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Objective To establish a prokaryotic expression system of interstitial lung disease associated autoantigen human bactericidal/permeability-increasing fold-containing B1 (BPIFB1), providing tools for the study on its function in immune responese. Methods The coding region of BPIFB1 gene was amplified with specific primers from recombinant pGEM-C20ORF114 plasmid and cloned into the pET28a-MBP-His and pGEX-5X-1 vectors. The recombinant pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST plasmids were transfected into Top10 cells. The positive clones were selected and sequenced. The correct clones of pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST were transfected into prokaryotic expression strain Rosetta (DE3) and induced by Isopropyl β-D-Thiogalactoside (IPTG). The expression of recombinant BPIFB1 fusion protein was analyzed by SDS-PAGE and Western blotting, and purified by urea modified and renaturation and affinity chromatography of nickel NTA-resin. Results The polymerase chain reaction (PCR) produced specific product with the molecular weight equivalent to that of BPIFB1. The recombinant pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST plasmids were cloned by double restriction enzyme digestion and ligation and confirmed by sequencing. The SDS-PAGE result showed that both BPIFB1-MBP and BPIFB1-GST fusion proteins were mainly expressed in the form of inclusion bodies. The Western blotting result revealed that the recombinant BPIFB1-MBP-His protein could be recognized by Anti-6 ×His antibody. The purified soluble BPIFB1-MBP fusion protein was obtained by urea denaturation, affinity chromatography of nickel NTA-resin and then renaturation after purification. Conclusion The BPIFB1 prokaryotic expression system is established by construct recombinant plasmid pET-BPIFB1-MBP-His, and an approach of renaturation after nickel resin affinity purification in denatured condition.
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Aim To explore the effect of miRNA-143 ( miR-1 4 3 ) on homocysteine ( Hcy ) induced-vascular smooth muscle cells ( VSMCs ) proliferation and the mechanism .Methods VSMCs were cultured and in-cubated with Hcy by using primary cultured method . Then, cells were treated with different concentrations of Hcy and folate .VSMCs proliferation was determined with MTT assay , miR-143 was measured by qRT-PCR, and methylation of miR-143 was determined with meth-ylated PCR.Results After cells were treated with dif-ferent concentrations of Hcy , the proliferation of VSMCs was significantly increased , mRNA expression of miR-143 was decreased and methylation of miR-143 was increased .The proliferation of VSMCs was signifi-cantly decreased when transfected VSMCs with miR-143 precursor , and cell proliferation was increased by using miR-143 inhibitor transfection .Conclusion Hy-pomethylation of miR-143 may inhibit VSMCs prolifera-tion.
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We obtained recombinant ferritin from Dermatophagoides f arinae ,and analyzed the characterization of the pro‐tein .A pair of primers was designed according to the known sequence of ferritin gene .The live mites identified and cultured lo‐cally were picked and the total RNA was extracted .The ferritin gene fragment was amplified by RT‐PCR ,and cloned into pET32a vector ,and then transferred into E .coli Top10 .The target gene obtained from the recombinant plasmid by digestion with Bam HⅠand Hind Ⅲ was connected to the prokaryotic expression vector pET‐32a .The expressed recombinant plasmid containing ferritin gene was constructed by cloning target gene into pET‐32a and transferred into E .coli Bl21 (DE3) .The ex‐pressed recombinant protein was analyzed by SDS‐PAGE ,and was purified by immobilized metal ion affinity chromatography (IMAC) .The ferritin expressed by dust mite was analyzed by the method of bioinformatics .The recombinant plasmid pET32a‐ferritin was constructed .SDS‐PAGE showed a correct molecular weight of the recombinant ferritin protein .After purification by affinity chromatography ,the protein showed only one strip on SDS‐PAGE gel .SDS‐PAGE showed a band at 20 kD .Dust mite ferritin contains 8 serine kinase ,7 threonine kinase ,7 tyrosine kinase ,and 0 histidine kinase phosphorylation sites .Hy‐drophilic region is larger than the hydrophobic region and it is an unstable protein .In conclusion ,the ferritin gene has been cloned and expressed .The purified ferritin has high purity . The study provides a basis for further study of composition and physicochemical properties of house dust mite allergen .
