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Objective To explore the interaction between HT036(hypothetical protein HT036)and P311 by co-immunoprecipitation.Methods HA-tagged fusion protein(HA-HT036)expression vector was constructed,identified and transfected into human embryo kidney 293(HEK293)cells alone or with Myc-tagged fusion protein(Myc-P311)expression vector pCMV-Myc-p311.The interaction between P311 and HT036 was detected by co-immunoprecipitation.Results Double restriction enzyme digestion showed that pCMV-HA-HT036 was constructed correctly.When Myc-P311 was immunoprecipitated by anti-Myc antibody,HA-HT036 was identified by Western blotting with anti-HA antibody from immunoprecipitated complex.Conclusion The recombinant vector pCMV-HA-HT036 was constructed successfully.The interaction between HT036 and P311 could be identified by co-immunoprecipitation after co-expression of pCMV-HA-HT036 and pCMV-Myc-p311.The result provides an important basis for further study of the intracellular signal transduction of P311.
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Objective To find the different plasma-associated proteins of rheumatoid arthritis (RA) by using two-dimensional gel electrophoresis for understanding the pathogenesis of RA. Methods The total protein from either RA patients or normal ones was prepared by means of immobilized pH gradient based on two-dimensional gel electrophoresis. After silver staining, gel-image analysis was performed by using PDQuest. The differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). Results 2-DE patterns of plasma from controls and RA patients were presented. The results showed that average number of protein spots was 592 and 563 respectively, and the corresponding average matching rate was 89% and 87% respectively. Gel-image analysis revealed that there were 24 differential protein spots. A total of 15 differential protein spots were successfully identified by MALDI-TOF-MS, of which 6 proteins were up-regulated as compared with control. Conclusion The differentially expressed proteins can be observed in plasma from RA and controls, which can be used to elucidate the pathogenesis of RA for further study.
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Objective To express human cytotoxic T lymphocyte associated antigen-4 (CTLA4) extracelluar domain in Bacmid-baculovirus expression system. Methods The cDNA of human CTLA4 extracelluar domain was isolated from plasmid pUC19-CTLA4Ig by PCR.The specific cDNA was subcloned into plasmid pFastBacl. The insertion was confirmed by DNA sequencing, and then transposed into Hz8 Bacmid in DH10B. The recombinant Bacmid was used to transform Hz insect cells. The supernatant and cellular lysate were analyzed by using SDS-PAGE. Results A specific protein in the cellular lysate of Hz insect cells containing human CTLA4 extracelluar domain with MW38 x 103 was found. Conclusion Human CTLA4 extracellular domain is expressed in the Bacmid-baculovirus expression system.
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<p><b>OBJECTIVE</b>To screen the hypertrophic scar related cytoskeletal genes during early postburn stage, so as to explore their roles in postburn scar contraction.</p><p><b>METHODS</b>cDNA microarray chips containing 4096 human cDNAs were employed to investigate the cytoskeletal gene expression of the scar samples from human postburn hypertrophic scar. Furthermore, the expression of one of the cytoskeletal genes in hypertrophic scar tissue was studied by in situ hybridization.</p><p><b>RESULTS</b>Thirteen up - regulated cytoskeletal genes in 3 early postburn hypertrophic scar samples were identified. Moreover, the cells expressing human tropomyosin TM30 mRNA, one of the up - regulated cytoskeletal genes, were found increased in the early postburn hypertrophic scar samples.</p><p><b>CONCLUSION</b>In this study up - regulated expression of many hypertrophic scar related cytoskeletal genes was found in the scar samples during early postburn stage, and they might be important factors leading to postburn hypertrophic scar formation and contraction.</p>
Subject(s)
Adolescent , Adult , Child , Child, Preschool , Humans , Male , Burns , Genetics , Cicatrix, Hypertrophic , Genetics , Cytoskeleton , Genetics , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , RNA, Messenger , Genetics , Metabolism , Tropomyosin , Genetics , Up-RegulationABSTRACT
Objective Vimentin is one of the proteins of cytoskele to n and cell movement. To investigate the effect of gene expression of fibroblast vimentin on the formation and contraction of hypertrophic scar in this report. Methods According to the results of differently expressed genes in hypertrophic scar by gene microarray, Vimentin, one of the most important g enes , was selected and made into oligonucleotide probe. 24 cases of hypertrophi c scar and 24 non-hypertrophic scar and 12 normal skin were used and these scar were taken on 3, 6, 9 and 12 months after burned. Frozen section and cultured f ibroblasts were made to detect the expression of the gene by in situ hybridizati on. Results Expression of vimentin was defected in scar tissue, but those in the hypertrophic scar on 3 and 6 months after burn were significan tly stronger than those in the hypertrophic scar on 9 and 12 months after burn a nd non-hypertrophic scar. Conclusions Over-expression of cyto skeletal relative genes causes the contraction of scar and vimentin acts leading and key functions.