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Objective To evaluate the repeatability and stability of double antibody sandwich ELISA kit for micro-quantitative soluble complement receptor 1(sCR1) in human serum and to understand its practical application effect .Methods 50 patients with middle and advanced liver cirrhosis and 50 individuals undergoing physical examination served as the liver disease group and normal control group respectively .The mouse anti-human CD35 monoclone antibody ,rabbit anti-human sCR1 polyclonal antibody and goat anti-rabbit IgG labeled by horseradish peroxidase served as the envelope antibody ,sandwich antibody and detection antiboby .The purified recombinant human sCR1 protein served as the standard substance .The human serum micro-quantitative double antibody sandwich CR1 ELISA kit was established .Then the repeatability and stability tests were performed .Then the sCR1 protein level of two group of serum was detected by this kit .Results The linear range of double antibody sandwich ELISA for detecting human se-rum micro-quantitative sCR1 protein was 15 .60 -250 .00 ng/mL ;the regression equation of sCR1 protein concentration to absor-bance value was Y=112 .10X2 +18 .21X+1 .694(r2 =0 .998);in the repeatability test ,the intra-batch relative standard deviation (RSD) in high and low concentrations of standard substance detection value was 6 .20% and 7 .40% respectively ,the inter-batch RSD was 6 .70% and 7 .90% respectively ;in the stability test ,RSD was not more than 0 .01;the serum sCR1 expression level in the liver disease group was significantly higher than that in the normal control group (P<0 .01) .Conclusion The human serum double antibody sandwich ELISA kit for detecting human sCR1 has wide linear range ,good repeatability ,is easy to be stored and suitable for clini-cal and scientific research detection work .
ABSTRACT
Complement system is a major effecter system of the innate immunity that bridges with adaptive immunity. The system consists of about 40 humoral and cell surface proteins that include zymogens, receptors and regulators. The zymogens get activated in a cascade fashion by antigen-antibody complex, antigen alone or by polymannans, respectively, by the classical, alternative and mannose binding lectin (MBL) pathways. The ongoing research on complement regulators and complement receptors suggest key role of these proteins in the initiation, regulation and effecter mechanisms of the innate and adaptive immunity. Although, the complement system provides the first line of defence against the invading pathogens, its aberrant uncontrolled activation causes extensive self tissue injury. A large number of humoral and cell surface complement regulatory protein keep the system well-regulated in healthy individuals. Complement profiling had brought important information on the pathophysiology of several infectious and chronic inflammatory disorders. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases that affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This brief review discusses on the complement system, its functions and its importance as biomarkers and therapeutic targets for autoimmune diseases with focus on SLE and RA.
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PURPOSE: Although the polymorphisms of erythrocyte complement receptor type 1 (CR1) in patients with malaria have been extensively studied, a question of whether the polymorphisms of CR1 are associated with severe malaria remains controversial. Furthermore, no study has examined the association of CR1 polymorphisms with malaria in Chinese population. Therefore, we investigated the relationship of CR1 gene polymorphism and malaria in Chinese population. MATERIALS AND METHODS: We analyzed polymorphisms of CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T in 509 patients with malaria and 503 controls, using the Taqman genotyping assay and PCR-direct sequencing. RESULTS: There were no significant differences in the genotype, allele and haplotype frequencies of CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T polymorphisms between patients with malaria and controls. Furthermore, there was no association of polymorphisms in the CR1 gene with the severity of malaria in Chinese population. CONCLUSION: These findings suggest that CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T polymorphisms may not be involved in susceptibility to malaria in Chinese population.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Alleles , Asian People , Case-Control Studies , China , Erythrocytes/parasitology , Genetic Predisposition to Disease , Genotype , Haplotypes , Malaria/ethnology , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , Receptors, Complement/blood , Taq PolymeraseABSTRACT
Objective To construct and express a small complement receptor type 1 (CR1) deriv-ative which contained two functional domains. Methods Total RNA was isolated from the peripheral blood mononuclear cell. The functional fragment Ⅰ of CR1 was amplified using the RT-PCR. The functional frag-ment Ⅱ was amplified with the plasmid of pET-32a-CR1-SCR15-18 as template which had been already con-structed in our laboratory. Then the chimeric gene that contained the two fragments was constructed with spli-cing overlap extention PCR. The chimeric gene was then inserted into the plasmid of pET-32a (+) and transformed into E. coli Rosetta(DE3). The inserted gene was verified by enzyme digestion and DNA se-quencing. After induced by IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. Then the fusion protein was purified by Ni-NTA affinity column and renatured through dialysis. The comple-ment inhibition activity was determined by CH50 method. Results The chimeric gene was successfully cloned into pET-32a(+). The result of SDS-PAGE and Western blot confirmed the expressed protein and showed that the molecule mass(Mr) of the expressed protein was 63×103. The purity of the recombinant protein was up to 92% after Ni-NTA column affinity chromatography. The bioactivity assay showed the fusion protein had a concentration-dependent complement inhibition activity within the concentration range of 0-200 μg/ml. Conclusion The two functional domains contained small CR1 derivative was successfully construc-ted and expressed in E. coli Rosetta. The fusion protein had a relative high bioactivity, providing a basis for further function experiment in vivo.
