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Objective: To optimize the expression of recombinant human monocyte chemoattractant protein (rhuMCP-1) in E.coli DE3. Methods:With NBS-MICROS 15 L T.DR fermentor, pGEX-IN/huMCP-1 was constructed by our laboratory. Four parameters including pH,temperature,agitation rate and concentration of IPTG were studied by orthogonal experimental design. Results: It was found that the expression level was greatly affected by the amount of dissolved oxygen. This indicated that the agitation rate and ventilation amount were the most important parameters during fermentation. Examined by SDS-PAGE and gel scanning, the expression level of total protein was over 40% when agitation rate was 300 r/min and ventilation amount was 10 L/min. Conclusion: A method for high-level expression of huMCP-1 on pilot-scale is established, and it will be useful for large-scale industrial production of target protein.
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Objective:To establish adoptive transfer model mice of type 1 diabetes for investigating the pathogenesis of type 1 diabetes.Methods:16 NOD.Scid mice were randomly divided into two groups.One was injected i.p. with splenocytes which were obtained from a diabetic NOD mouse and the other was injected with splenocytes obtained from a normal one.The blood glucose levels and weight were detected daily.The mice were killed when they had significant clinic appearance or 10 weeks after cell injection,followed by pathologic studies and cytokine assays.Results:The incidence of adoption group was 8/8 whereas that of control group was 0/8.The pathology scores of 2 groups were 2.5?0.2,0 separately.The amounts of IL-2,IL-10,IFN-? were 60.7 pg/ml,15.5 pg/ml and 20.2 ng/ml respectively in adoption group,and those in control group were 2.4 pg/ml,17.5 pg/ml,3.2 ng/ml.Conclusion:Model mice of type 1 diabetes can be established in NOD.Scid mice during the 5-10 week after adoptive transfer of splenocytes of diabetic NOD mice.
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Objective:To screen for simulation peptide binding specifically to the first and the second extra-cellular domain of CC chemokine receptor 5(CCR5),and to observe their therapeutic effect on mice with experimental autoimmune encephalomyelitis(EAE).Methods:Phage display peptide library was used to screen for peptide sequence binding specifically to CCR5;ELISA was used to identify its binding activity and analyze its DNA sequence.The simulation peptide was synthesized and was injected into abdominal cavity of the EAE mice.Spinal cord tissues were obtained and the pathologic changes were studied by H-E staining in EAE control group and simulation peptide group.Results:Twenty phage clones were randomly chosen for identification and ELISA showed that there were eighteen clones had a strong binding activity with CCR5.The positive clones were sequenced and four peptides of high frequency were obtained:STFTTTL,TPIPQLL,SLPLPKP,and QTSSAAL.Mean clinical score of mice in the EAE model group was 3 and that of the simulation peptide group was 1.H-E staining found that the spinal cord tissues in EAE model group had great number of inflammatory cells and evident demyelination changes;the simulation peptide group had less inflammatory cells and no demyelination changes.The four short peptides had an effect of suppressing and delaying the development of EAE,with the average inhibition rate being 43%(P
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Objective:To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library.Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph.D.-7 peptide library with phage display technique,and the positive clones were identified by ELISA.Results: After three rounds of biopanning,the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA.Sequencing result showed that the binding peptides had high affinity and specificity.Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library,which paves a way for the diagnosis and treatment of hepatitis B infection.
