Construction of eukaryotic expression vector for human platelet CD36 gene 220C>T and 429+4insg variants and analysis of their expressions in HEK293T cells / 中华医学遗传学杂志
Chinese Journal of Medical Genetics
; (6): 124-127, 2019.
Article
en Zh
| WPRIM
| ID: wpr-775798
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WPRO
ABSTRACT
OBJECTIVE@#To construct eukaryotic expression vectors for human platelet CD36 gene 220 C>T and 429+4insg variants and analyze their expressions in HEK293T cells.@*METHODS@#RNA was isolated from human platelets and reversely transcribed into cDNA. Sequences of 220C>T and 429+4insg variants were derived by PCR amplification. The target sequence was ligated into a pcDNA3.1/V5-His-TOPO vector by TA cloning, which was transformed into TOP10 E. coli. Positive plasmids were screened by blue-white selection. After sequencing, plasmid DNA carrying 220C>T or 429+4insg variant was used to transfect HEK293T cells with the help of effectene. Expression of CD36 protein was then analyzed by flow cytometry and Western blotting.@*RESULTS@#An eukaryotic expression vector pcDNA3.1/V5-His-CD36 (220C>T/429+4insg) was constructed by TA cloning. After transfected into HEK293T cells, the 220C>T and 429+4insg variants resulted in CD36 deficiency in HEK cells, which was confirmed by flow cytometry and Western blotting.@*CONCLUSION@#The 220C>T and 429+4insg variants can cause CD36 deficiency in human platelets. This system may be used for assessing the effect of 220C>T, 429+4insg, and other variants on the expression of CD36.
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WPRIM
Asunto principal:
Plásmidos
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Plaquetas
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Transfección
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Clonación Molecular
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Antígenos CD36
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Escherichia coli
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Eucariontes
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Células HEK293
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Vectores Genéticos
Límite:
Humans
Idioma:
Zh
Revista:
Chinese Journal of Medical Genetics
Año:
2019
Tipo del documento:
Article