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Objective To prepare highly expressed, purified and refolded SCR15-18 of human soluble complement receptor type 1 (sCR1-SCR15-18) protein. Methods The expression of recombinant pET32-sCR1-SCR15-18 in E.coli.. BL21 was induced by IPTG of different concentrations for different time period under different temperatures and the bacteria were split by sonication. The sCR1-SCR15-18 protein was purified by Ni2+-NTA resin affinity chromatography. The purified protein was refolded under different conditions. Then the bioactivity of the protein was analyzed. Results The sCR1-SCR15-18 protein of high expression, purity and bioactivity was attained. Conclusion The parameters of expression, purification and refolding of sCR1-SCR15-18 protein were optimized, which may pave a way for further studies.
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Objective:To investigate the correlationship between levels of erythrocyte receptor type1(E-CR1) and disease activity and abnormal criteria of immunology in systemic lupus erythematosus (SLE)and to study the E-CR1 levels in pathogenesis of SLE.Methods:The fluorescence intensity in sera from 72 patients with SLE and 20 cases of normal controls was detected by flucytometry.Complement(C3,C4),immunoglobulins(IgG,IgA and IgM),gammaglobulins(?-G),erythrocyte sediment rate(ESR) and blood cell counts,including white blood cell(WBC),red blood cell(RBC),hemoglobin(HGB),hematocrit(HCT),mean corpuscular volume(MCV)and platelet(PLT) were detected at the same time.The scoring instruments of systemic lupus activity measure (SLAM) were used to evaluate disease activity.Results:The E-CR1 levels in patients with SLE were lower significantly than that in normal controls (P
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Objective To express the short consensus repeat 15-18 (SCR15-18) domain of human complement receptor 1 (CR1) in Pichia pastoris as a secreted protein in order to found a base for large-scale industry fermentation. Methods The gene fragment of CR1-SCR15-18 was amplified by PCR from plasmid pET32a-sCR1-SCR15-18. The obtained sequence was subcloned into Pichia pastoris secretory expression vector pPIC9. After identification the recombinant plasmid pPIC9-CR1-SCR15-18 was electrotransported into the yeast. Positive clones were identified using colony PCR. Positive recombinants were fermented in shake flaskes and induced with methanol. The recombinant proteins were identified with SDS-PAGE and Western blot analysis. After the recombinant protein was purified by Ni-NTA agarose metal chelate affinity chromatography, inhibiting complement hemolysis testing was used to detect the biological activity. Results Recombinant expression plasmid pPIC9-CR1-SCR15-18 was successfully constructed. SDS-PAGE and Western blot analysis revealed that the target gene was successfully expressed in the yeast and the recombinant protein was successful secreted in the culture supernatant. After purification, the protein inhibited complement hemolysis in vitro. Conclusion CR1-SCR15-18 is successful expressed in Pichia Pastoris with high activity of inhibiting complement hemolysis.
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Objective:To evaluate the protective effects of adenovirus-mediated gene transfer of soluble complement receptor type 1(sCR1) on acute myocardium ischemia in mice.Methods: Twenty-seven SD rats were subjected to left anterior descending coronary artery(LAD) occlusion(30 min) and reperfusion.In the treatment group(n=14),a mixture of adenovirus carrying sCR1 and LacZ was injected into the ischemic zone(100 ?l,10~(10)pfu)5 min before reperfusion;in the control group,only adenovirus carrying LacZ was injected(n=13).Echocardiography was performed 2 weeks later,followed immediately by pathologic examination.Results: Echocardiographic results of the treatment group were better than those of the control group((P