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Objective:To study the expression of chemokine receptor CCR5 in follicular thyroid carcinoma and the serum level of CCR5 ligand,so as to assess the role of CCR5 in progression and metastasis of follicular thyroid carcinoma.Methods:Fifteen samples of follicular thyroid carcinoma,17 samples of follicular thyroid adenoma and 12 adjacent normal samples were analyzed immunohistochemically for CCR5 expression.The sera concentrations of CCL3,CCL4 and CCL5 were measured by ELISA in all patients.Results:CCR5 was positive in follicular thyroid carcinoma samples,with the positive rate being 73.33%,and was not detected in the follicular thyroid adenoma and the normal samples(P
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Objective:To explore the anti-diabetogenic effect of antibody blocking portion of chemokine receptor-5 antibody(CCR5) molecule.Methods: The severe combined immunodeficient NOD(NOD.Scid) mice,injected i.p.with splenocytes from nonobese diabetic mouse,were randomly divided into 2 groups(8 of each group): anti-CCR5 Ab group and PBS group.The mice in anti-CCR5 Ab group were treated with a specific polyclonal antibody,targeting the first extracellular loop of CCR5,and PBS group was treated with PBS.Blood glucose levels were measured to observe the anti-diabetogenic effect of the antibody.Histological examination and ELISA analysis were also performed.Results: Within 70 d,all of the control mice developed diabetes while 5 of the 8 mice treated with anti-CCR5 Ab were diabetes-free.The difference of inflammation score between 2 groups was significant(P
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Objective:To prepare the first extracellular domain of human ? chemokine receptor 5(huCCR5)NH 2 terminal's specific antibody F(ab′) 2 and its detection method Methods:Use computer analyzed and located the least homologous domain of the extracellular first loops The gene fragment was amplified by PCR and cloned into the pGEX IN vector A recombinant GST fusion protein was constructed After comfirming the correctness of the inserted sequence,the transformation and expression of this fusion protein were performed in E coli The expression products of the fusion protein were purified and 2 New Zealand rabbits were immunized An anti huCCR5 NH 2 terminal antibody F(ab′) 2 was prepared by protein A Sepharose CL 4B affinity chromatography ,pepsin digestion and S 200 column seperation.Results:Reduced,unreduced SDS PAGE and ELISA block examination analysis demonstrated that this F(ab′) 2 had high specificity to combine with huCCR5 Conclusion:In this paper,not only introduce a simple and quick method to get a specific antibody F(ab′) 2 of certain functional domain but also a good idea and technique to study other high similar superfamily members
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Objective:To detect the expression of chemokine receptor CCR5 in human invasive ductal breast carcinoma and explore the role of CCR5 in breast cancer.Methods: Thirty samples of invasive ductal breast carcinoma and 10 samples of breast fibroadenoma were analyzed separately by immunohistochemical staining for CCR5 expression. MIP1?,MIP1? and RANTES concentrations were measured by ELISA in the sera of patients. Results: The expression of CCR5 was detected in invasive ductal breast carcinoma (the positive rate was 53.3%) and was not detected in breast fibroadenoma. RANTES concentrations in the serum of breast carcinoma patients increased significantly (P
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This paper reports four enhancing agents of interferon induction promoter in promoting induction of Namalwa cell interferon. In addition to the confirmation of the effects of the previously reported sodium butyrate and BrdU in raising interferon production by Namalwa cells, our study also shows that Acanthopanax senticosus polysaccharide, a classical Chinese herb medicine and sodium carboxyme- thyl starch possess the property of enhancing interferon production. When Namalwa cells are pre-treated with these two preparations, the mean titre of interferon is increased to 5 folds and 8-20 folds respectively. Possible operative mechanisms of this enhancement are discussed.
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Objective:To investigate the role of CCR5 key residues as a co-recptor for the cellular entry of human immunodeficiency virus type Ⅰ(HIV-Ⅰ).Methods: Mutation of amino acids was introduced in different extracellular loops of CCR5 by site-directed mutagenesis technique,turning the non-polar amino acids into polar ones,the non-hydrophilic into hydrophilic,and the aromatic into non-aromatic.The mutants of CCR5 were expressed in BamHⅠ/XhoⅠ and were allowed to bind with gp120,and the binding activity of the mutants was compared with that of wild-type CCR5.Results: The coreceptor activity of CCR5 was reduced greatly when Cys in the first extracellular loop was replaced by Tyr and Pro in the third extracellular loop was replaced by Ser.There was no obvious change in the coreceptor activity of CCR5 when other replacements were introduced.Conclusion: HIV-Ⅰ virus needs receptor and co-receptor to achieve its cellular entry.CCR5 is a co-receptor and some of its extracellular loop amino acids are essential for gp120 recognition of HIV-Ⅰ.
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Objective:To study the therapeutic effect of 2 artificially synthesized fusion peptides on scalded mice. Methods: Two new fusion peptides, M-T508 and M-T?1, were synthesized based on amino acid sequences selected according to the characteristics of scald (including 3 stages: inflammation, infection and repair by cell proliferation). The 2 peptides were applied for second degree scald burn in mice and the healing of the scald burn was observed. The expression ofⅠcollagen, vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), and cytokeratin (CK) was detected by immunohistochemical staining. Serum TNF-? concentration was determined by ELISA in the animals and the blood vessel permeability was analyzed by Evans blue staining. Results: The healing periods of scald burns in the 2 peptide groups were 1 week earlier than that in the control group. Immunohistochemical results showed that the expression of collagenⅠ, VEGF, PCNA and CK was all positive in the 2 peptide groups 10 days after scald and negative in the control group. Examination of blood vessel permeability and serum TNF-? concentration in the M-T?1 and M-T508 groups showed that the 2 fusion peptides had anti-inflammation and anti-infiltration effects in scalded mouse. Conclusion: The synthesized fusion peptide M-T?1 and M-T508 can promote the healing of scald burns in mice.