being induced with IPTG possesses good immunoreactivity.
ABSTRACT
Objective To construct eukaryotic expression plasmid of human B7-H1 extracellular region-hIgG1Fc-pCI-neo eukaryotic expression vector,and express the functional fusion protein in mammalian CHO cell.Methods Full-length human B7-H1 encoding sequence was amplified from human activated T cell cDNA library by PCR,fused with hIgG1Fc,then transformed into pCI-neo expression vector and verified by sequencing.The validated recombinant was transfected into mammalian CHO cell by lipofectamine reagent.The supernatant of the cultured cell was collected and analyzed by the sandwhich ELISA to detect if there was the fusion protein,and the fusion protein was purified by HiTrap recombination protein Protein A affinity chromatography.The concentrated supernatant or purified fusion protein were used for Western blotting after SDS-PAGE to identify the molecular weight and immune activity of the fusion protein of B7-H1.Results The extracellular region of hB7-H1 about 727 bp was cloned from human T cell cDNA library and was inserted with hIgG1Fc into the eukaryotic expression vector pCI-neo.After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent,the expression of B7-H1 fusion protein was detected in the cultured CHO cell supernatant by the sandwhich ELISA.The immune activity of the fusion protein was verified by Western blotting,and its molecular weight was about 51.76?10~(3),very close to the expected value.Conclusion The hB7-H1-Fc chimeric molecule was successfully constructed and the expression of its functional fusion protein in mammalian CHO cells lays a foundation for further research on the role of B7-H1 in immune tolerance,autoimmune diseases.