ABSTRACT
The aim of this study is to construct a SARS-CoV S protein-specific phage displayed antigen library for the epitope characterization of anti-S monoclonal antibodies (mAbs). First, the full-length gene of SARS-S protein was PCR amplified, purified and then digested with DNase I to obtain DNA fragments in the size range of 50-500 bp. The resulting fragments were blunt-end ligated to the modified phage display vector pComb3XSS. The reactions were electrotransformed into XL1-Blue and infected with VCSM13 helper phage. The SARS-CoV S protein-specific phage displayed antigen library was biopanned and screened against two anti-S mAbs, S-M1 and S-M2. The results showed that we successfully constructed the phage displayed antigen library with a size of 5.7 x 10(6). After three-rounds of biopanning, 14 positive phage clones for S-M1 and 15 for S-M2 were respectively identified. Sequence analyses revealed the possible epitopes of two mAbs. Therefore, the S protein-specific phage displayed antigen library provides a crucial platform for the epitope characterization of anti-S antibodies and it is highly valuable for development of SARS vaccines and diagnostics.
Subject(s)
Humans , Antibodies, Viral , Allergy and Immunology , Bacteriophages , Genetics , Metabolism , Epitopes , Genetics , Allergy and Immunology , Membrane Glycoproteins , Genetics , Allergy and Immunology , Peptide Library , Severe acute respiratory syndrome-related coronavirus , Genetics , Allergy and Immunology , Severe Acute Respiratory Syndrome , Allergy and Immunology , Virology , Spike Glycoprotein, Coronavirus , Viral Envelope Proteins , Genetics , Allergy and ImmunologyABSTRACT
<p><b>BACKGROUND</b>It is necessary to develop some innovative methods to reveal and discover the novel (SLE)-related protein molecules. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was employed to detect the differential expression of serum polypeptides in the patients with systemic lupus erythematosus (SLE) presenting with a family history or complicating with kidney injury so as to identify the proteins associated with the genetic factors and kidney injury in SLE.</p><p><b>METHODS</b>The subjects recruited were divided into four groups, that is, a group of SLE patients with both family history and kidney injury, a group of SLE patients with only kidney injury but no family history, a group of SLE patients with neither family history nor kidney injury, and a control group consisting of healthy volunteers. By adopting MALDI-TOF MS analysis, the serum samples obtained from the three groups of SLE patients were examined and compared with those from the control group; the categorized peptide fingerprint profile was established via the biological data collected from the samples.</p><p><b>RESULTS</b>The expression of protein with a m/z of 4207 Da increased significantly in SLE patients; the protein with a m/z of 2658 Da was expressed in all SLE patients; three proteins (with m/z of 1465, 5332, and 5900 Da respectively) were expressed in the SLE patients complicated with kidney injury and the protein with a m/z of 1943 Da was expressed in SLE patients with family history.</p><p><b>CONCLUSION</b>A number of differential proteins were successfully detected and identified through MALDI-TOF MS detection and these proteins may be associated with the genetic basis of SLE and the complicating kidney injury.</p>
Subject(s)
Adolescent , Adult , Female , Humans , Middle Aged , Kidney Diseases , Genetics , Lupus Erythematosus, Systemic , Genetics , Peptide Mapping , Methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
<p><b>BACKGROUND</b>Low potassium dextran (LPD) solution can attenuate acute lung injury (ALI). However, LPD solution for treating acute kidney injury secondary to ALI has not been reported. The present study was performed to examine the renoprotective effect of LPD solution in ALI induced by oleic acid (OA) in piglets.</p><p><b>METHODS</b>Twelve animals that suffered an ALI induced by administration of OA into the right atrium were divided into two groups: the placebo group (n = 6) pretreated with normal saline and the LPD group (n = 6), pretreated with LPD solution. LPD solution was injected intravenously at a dose of 12.5 ml/kg via the auricular vein 1 hour before OA injection.</p><p><b>RESULTS</b>All animals survived the experiments with mild histopathological injury to the kidney. There were no significant differences in mean arterial pressure (MAP), creatinin and renal damage scores between the two groups. Compared with the placebo group, the LPD group had better gas exchange parameters at most of the observation points ((347.0 ± 12.6) mmHg vs. (284.3 ± 11.3) mmHg at 6 hours after ALI, P < 0.01). After 6 hours of treatment with OA, the plasma concentrations of NGAL and interleukin (IL)-6 in both groups increased dramatically compared to baseline ((6.0 ± 0.6) and (2.50 ± 0.08) folds in placebo group; and (2.5 ± 0.5) and (1.40 ± 0.05) folds in LPD group), but the change of both parameters in the LPD group was significantly lower (P < 0.01) than in the placebo group. And 6 hours after ALI the kidney tissue concentration of IL-6 in the LPD group ((165.7 ± 22.5) pg×ml(-1)×g(-1) protein) was significantly lower (P < 0.01) than that in placebo group ((67.2 ± 25.3) pg×ml(-1)×g(-1) protein).</p><p><b>CONCLUSION</b>These findings suggest that pretreatment with LPD solution via systemic administration might attenuate acute kidney injury and the cytokine response of IL-6 in the ALI piglet model induced by OA injection.</p>