[Cloning of human RHD gene and its expression in K562 cells].
The aim of this study was to clone human RHD gene and to investigate its expression in transduced K562 cells. Total RNA was extracted from reticulocyte of cord blood. RHD and RHCE genes were amplified using RT-PCR method. The amplified products were cloned into pGEM-T plasmid by TA ligation and several clones were screened by direct sequencing method in order to obtain the RHD gene. RHD gene was subcloned into pcDNA3.1(-) expression vector, then the recombined plasmids were transduced into K562 cells with superfect transfection reagent kit. Finally transcription and expression of RHD gene in K562 cells were detected. The result showed that RHD gene has been cloned sucessfully, the inserted sequence and direction of RHD cDNA in its recombined pcDNA3.1(-) vector were identified using enzyme cutting and sequencing method. After transduced with recombined pcDNA3.1(-) vector, K562 cells could transcribe RHD mRNA in its cytoplasm and express RhD antigen on its membrane surface. In conclusion, RhD antigen can expressed in K562 cells with RHD cDNA transduction, and the expression system in vitro may be helpful to further investigate the molecular basis of RhD variants.
Yan
Li-Xing
LX
Blood Center of Zhejiang Province, Hangzhou 310006, China.
Xu
Xian-Guo
XG
Zhu
Fa-Ming
FM
He
Ji
J
D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012204:Rh-Hr Blood-Group System / Q000096:biosynthesis / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014162:Transfection
D018076:DNA, Complementary; C066468:RHCE protein, human; D012333:RNA, Messenger; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen