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
in Chinese
| WPRIM
| ID: wpr-775798
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Blood Platelets
/
Transfection
/
Cloning, Molecular
/
CD36 Antigens
/
Escherichia coli
/
Eukaryota
/
HEK293 Cells
/
Genetic Vectors
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Medical Genetics
Year:
2019
Type:
Article
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