1.Detection of homodimer formation of CD99 through extracelluar domain using bimolecular fluorescence complementation analysis.
Gowoon CHOI ; Sang Wook LEE ; Kyoung Cheon JUNG ; Eun Young CHOI
Experimental & Molecular Medicine 2007;39(6):746-755
Although various functions of CD99 have been reported, such as apoptosis and homotypic aggregation of thymocyte and transendothelial migration of immune cells, biochemical/molecular natures of CD99 are still elusive. Using mouse CD99 gene, we show that CD99 forms homodimer through its extracellular domain. Expression of mouse CD99 is up-regulated on T cells after CD3-mediated activation, like the case for human CD99. The potential of CD99 to form homodimer was tested with a recently developed bimoleular fluorescence complementation analysis (BiFC). In BiFC analysis, the dimerization-induced fluorescence was strong near the perinuclear region and was faded at the cell membrane. However, surface expression of CD99 was still detected by flow cytometry, suggesting that CD99 either in monomer form or in association with other molecules exists on the cell surface. In BiFC analysis using CD99 mutants with its extracellular, transmembrane, or cytosolic domains changed to corresponding human CD4 domains, the mutant replaced with human CD4-extracellular domain did not produce fluorescence. Purified soluble CD99-Fc fusion proteins bound to CD99-Fc immobilized onto the gold sensor chip in surface plasmon resonance analysis, confirming that the extracellular domain was responsible for dimer formation. Intracytoplasmic staining for CD99 expression in the thymocytes and mature T cells showed that most of the cells, even the cells with low surface level of CD99, contained the molecule inside the cell. Our results suggest that majority of CD99 homodimers may exit in the cell and be exported to the cell surface, dissociating from each other, after a certain regulatory signal is delivered.
Animals
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Antigens, CD/chemistry/*isolation & purification
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Cell Adhesion Molecules/chemistry/*isolation & purification
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Flow Cytometry
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*Fluorescence
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Luminescent Measurements/*methods
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Mice
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Molecular Biology/*methods
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T-Lymphocytes/immunology
2.Sequence Diversity in MIC6 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations.
Zhong Yuan LI ; Hui Qun SONG ; Jia CHEN ; Xing Quan ZHU
The Korean Journal of Parasitology 2015;53(3):341-344
Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.
Amino Acid Sequence
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Animals
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Base Sequence
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Cats
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Cell Adhesion Molecules/chemistry/*genetics/metabolism
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Deer
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*Genetic Variation
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Genotype
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Goats
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Humans
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Molecular Sequence Data
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Phylogeny
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Protozoan Proteins/chemistry/*genetics/metabolism
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Sequence Alignment
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Sheep
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Swine
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Toxoplasma/classification/*genetics/isolation & purification/physiology
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Toxoplasmosis/*parasitology
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Toxoplasmosis, Animal/*parasitology
3.Cloning of human migfilin N-terminal domain and preparation of anti-migfilin polyclonal antibody.
Wei GONG ; Jie LI ; Yun-Ling WANG ; Qing-Zhen NAN ; Bo JIANG ; Hong-Quan ZHANG
Journal of Southern Medical University 2008;28(6):915-918
OBJECTIVETo clone migfilin-N terminal sequence into E.coli and obtain a fusion protein for preparing rabbit polyclonal antibody against migfilin, thereby facilitating the study of the role of migfilin in the biological behavior of colon cancer.
METHODSBased on human migfilin cDNA sequence, a pair of primers was designed to amplify migfilin-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 with EcoRI/XhoI sites, and the target recombinant plasmids were identified with enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL(21) cells, the GST-migfilin-N fusion protein was expressed with IPTG induction. Glutathione-sepharose beads were used to purify the fusion protein, and anti-migfilin polyclonal antibody was produced by immunization of rabbits with the purified GST-migfilin N-terminal fusion protein. The resultant anti-migfilin polyclonal antibody was purified by protein A beads and used for Western blotting for detecting migfilin expression in different cell lines.
RESULTS AND CONCLUSIONThe migfilin-N terminal gene fragment was cloned successfully, and purified GST-migfilin N-terminal fusion protein and anti-rabbit migfilin polyclonal antibodies were obtained. Western blot analysis demonstrates that the antibodies specifically detected migfilin expression in the cell lines, which may facilitate further investigation of the role of migfilin in the biology of colon cancer.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; isolation & purification ; Base Sequence ; Blotting, Western ; Cell Adhesion Molecules ; genetics ; immunology ; Cell Line, Tumor ; Cloning, Molecular ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Cytoskeletal Proteins ; genetics ; immunology ; DNA, Complementary ; chemistry ; genetics ; Escherichia coli ; genetics ; Humans ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA