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1.
PLoS One ; 12(9): e0185593, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28957394

RESUMO

Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impacts. Like other chlamydial species, Chlamydia pneumoniae possesses a unique developmental cycle, the infectious elementary body gains access to the susceptible host cell, where it transforms into the replicative reticulate body. The cytoplasmic vacuole where Chlamydia pneumoniae replicates is called an inclusion, which is extensively modified by the insertion of chlamydial effectors known as inclusion membrane proteins (Incs). The C. pneumoniae-specific inclusion membrane protein (Inc) Cpn0147 contains domains that are predicted to be exposed to the host cytoplasm. To map host cell binding partners of Cpn0147, a yeast two-hybrid system was used to screen Cpn0147 against a HeLa cell cDNA library, which led to the finding that Cpn0147 interacted with the host cell protein cyclic adenosine monophosphate (cAMP)-responsive element (CRE)-binding protein (CREB3). The interaction was validated by co-immunoprecipitation of Cpn0147 with CREB3 from HeLa cells ectopically expressing both. Furthermore, Cpn0147 and CREB3 were co-localised in HeLa cells under confocal fluorescence microscopy. The above observations suggest that CREB3 may directly bind to the cytoplasmic domain of Cpn0147 to mediate the interactions of chlamydial inclusions with host cell endoplasmic reticulum.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Membrana/metabolismo , Western Blotting , Células HeLa , Humanos , Microscopia de Fluorescência , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 32(1): 49-53, 2016 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-26728378

RESUMO

OBJECTIVE: To construct an eukaryotic expression vector of mannose-binding lectin associated protein 19 (MAp19) and further express MAp19 fusion proteins. METHODS: MAp19 gene fragment was amplified by PCR. The eukaryotic expression vector pcDNA3.1/Myc-His A-MAp19 was constructed by gene cloning technology, identified by restriction enzyme digestion and further confirmed by sequencing. The recombined plasmid was then transfected into HeLa cells by liposome-mediated method, and the positive clones with vector pcDNA3.1/Myc-His A-MAp19 were selected by G418. Location of MAp19 fusion proteins in the transfected HeLa cells was observed under the fluorescence microscope, and the expression levels of MAp19 fusion proteins were detected by Western blotting. RESULTS: The recombined vector pcDNA3.1/Myc-His A-MAp19 was successfully constructed. The result of DNA sequencing was in accordance with NCBI data bank. By G418 selecting, we obtained the HeLa cells which could stably express exogenous MAp19 fusion proteins. The protein was located in the cytoplasm. Western blotting also suggested that the MAp19 was secretory protein. CONCLUSION: The vector expressing MAp19 has been prepared successfully, and can express the target protein (MAp19) in the eukaryotic cells (HeLa cells).


Assuntos
Clonagem Molecular/métodos , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Expressão Gênica , Vetores Genéticos/genética , Gentamicinas/farmacologia , Células HeLa , Humanos , Lipossomos , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/genética
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