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Objective To investigate the correlations between miRNA and mRNA ( the regulatory effects of miRNA) in a rat model of trinitro-benzene-sulfonic acid (TNBS)/ethanol induced ulcerative colitis ( UC) .Methods TNBS and ethanol were used to induce the development of UC in rats .After the modeling procedure and oral administration of normal saline ( NS) for 14 days, rats from the control and model groups were dissected to collect the samples of colonic mucosa .General and histological evaluations were performed to validate the modeling of UC .The expression of miRNA was profiled using miRNA microarray .The target miRNAs that were closely related to the pathogenesis of UC were selected out according to the results of mi -croarray and related literatures .RT-PCR was performed to verify the differentially expressed miRNAs .The mirWalk database was used to predict the target genes of miRNAs .In order to verify whether the predicted results were in accordance with the actual results , the microarray technology was used for mRNA expression profiling .The genes that showed interactions with those miRNAs were screened out .The David database was used for gene annotation .An interaction net between miRNA and mRNA was formed .Results General and histological manifestation of colon tissue samples from the model group were in accordance with the features of UC.Sixty-eight miRNAs were identified to be differentially expressed in rats from the model group and the control group (fold change>2, P7).Six candidate miRNAs were selected as hav-ing close relations to the pathogenesis of UC referring to reported literatures , the expression of which was checked and verified by real-time polymerase chain reaction (PCR).Compared with the control group, 4 miRNAs (miR-146a-5p, miR-146b-5p, miR-126a-3p and miR-21-5p) were up-regulated (P<0.01, P<0.05) and 2 miRNAs (miR-200b-3p and miR-145-5p) were down-regulated (P<0.01) in rats with TNBS/ethanol induced UC.Four mRNAs (IL-6, Ccl5, Mapk3 and Smad7) that interacted with the 6 miRNAs were identified based on the results of target gene prediction of the above 6 miRNAs and gene expression pro-filing.The David database was used to annotate the interactions for elucidating their significance in the path -ogenesis of UC .Conclusion A miRNA can regulate many signaling pathways and a signaling pathway can also be regulated by many miRNAs .Therefore , simply inhibiting certain pathways may not radically stop the process of inflammation .Studying the functions of miRNAs and elucidating the correlations between miRNA and mRNA might fundamentally inhibit the development of UC .
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Aim To isolate and characterize the human circulating fibrocytes from human peripheral blood and explore the effects of curcumin on human circulating fi-brocytes.Methods The cells were isolated and puri-fied by density gradient centrifugation,and identified by flow cytometry and immunocytochemistry .Then , CCK-8 and flow cytometry were used to study the effect of curcumin on the proliferation as well as COL I ex-pression of human circulating fibrocytes,respectively. Results After being isolated the cells expressed CD34,CD45 and COLⅠ,among which 79.7% were both CD45 and collagen I positive,typical of human circulating fibrocytes.Curcumin could exert regulatory effects on proliferation of human circulating fibrocytes. Exposure of the cells to curcumin for as short as 24 hours promoted their growth,while prolonged treatment (72 h ) significantly inhibited cell propagation and downregulated the COLⅠ levels,best manifested at a concentration as high as 20 μmol · L-1 .Conclusion The proliferation of cells and COLⅠexpressions can be effectively inhibited by curcumin with the prolonged action period and high concentrations.
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Objective To construct the micro-invasive immune rejection monitoring methods with peripher-al blood mononuclear cell gene expression detection and evaluate the clinic rejection estimation value. Methods The SYBR Green Ⅰ was used as fluorescent dye and the GAPDH as house keeping gene control in the quantitatiun RT-PCR technique to observe the 16 immune rejection relative genes expression features after heart transplantation. results were also compared with that of the normal people. Results The 16 immune rejection relative genes expres-sion were no different between normal people and the transplantation recipients before surgery (P>0.05). After heart transplantation the expression of ITGA4, FKB, ILI R-2 up regulated and the level of PF4、ITGAM、TGFβ1、 RHOU down regulated. The results were similar with the clinic observation that the immune rejection often occurs in the first 3 months after heart transplantation. It implied that these 7 genes may play an important role in the acute im-mune rejection after transplantation. Conclusion The real time quantitation RT-PCR methods were constructed suc-cessfully to detect the multiple immune relative genes expression and is of chnic applicable.
