Effects of Different Stimulators on Proliferation of Peripheral Blood Lymphocyte Subsets / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of different stimultors (PHA, PMA and IL-2) and culture systems (PBMC and whole blood) on the proliferation of human peripheral blood lymphocyte subsets, so as to provide the experimental basis for selecting the appropriate system according to the experimental purposes.@*METHODS@#A total of 10 ml serum samples were collected from healthy volunteers (n=6). The 300 μl whole blood was directly used to detect lymphocyte subsets by flow cytometry. The 400 μl whole blood were inoculated respectively with 3 different stimulators at 37℃ and 5% CO2 for 60 h; Three different stimulators were also added to the PBMC which were isolated from 2 ml whole blood. Then the proliferation ability of lymphocyte subsets was analyzed by flow cytometry.@*RESULTS@#After the PBMC were stimulated with PHA, CD4CD8CD3 lymphocytes were the most subset; The proportion of CD3CD4 T lymphocytes and CD3CD19 B lymphocytes decreased after being stimulated by PMA (P<0.01, P<0.05); the lymphocyte subset ratio had no significant change after being stimulated by IL-2. After the whole blood system was stimulated with PHA, the CD4/CD8 T lymphoblasts were main subsets, the counts of B lymphocytes and NK cells were reduced; after being stimulated with PMA, the number of CD8CD3 T lymphoblast and CD4CD8T lymphocytes increased, the B/NK cells were not distinguished with the surface markers; after the whole blood system was stimulated with IL-2, the proportion of NK cells significantly increased (P<0.05).@*CONCLUSION@#Lymphocyte proliferation stimulated by PMA is the fastest, while the effect of IL-2 on the lymphocyte subset proportion stimulated by IL-2 is the minimal. After being stimulated by PHA the division cycles of lymphocyte are the most.

Isolation and characterization of antimicrobial peptides from healthy male urine

A complex of low-molecular cationic peptides, extracted from human urine by a combination membrane ultrafiltration and weak cation exchange chromatography, was characterized in this study. It provides a simpler solution for the development of novel antimicrobial peptides from biological liquid waste

Pilot-scale production and quality control of multiepitope hepatitis B virus DNA vaccine / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method for pilot-scale production and quality control of multiepitope hepatitis B virus (HBV) DNA vaccine (PVAX-HS).</p><p><b>METHODS</b>Recombinant DH5alpha/pVAX-HS was obtained by fed-batch fermentation, and the plasmid was extracted by alkaline lysis and concentrated by ultrafiltration. The plasmid DNA was purified by a three-step column chromatography to obtain the DNA vaccine, and quality control tests were performed on the final product.</p><p><b>RESULTS</b>The quantity of the fed-batch product reached 50-60 g/L, and the final plasmid output was 1.0 mg per gram of the bacteria. The quality of the DNA vaccine met the requirements for medical use.</p><p><b>CONCLUSION</b>A simple and stable procedure was established for pilot-scale production of multiepitope HBV DNA vaccine, which allows potential large-scale production of the DNA vaccine.</p>

Gene cloning, prokaryotic expression and functional evaluation of intimin from enterohemorrhagic Escherichia coli O157:H7 / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain highly purified intimin encoded by the eae gene and study its adhesion activity.</p><p><b>METHODS</b>The eae gene was amplified from enterohemorrhagic Escherichia coli O157:H7 (EHEC) chromosome by PCR and cloned into pMD19-T vector. The eae gene was cut from pMD19-T vector and subcloned into the prokaryotic expression plasmid pET28a(+), and expressed in E.coli BL21(DE3). The recombinant protein was purified with Ni(2+)-chelating affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purified intimin was detected by immunofluorescence staining to test its adhesion.</p><p><b>RESULTS</b>The 2805-bp eae gene fragment was obtained, and the recombinant expression plasmid pET28a(+)-eae was successfully expressed in E.coli BL21 (DE3). The molecular weight of the recombinant protein was 97 000. Purified recombinant intimin was recognized by rabbit anti-O157 antiserum, and bound to the surface of HEp-2 cells as revealed by immunofluorescence staining.</p><p><b>CONCLUSION</b>Highly purified and immunoreactive intimin has been successfully obtained, which can adhere to the surface of HEp-2 cells. The acquisition of recombinant intimin provides the basis for studying its interaction with the host receptors during EHEC O157:H7 infection.</p>

Purification of E. coli invasin IbeA-binding protein in intestinal epithelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To purify IbeA-binding protein from intestinal epithelial Caco-2 cells.</p><p><b>METHODS</b>Recombinant IbeA was purified, and 1, 5, and 10 microg/ml His-IbeA and bovine serum albumin (control) were preincubated with confluent Caco-2 monolayer for 30 min at 4degrees celsius;. Gentamicin protection assay was used to test the invasion of E. coli K1 pathogenic isolate E44 in Caco-2 cells. The binding proteins were purified from Caco-2 by IbeA-Cu(2+) sepharose affinity chromatography, and validated by Far-Western blotting. The N-terminal amino acid sequence of the binding protein was determined using Edman assay.</p><p><b>RESULTS</b>E44 invasion in Caco-2 cells was blocked by the recombinant IbeA in a dose-dependent manner. Two binding bands were obtained with His pull-down, and the binding specificity was demonstrated by Far-Western blotting. The N-terminal amino acid sequence of IBP200 was MASITKLP with an isoelectric point of about 5.0.</p><p><b>CONCLUSION</b>Two novel Caco-2 proteins interacting with IbeA of E. coli have been purified and identified.</p>