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Objective To evaluate the performance of homocysteine(Hcy)test kit produced by a domestic company and to inves-tigate its feasibility in clinical application.Methods A series of methodological evaluation experiments including the accuracy,re-peatability,stability and linear range of the Hcy reagent kit were carried out on the Olympus Au640 biochemistry analyzer.Results The experiments showed that the relative biases of detection results of normal value quality control serum and pathological value quality control serum were 0.2% and 4.9%,respectively,which were far less than 15% of the reagent instruction.The intra-assay coefficients of variation(CV)of normal and pathological quality control serum were 2.6% and 2.3% respectively,which were less than 5% of the reagent instruction.While the inter-assay CV of normal and pathological quality control serum were 4.4% and 4.1% respectively,which were less than 10% of the reagent instruction.The experiments for the linear range evaluation showed that R 2 was 0.997 in the concentration range of 0 -66 μmol/L,which suggested that the correlation between expected value and measured value was very well.Otherwise,the slope rate b1 of 0.979 9 and the intercept b0 of 0.151 8 were both showed that the de-tection results of Hcy reagent were linear in the concentration of 0-66 μmol/L.Conclusion The performance of Hcy kit produced by a domestic company is very good in the performance indexes of accuracy,repeatability,stability,and linear range and suitable for the clinical application.
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Objective:To screen and clone a carrier molecule for the expression of small bioactive peptides at high levels. Methods: A carrier molecule, PaP3.30, was screened out from the genome of Pseudomonas aeruginosa phage PaP3 and its gene was cloned by PCR method and inserted into pQE 32 expression plasmid, this recombinant plasmid was named pQE PaP30. The peptide antibiotics hPAB ? gene was then inserted into pQE PaP30 and induced to express the fusion protein in Escherichia coli . The ability of PaP3.30 to express other bioactive peptides was evaluated by fusing 6 different origins, varies in sizes and isoelectric points selected peptides to it. Results: After fused to PaP3.30, the peptide antibiotics hPAB ? could express as fusion protein above 30% of total bacterial proteins. Six selected peptides were also expressed by the level of 35%～44% total bacterial proteins when fused to carrier molecule, PaP3.30. Conclusion: The new carrier molecular, PaP3.30, is versatile in the expression of small bioactive peptides.
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Objectives: To design the mutants of peptide antibiotics hPAB ? based on its molecular structure. Methods: The three dimension structure of hPAB ? was constructed by protein homology modeling method. The mutant molecules were designed and generated by PCR and inserted into pQE CP4 expression plasmid. The recombinant plasmids were identified by PCR and DNA sequencing and then transformed into Escherichia coli JM109 to express target fusion proteins. Results:Peptide hPAB ? shows one ? helical and three ? sheet in its structure. Its ? helical regions seem play a key role in the formation of active oligomer. Aside from positioning Thr 7 and Lys 10 into contact positions, the orientation of the ? helix is conserved about the oligome core, forming a ridge around it. Additionally, the dipoles of the helices would overlap to create a positively charged region near the core. These dipoles may be offset, however, by the presence of Asp 4 at the base of the helix. Two mutant molecules, hPAB ? 38 and hPAB ? 34, were designed by deleting N or/and C terminal 2～5 amino acid residues based on hPAB ? structure. The recombinant plasmids containing the mutants gene can express interest fusion proteins in E. coli JM109 successfully. Conclusions: Design, cloning and expression of the mutants of peptide antibiotics hPAB ? lay down the foundation for screening of the mutant of shorter peptide chain and having high or same antimicrobial activity.