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Objective To clone fliH, fliⅠ, fliY and fliN genes that encoding flagellum-associated proteins of L. interrogans for construction of their prokaryotic expression systems, and to determine the loca- tions of Flirt, FliⅠ, FIiY and FIiN. Methods The fliH, fliⅠ, fliY andfliN genes were amplified by PCR and sequenced after T-A cloning. Prokaryotie expression systems of the target genes were constructed subsequently. Expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzer, and Ni-NTA affinity chromatography was performed to extract the target expression prod- ucts. Rabbits were subcutaneously immunized with the four recombinant proteins respectively to obtain anti- sera. ELISA was performed to measure the titers of antisera and Western blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of FliH, FliⅠ, FliY and FIiN. Results Segments of fliH, fliⅠ, fliY andfliN genes with 924, 1365, 1065 and 318 bp in size were successfully obtained by PCR. Similarities of nucleotide and puta- tive amino acid sequences from the four genes were 100% compared with the reported sequences. The con- structed prokaryotic systems efficiently expressed rFliH, rFliⅠ, rFliY and rFliN with the outputs of approxi- mate 20% of the total bacterial proteins. The rabbits immunized by rFliH, rFliⅠ, rFliY or rFliN could pro- duce antibody. The antisera had the titers above 1:100 000, and could recognize the corresponding recombi- nant proteins and membrane proteins of L interrogans to display positive Western hybridization bands. Flirt, FliⅠ, FliY and FliN were found to distribute on the external surface of inner envelope, the internal surface of outer envelope or the interspaces between the two layers of L. interrogans envelope. Conclusion The pro- karyotic expression systems was successfully constructed in this study, which could efficiently express flagel-lum-associated proteins FliH, FliⅠ, FliY and FliN of L. interrogans. The antisera with high titers to recognize their protein antigens were also obtained. Flagellum-associated proteins Flirt, FliⅠ, FIiY and FIiN are the inner envelope proteins and/or outer envelope proteins of L. interrogans.
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Differential gene expression profiles in Balb/cJ mouse model of acute hepatic failure in- fected with MHV-3 virus intervened by anti-hepatic failure compound (AHFC) and the changes of cytokines regulated by genes were investigated. The Balb/cj mice were divided into AHFC-intervened group and control group randomly. Acute hepatic failure model of Balb/cJ mice infected with MHV-3 virus was established. The survival rate in the two groups was observed. It was found that the survival rate in the AHFC-intervened group and control group was 90% and 50% re- spectively 48 h after intrapefitoneal injection of MHV-3 (P<0.05). Before and after the experiment, the cytokines in peripheral blood of the survival mice were determined, and RNA was extracted from survival mouse liver tissue for the analysis of the differential gene expression by a 36 kb mouse oli- gonuleotide DNA array. In all the genes of microarray there were 332 genes expressed differently in the two groups, in which 234 genes were up-regulated and 78 genes down-regulated. Through clustering analysis, the differential expression of immune related genes, including TNF receptor superfamily, Kctd9, Bcl-2, Fg12, IL-8, IL-6, IFN-γ, TNF-α etc. might be related with the curative effectiveness of AHFC. It was suggested that AHFC can balance the immune state of mouse model of acute hepatic failure infected with MHV-3 virus mainly through regulating the expression of immune related genes, decrease the immune damage and inhibit liver cell apoptosis of mouse acute hepatic failure model obviously so as to increase the survival rate of mouse models of acute hepatic failure.
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A model of chronic hypoxic pulmonary hypertension (HPH) was reconstructed in rats exposed to hypoxia with normal pressure for 14 d. Chronic HPH was prevented by giving Ligustrazine (LTZ) in an intraperitoneal dose of 80 mg ?kg-1 wt. twice daily. The mean pulmonary arterial pressure (mPAP), plasma and pulmonary small arterial cGMP level, and mRNA expression of nitric oxide synthase (NOS) gene in lung tissues were studied. The results showed that the mPAP was significantly higher, but plasma and pulmonary small arterial cGMP level as well as mRNA expression of NOS gene in lung tissues were markedly lower in chronic hypoxic rats than those in control animals. LTZcould reverse those changes of above indexes in hypoxic rats, but did not affect those indexes in normal rats. There was a strong negative correlation between cGMP level and mPAP. It is suggested that the lower mRNA expression of NOS gene in hypoxic rats may be one of pathogenetic mechanisms in chronic HPH, and LTZ can increase the mRNA exprssion of NOS gene and the production of nitric oxide in hypoxic rats, which may be an important mechanism in LTZ preventing chronic HPH.