Construction of eukaryotic expression vector for human platelet CD36 gene 220C>T and 429+4insg variants and analysis of their expressions in HEK293T cells.
10.3760/cma.j.issn.1003-9406.2019.02.007
- Author:
Xiuzhang XU
1
;
Haoqiang DING
;
Jing LIU
;
Wenjie XIA
;
Jing DENG
;
Yangkai CHEN
;
Jiali WANG
;
Yuan SHAO
;
Dawei CHEN
;
Xin YE
Author Information
1. Institute of Blood Transfusion, Guangzhou Blood Center, Guangzhou Municipal Key Laboratory for Medical Research, Guangzhou, Guangdong 510095, China. 779684055@qq.com.
- Publication Type:Journal Article
- MeSH:
Blood Platelets;
CD36 Antigens;
Cloning, Molecular;
Escherichia coli;
Eukaryota;
Genetic Vectors;
HEK293 Cells;
Humans;
Plasmids;
Transfection
- From:
Chinese Journal of Medical Genetics
2019;36(2):124-127
- CountryChina
- Language:Chinese
-
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.
