[Construction of mammalian co-expression plasmid pIRES -LMP1-HSP90 beta and its expression in vitro].
OBJECTIVE
This study sought to clone Epstein-Barr virus latent membrane protein 1 (LMP1) and heat shock protein 90 beta (HSP90 beta) of nasopharyngeal carcinoma (NPC), to construct the mammalian co-expression plasmid pIRES-LMP1-HSP90 beta, and to detect the expression of the plasmid in vitro.


METHODS
Total RNA was isolated from human NPC by cloning technique, their cDNA fragments of LMP1 gene and HSP90 beta gene were gained by RT-PCR, and their cDNA fragments were constructed into the mammalian co-expression plasmid vector pIRES. The inserted target genes in the mammalian co-expression plasmid were verified by nucleotide sequencing. COS cell line was transfected with this mammalian co-expression plasmid using lipofectin reagent. The expression of LMP1 and HSP90 beta molecules were detected by RT-PCR and Western blot technique.


RESULTS
The mammalian expression plasmid pIRES-LMP1-HSP90 beta was obtained by cloning technique. The nucleotide sequences of LMP1 gene and HSP90 beta gene in this mammalian co-expression plasmid had high homology with EBV-LMP1 (100%) and human HSP90 beta (100%) respectively. After transfection with this mammalian co-expression plasmid, the LMP1 and HSP90 beta molecules were expressed in COS cells.


CONCLUSIONS
The constructed mammalian co-expression plasmid pIRES-LMP1-HSP90 beta can express LMP1 and HSP90 beta molecules in vitro at the same time.
Zhao
Ju-mei
JM
State Key Laboratory of Biotherapy Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China.
Liu
Tao
T
Tian
Ling
L
Wei
Yu-quan
YQ
Wen
Yan-jun
YJ
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D005057:Eukaryotic Cells / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D009303:Nasopharyngeal Neoplasms / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014763:Viral Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics
D018076:DNA, Complementary; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D018841:HSP90 Heat-Shock Proteins; D014763:Viral Matrix Proteins