Animals ; Antigens, CD ; biosynthesis ; genetics ; immunology ; B7-H1 Antigen ; CD28 Antigens ; biosynthesis ; genetics ; Humans ; Immune Tolerance ; Liver ; immunology ; RNA, Messenger ; biosynthesis ; genetics ; T-Lymphocytes ; immunology

Adult ; Aged ; Antigens, CD ; biosynthesis ; genetics ; B7-H1 Antigen ; CD28 Antigens ; biosynthesis ; genetics ; Female ; Hepatitis B, Chronic ; immunology ; Humans ; Leukocytes, Mononuclear ; immunology ; Male ; Middle Aged

This study was aimed to establish downstream purification procedure by which the protein of interest can be purified to higher purity rapidly and efficiently. The different combinations of various purification strategies, methods and conditions were compared, including reversed phase chromatography, metal chelating chromatography, anion exchange chromatography, blue dye affinity chromatography, filtration chromatography and so on. The results showed that in reversed phase chromatography, isolated protein of interest was denatured and precipitated immediately after chromatography because methanol or acetonitrile were adopted as the organic phase. In blue dye affinity chromatography expecting to purify the protein of interest in one step, protein of interest was difficultly differentiated from mixed protein as much proteins bound to the chromatography media by non-specific affinity. While there is a translation-enhancing sequence T7-g10 in the PRSETA-B7-2-PE40KDEL expression vector, so it adds 6 histidines to the N terminus of the protein of interest, this allows to purify the protein of interest by metal chelating chromatography. Based on this characteristic, a three-step chromatography line including metal chelating chromatography, anion exchange chromatography and filtration chromatography was finally established after repeated experiments. By this way the purity of protein of interest reached 95% and the total recovery rate was 8%. The result of Western blot indicated that the expressed and purified recombinant B7-2-PE40KDEL could specifically bind with mAb against human B7-2 and multiple antibody against PEA. The cytotoxicity of the recombinant toxin tested by MTT method showed that the B7-2-PE40KDEL could selectively kill Jurkat cell line expressing CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor. It is concluded that a high efficient and speedy three-step purification procedure for the purifying recombinant protein B7-2-PE40KDEL was established, and this procedure possess selective killing activity on CD28 positive T lymphocytes.
B7-2 Antigen ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; CD28 Antigens ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

It is known that CD28, a positive costimulatory receptor, plays a very important role in inducing the optimal stimulation of T lymphocytes. CTLA-4 (CD152), however, acts as a negative regulator in T lymphocyte activation. The effect of an feline immunodeficiency virus (FIV) infection on the expression of feline CD28 and CTLA-4 was studied with FIV-infected and uninfected peripheral blood mononuclear cells (PBMC) using a competitive PCR assay. The nature of CD28 and CTLA-4 expression was also examined with fresh and antigen-stimulated PBMC. FIV infection induced a lower expression of CD28, but a higher expression of CTLA-4 in the infected PBMC than in the uninfected PBMC. Relatively high levels of CD28 expression were demonstrated in both the fresh and the antigen-stimulated PBMC. The expression level of CTLA-4 in the freshly isolated PBMC was rather low, however, FIV antigen stimulation induced a relatively high expression of CTLA-4 in feline PBMC.
Animals ; Antigens, CD ; Antigens, CD28/*biosynthesis ; Antigens, Differentiation/*biosynthesis ; Antigens, Viral/*immunology ; Cats ; Cell Survival ; Cells, Cultured ; Gene Expression ; Immunodeficiency Virus, Feline/immunology/*physiology ; Leukocytes, Mononuclear/immunology/*virology ; Polymerase Chain Reaction/veterinary

In order to study how to activate T cells and their immunological characteristics, the anti-CD3 and anti-CD28 McAbs were used to stimulate PBMNC, then their related immunological changes, such as lymphocyte transformation function, the percentage of CD8(+)CD25(+) cells and TGF-beta expression were deleted by lymphocyte transformation assay, flow cytometry and RT-PCR respectively. The results showed that in costimulation with anti-CD28 antibody stimulation, the activity of anti-CD3 antibody was significantly enhanced, the ratio of CD8(+)CD25(+) cells of T cells was obviously increased, while TGF-beta expression was down-regulated. It was concluded that the anti-CD28 antibody costimulation could provide stimulatory signal II, which make T cells more active, while the expression of TGF-beta significantly down-regulated.
Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; immunology ; CD3 Complex ; immunology ; Down-Regulation ; Humans ; Leukocytes, Mononuclear ; immunology ; metabolism ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; immunology ; Transforming Growth Factor beta ; biosynthesis

Adolescent ; Adult ; Aged ; CD28 Antigens ; biosynthesis ; genetics ; Female ; Genetic Predisposition to Disease ; Hepatitis B, Chronic ; genetics ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Promoter Regions, Genetic ; genetics

