[Cloning of human NKG2D gene and its expression in CHO cells].
OBJECTIVE
To construct a recombinant eukaryotic expression vector of human NK cell receptor NKG2D, and express the recombinant human NKG2D in CHO cells.


METHODS
A NKG2D gene fragment, with a length of about 650 bp, was amplified from the NK-92 cell line by RT-PCR and was cloned to plasmid pGEM-T Easy. Then the cloned DNA fragment was sequenced. The recombinant plasmid pGEM-T Easy/NKG2D was digested with EcoR I and BamH I, and then NKG2D fragment was isolated and inserted into the corresponding restriction site on eukaryotic expression vector pEGFP-N1. The Lipofectin was used to transfect the recombinant eukaryotic expression plasmid in CHO cells. The expression level of NKG2D gene in transfected CHO cells was detected by fluorescence microscope, RT-PCR, Western blot and immunohistochemical staining.


RESULTS
The length of cDNA fragment amplified by RT-PCR was consistent with that of NKG2D. DNA sequencing of pGEM-T Easy/NKG2D revealed that the cloned DNA sequence was identical to that of reported NKG2D. Green fluorescence was seen in transfected CHO cells by fluorescence microscope. Human NKG2D mRNA was highly expressed in transfected CHO cells. Western blot and Immunohistochemical staining detection showed that NKG2D was expressed in transfected cells.


CONCLUSIONS
A recombinant eukaryotic expression vector of human NKG2D can be constructed and it can be expressed successfully in CHO cells.
Xu
Xiao-qun
XQ
Institute of Basic Medicine, Shandong Academy of Medical Science, Jinan 250062, China.
Chen
Xue-mei
XM
Zhang
Cai
C
You
Li
L
Zhao
Chang-sheng
CS
Wang
Jun-fu
JF
Zhang
Jian-hua
JH
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018076:DNA, Complementary; C527085:KLRK1 protein, human; D055655:NK Cell Lectin-Like Receptor Subfamily K; D012333:RNA, Messenger