ABSTRACT
<p><b>OBJECTIVE</b>To investigate the presence of Cosmc gene mutation in children with Henoch-Schönlein purpura (HSP) and the association between Cosmc gene mutation and the susceptibility to HSP.</p><p><b>MESULTS</b>Eighty-four children who were diagnosed with HSP between March 2014 and December 2015 were selected as the HSP group. Fifty-eight healthy volunteers matched for age and sex were enrolled as the control group. Fasting venous blood (5 mL) from the two groups was collected in EDTA anticoagulated tubes, followed by the isolation of peripheral blood mononuclear cells (PBMCs) through density gradient centrifugation. Genomic DNA was extracted from PBMCs according to the manufacturer's protocol, and the whole exon region of Cosmc gene was amplified by touch-down polymerase chain reaction (touch-down PCR). The PCR products were identified by 1% agarose gel and sequenced in order to further examine the association between Cosmc gene mutation and the susceptibility to HSP.</p><p><b>RESULTS</b>Sequencing results showed two mutations (c.393T>A and c.72A>G) of Cosmc gene in children with HSP. There were no significant differences in the genotype and allele frequencies at the two loci between the HSP and control groups, and this distribution was not associated with sex.</p><p><b>CONCLUSIONS</b>The mutations c.393T>A and c.72A>G in the exon region of Cosmc gene in children with HSP are not associated with the onset of HSP.</p>
Subject(s)
Child , Child, Preschool , Female , Humans , Male , Genetic Predisposition to Disease , Molecular Chaperones , Genetics , Mutation , IgA Vasculitis , GeneticsABSTRACT
Orjective:To obtain recombinant CHO-K1 with expressing sPDGFR? and to identify the biological activities of sPDGFR? secreted in non-serum medium.Methods:Recombinant human sPDGFR? expression vector pIRES-Neo3-sPDGFR?-Fc was constructed and then transfected into CHO-K1 cells by using LipofectamineTM 2000.After screened with G418 in 8 weeks,some monoclone cells were selected randomly to amplify in 96-well-plate to 24-well-plates,and then to identify positive cell clones by RT-PCR.Furthermore,the candidate cell clones were test by Real-Time PCR and Western blot assays.Finally,anti-proliferation activities of the expressed sPDGFR? were analyzed by MTT.Results:sPDGFR?-Fc was cloned into pIRES-Neo3 correctly.The sPDGFR?-Fc expression level in recombinant CHO-K1 cell clones were concordant in between Realtime PCR and Western blot assay.sPDGFR?-Fc obtained from cultured non-serum medium of positive CHO-K1 could significantly inhibit proliferation of vascular endothelial cell.Conclusion:Successed to select recombinant CHO-K1 cell lines with high expressed sPDGFR?-Fc.The sPDGFR?-Fc can inhibit the cell proliferation significantly and it means sPDGFR?-Fc might be a new anti-cancer drug in the future.
ABSTRACT
Objective:To produce rhBMP-4 with bioactivity in E.coli. Methods: The full-length human BMP-4 gene was mutated by PCR without changes in amino acid sequence, then the synthesized gene was cloned into plasmid pET-3c, transducted into BL21(DE)plysS, and induced by adding IPTG to a final concentration of 1.0 mmol/L. The protein product was purified using ion-exchange chromatography method and then renaturated, bioactivity was checked by C2C12 differentiation in vitro and mouse ectopic bone formation in vivo. Results: A 438 bp gene fragment encoding mature peptide of hBMP-4 was cloned , the protein product was mostly in the form of inclusion body, after renaturation, the engineering protein shows better bioactivity. Conclusion:The mutant strategy can enhance the expression of bioactive rhBMP-4 in E.coli expression system.