[Construction of human TAP1 expression vector and the effects of it on HLA-I expression in GES-1 cells in vitro].
OBJECTIVE
To construct the human TAP1 expression vector and to evaluate its effects on HLA-I expression in GES-1 cells in vitro.


METHODS
The human TAP1 expression vector (pcDNA3.1/V5-His-TAP1) was constructed by gene recombination technology. The expression of HLA-I on human gastric epithelial cell line (GES-1 cells) after transfection was detected by RT-PCR, Western blot and flow cytometry (FCM).


RESULTS
Human full-length TAP1 gene was obtained from human peripheral blood mononuclear cells by RT-PCR reaction, then TAP1 gene was inserted into pcDNA3.1/V5-HisB vector to get the TAP1 expression vector (pcDNA3.1/V5-His-TAP1) by recombination technology including digestion with restriction enzymes, ligation and transformation. The vector was sequenced to ensure the sequence fidelity.To further evaluate the function of the TAP1 plasmid we constructed, GES-1 cells were selected as the target cell to be transfected. Firstly RT-PCR and Western blot results showed that the expression of TAP in GES-1 cells was increased after pcDNA3.1/V5-His-TAP1 transfection. Based on the high efficiency of transfection in GES-1 cell, we then detected the expression of HLA-I. The results showed that the expressions of HLA-A, HLA-B and HLA-C at mRNA level were all increased by TAP1 transfection, but no change was found in beta2m mRNA. HLA-I protein level was increased correspondingly with the TAP expression in cells by FCM and Western blot assay.


CONCLUSIONS
The TAP1 expression vector was successfully constructed, and it can induce the expression of HLA-I on the GES-1 cells after TAP1 transfection. The results confirm that the TAP1 plays a cruical role in the HLA-I antigen expression and antigen presentation pathway.
Xing
Ling-xiao
LX
Lab Exp Pathology, Hebei Medical University, Shijiazhuang 050017, China. xinglingxiao@hotmail.com
Xing
Xin
X
Wan
Xiao-wei
XW
Xue
Li-ying
LY
Zhang
Xiang-hong
XH
Yan
Xia
X
Wang
Jun-ling
JL
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007167:Immunotherapy; D009369:Neoplasms / Q000628:therapy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D015395:Histocompatibility Antigens Class I; D012333:RNA, Messenger; C073119:TAP1 protein, human