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Objective: To screen the best genetic engineering bacterium for the production of peptide antibiotic hPAB-? and evaluate its fermentation level in bottle. Methods:After analysis of the interest fusion protein expression levels of 8 recombinant bacteria containing 1-8 copies of human peptide antibiotic hPAB-? expressing plasmid respectively,2-5 copies expressing bacteria were chosen for the further study of their bacteria yield,expression forms of the target protein, dissolution of the inclusion bodies and the efficiency of fusion protein purification by affinity chromatography, then the best engineering bacterium with the certain copies of interest peptide expressing plasmid was screened out and its optimal fermentation parameters in bottle were also studied. Results:The recombinant bacterium transformed by 3 copies of interest peptide expressing plasmid was the best candidate for its bacteria yield (3.153 g/L) and fusion protein expression level (27.7%) were the highest among 1-8 copies candidates. The inclusion bodies of 3 copies target fusion protein could be easily dissolved by 8 mol/L urea and captured by Ni-NTA column. The elution of the fusion protein could be directly cleaved to monomer by adding 2 mol/L hydroxylamine, adjusting pH to 9.0 and incubating at 45℃ for 2 h. The optimal fermentation conditions of the selected recombinant bacteria were: culture the organisms with modified M9-CAA media at 37℃ and 160 r/min to (A 600 )≈2.5, then add IPTG to the final concentration 100 ?mol/L to induce the expression of target fusion protein for 5 h. Conclusion:The engineering bacterium containing 3 copies interest peptide recombinant expressing plasmid is the best candidate for the production of peptide antibiotic hPAB-?,and its fermentation parameters are confirmed.
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Objective To construct the recombinant plasmid with a human peptide antibiotic hPAB-? gene and to make it expressed in E. coli. Methods To replace the CNBr cleavage site in plasmid pFAST-hPAB-? (CNBr), a pair of primers containing the hydroxylamine cleavage site were designed, and the amplified PCR fragments were cloned into pFAST-HTa plasmid to produce pFAST-hPAB-? (HA), which was then transformed into E. coli DH10B. The constructed plasmid was identified by Ehe Ⅰ/Hind Ⅲ digestion and direct DNA sequencing. An Ehe Ⅰ/Hind Ⅲ digested fragment from pFAST-hPAB-? (HA) was subcloned into pQE32-CP to construct pQE32-CP-hPAB-?, which was transformed into E. coli JM109. The bacteria containing the expression plasmid were induced to express the fusion protein by IPTG. SDS-PAGE was carried out to analyze the molecular weight, expression quantity and expression form of the target fusion protein. After captured by Ni-NTA affinity column, the fusion protein was subjected to hydroxylamine cleavage analysis. Results An expected 230bp fragment was obtained by digesting pFAST-hPAB-? with Ehe Ⅰ/Hind Ⅲ. After this fragment was cloned into pQE32-CP, the recombinant plasmid was confirmed to contain the correct target sequence by DNA sequencing. The recombinant plasmid pQE32-CP-hPAB-? could express a desired protein with a relative molecular weight about 27kD, and its expression level reached 43 percent of the total bacterial proteins. The inclusion bodies were lysed by 8mol/L urea, and the fusion protein could then be captured by Ni-NTA column and cleaved by 2mol/L hydroxylamine at pH9.0. Conclusion The recombinant plasmid pQE32-CP-hPAB-? has been successfully constructed, and it can express the desired hPAB-? fusion protein in E. coli JM109 at high level. These results provide the foundation for future research.
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Objective To investigate the effects of sea voyage on cellular immune function and nutrition status of naval servicemen. Methods A total of 173 male naval servicemen were included in the present study. The percentage of T, B and NK cell subpopulations from peripheral blood were assessed by flow cytometry. CD3+, CD3+CD4+, CD3+CD8+ and CD4+/CD8+ were employed as the markers of T cell subpopulation, CD3-CD19+ served as the marker of B cell subpopulation, and CD3-CD56+ served as the marker of NK cell subpopulation. The serum total protein (TP), albumin (ALB), glucose (Glu), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HLD-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A1 (ApoA1), apolipoprotein A2 (ApoA2), apolipoprotein B (ApoB), apolipoprotein C2 (ApoC2), apolipoprotein C3 (ApoC3), apolipoprotein E (ApoE), lipoprotein a (LPa), Fe2+, total iron-binding capacity (TIBC), Hb, K+, Na+, Cl-, Ca2+ and Mg2+ levels were determined by 7600DDP. Results Compared with that before sailing, the percentage of CD3+, CD3+CD4+ and CD4+/CD8+ elevated significantly during voyage (P