The expression of the costimulatory molecules B7/CD28 in peripheral blood mononuclear cells (PBMC) of the patients with systemic lupus erythematosus (SLE) and its relation to the pathogenesis of SLE were studied. The expression of the costimulatory molecules in PBMC in 30 patients with active SLE and 20 cases of healthy controls was detected by using the techniques of immunofluorescence and flow cytometer. The result showed that the expression percentage of CD28+, CD4+ CD28+ in T cells of PBMC from the patients with SLE decreased significantly as compared with that in healthy control group, while the expression percentage of CD80+, CD19+ CD80+ in B cells was significantly increased than that in healthy control group (P<0.01). It suggested that the abnormal expression of costimulatory molecules B7/CD28 played a role in the pathogenesis of SLE.
Adolescent ; Adult ; B7-1 Antigen ; biosynthesis ; genetics ; CD28 Antigens ; biosynthesis ; genetics ; Female ; Flow Cytometry ; Humans ; Leukocytes, Mononuclear ; metabolism ; Lupus Erythematosus, Systemic ; blood ; etiology ; immunology ; Lymphocyte Activation ; Male ; Middle Aged

OBJECTIVETo explore the relationship between CD8+ T-cell CD28 molecular expression in peripheral blood and TCM type in patients with chronic aplastic anemia (CAA).

METHODSUsing flow cytometry to detect the CD28 expression in 45 in-patients or out-patients and 24 healthy subjects for control. And the relation with TCM type was analyzed from the immunological aspect.

RESULTS(1) The levels of CD8, CD28, CD8+ CD28+ expression and CD8+ CD28+/CD8+ CD28- were all higher in the CAA patients than those in the healthy subjects (P < 0.05 or P < 0.01). (2) The levels of CD28, CD8+ CD28+ expression and CD8+ CD28+/CD8+ CD28- were all higher in the CAA patients of Shen-Yin deficiency type than those in the CAA patients of Shen-Yang deficiency type (P < 0.05 or P < 0.01).

CONCLUSION(1) The abnormal high expression of peripheral blood co-stimulatory molecules CD28 suggested CD28 disorder may play an important role in immuno-pathogenesis of CAA. (2) The levels of peripheral CD28, CD8+ CD28+ expression and CD8+ CD28+/CD8+ CD28- can be taken as an objective indexes for TCM typing of CAA, which was disordered more severe in patients of Shen-Yin deficiency type than in those of Shen-Yang deficiency type.

Adult ; Anemia, Aplastic ; classification ; diagnosis ; immunology ; CD28 Antigens ; biosynthesis ; CD8-Positive T-Lymphocytes ; immunology ; Chronic Disease ; Diagnosis, Differential ; Humans ; Kidney Diseases ; immunology ; Male ; Medicine, Chinese Traditional ; Yang Deficiency ; immunology ; Yin Deficiency ; immunology

CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. The data which have published indicate that the monoclonal antibody against CD28 can decrease curative effects when it was applied in vivo for a long time. In order to avoid the human-anti-mouse action, anti-CD28 mAb must be humanized before it can be used in clinical study. Chimeric antibody, consisting of variable regions of mouse antibody and the constant regions of human IgG1, is often chosen by designers in generating humanized antibody. In this study, to prepare the anti-human CD28 chimeric antibody, the genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were cloned by PCR method. Then, the target genes were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both end of the target genes, and inserted into the baculovirus transfer vector pAcUW3 respectively. Thus, the recombinant baculovirus transfer vector with two strong promoters, ph and p10 was successfully constructed, which can express two different foreign genes at the same time. The recombinant vector was identified by the methods of restriction digesting, electrophoresis, PCR amplification and further verified by DNA sequence analysis. This work will contribute to expressing the chimeric CD28 antibody in insect cells.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Baculoviridae ; genetics ; metabolism ; Base Sequence ; CD28 Antigens ; genetics ; immunology ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Immunoglobulin G ; biosynthesis ; genetics ; immunology ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Trinucleotide Repeat Expansion

The V(H) and V(L) gene fragments of anti-CD28 mAb were combined to form anti-CD28 ScFv gene by using TP-PCR method. Sequence analysis showed that 6 x His tag was added to it for the ease of purification and the V(H), and V(L) gene fragments were connected by a linker containing 15 amino acids which are biased by the baculovirus promoter, ph. Then ScFv gene fragment was inserted into baculovirus transfer vector pBacPAK8. The recombinant transfer vector, pBacPAK8/CD28-ScFv was constructed successfully. The pBacPAK8/CD28-ScFv and the linear Bm-BacPAK6 were co-transfected into the cell line of Bombyx mori (BmN) with the help of Lipofectin,then the product was purified by plaque assay and identified by PCR method. The recombinant virus, Bm-BacPAK6 CD28-ScFv, was obtained successfully. The BmN cells and the larvae of Bombyx mori were infected by the recombinant baculovirus and harvested every 24h postinfection. SDS-PAGE and Western Blotting analysis confirmed the expression of ScFv with the molecular weight of about 28 kD. The expression in BmN cells was detected 24h post infection and it peaked at 72 h, while in the larvae of Bombyx mori, the expression was detected 48 h post infection and it peaked at 120 h.
Animals ; Antibodies, Anti-Idiotypic ; biosynthesis ; genetics ; Antibodies, Monoclonal ; immunology ; Baculoviridae ; genetics ; metabolism ; Bombyx ; cytology ; genetics ; metabolism ; CD28 Antigens ; genetics ; immunology ; Cell Line ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Larva ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection