[人NKG2D基因的克隆及其在CHO细胞中的表达]

[Cloning of human NKG2D gene and its expression in CHO cells].

作者信息

Xu Xiao-qun, Chen Xue-mei, Zhang Cai, You Li, Zhao Chang-sheng, Wang Jun-fu, Zhang Jian-hua

机构信息

Institute of Basic Medicine, Shandong Academy of Medical Science, Jinan 250062, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Jul;22(4):447-9.

PMID:16806005

Abstract

AIM

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.

CONCLUSION

A recombinant eukaryotic expression vector of human NKG2D can be constructed and it can be expressed successfully in CHO cells.

摘要

目的

构建人NK细胞受体NKG2D的重组真核表达载体，并在CHO细胞中表达重组人NKG2D。

方法

通过RT-PCR从NK-92细胞系中扩增出长度约为650 bp的NKG2D基因片段，并克隆至质粒pGEM-T Easy。随后对克隆的DNA片段进行测序。用EcoR I和BamH I双酶切重组质粒pGEM-T Easy/NKG2D，分离出NKG2D片段并插入真核表达载体pEGFP-N1的相应酶切位点。利用脂质体将重组真核表达质粒转染至CHO细胞。通过荧光显微镜、RT-PCR、Western blot和免疫组织化学染色检测转染的CHO细胞中NKG2D基因的表达水平。

结果

RT-PCR扩增的cDNA片段长度与NKG2D一致。pGEM-T Easy/NKG2D的DNA测序显示，克隆的DNA序列与报道的NKG2D序列相同。荧光显微镜下可见转染的CHO细胞发出绿色荧光。人NKG2D mRNA在转染的CHO细胞中高表达。Western blot和免疫组织化学染色检测显示，NKG2D在转染细胞中表达。