[Construction of mammalian co-expression plasmid pIRES -LMP1-HSP90 beta and its expression in vitro]

Sichuan Da Xue Xue Bao Yi Xue Ban. 2006 May;37(3):339-43.
[Article in Chinese]

Abstract

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.

Conclusion: The constructed mammalian co-expression plasmid pIRES-LMP1-HSP90 beta can express LMP1 and HSP90 beta molecules in vitro at the same time.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • DNA, Complementary
  • Eukaryotic Cells / metabolism
  • HSP90 Heat-Shock Proteins / biosynthesis*
  • HSP90 Heat-Shock Proteins / genetics
  • Herpesvirus 4, Human / genetics*
  • Humans
  • Nasopharyngeal Neoplasms / virology*
  • Plasmids / genetics
  • Transfection*
  • Viral Matrix Proteins / biosynthesis*
  • Viral Matrix Proteins / genetics

Substances

  • DNA, Complementary
  • EBV-associated membrane antigen, Epstein-Barr virus
  • HSP90 Heat-Shock Proteins
  • Viral Matrix Proteins