Enhancing hGH expression level in insect cells by shortening the 5'-UTR of hGH cDNA.
- Author:
Zhao-Hui GENG
1
;
Ying LIU
;
Peng GAO
;
Dong-Ming ZHAO
;
Shu LI
;
Xin-Da YU
;
Bao-Zhu ZHANG
Author Information
1. Department of Biochemistry & Molecular Biology, Nankai University, Tianjin 300071, China.
- Publication Type:Journal Article
- MeSH:
5' Untranslated Regions;
genetics;
Animals;
Base Sequence;
Cells, Cultured;
DNA, Complementary;
genetics;
Gene Expression Regulation;
Genetic Vectors;
genetics;
Human Growth Hormone;
genetics;
metabolism;
Humans;
Immunoblotting;
Insecta;
cytology;
genetics;
Recombinant Proteins;
isolation & purification;
metabolism
- From:
Chinese Journal of Biotechnology
2002;18(4):505-508
- CountryChina
- Language:Chinese
-
Abstract:
The regulation of foreign gene expression in Insect-Baculovirus Expression System is very complex. In this report, the effect of 5'-UTR in the expression of hGH gene in cultured Sf9 cells was examined. A 18 bp length in the end of 5'-UTR of hGH (human Growth Hormone, hGH) cDNA including a stem-loop structure was deleted by PCR. The truncated hGH cDNA, delta 1hGH was cloned in pFastBac1, named pFast-Bac-delta 1hGH. After transforming into E. coli. DH10Bac, which have a shuttle vetor-Bacmid, the delta 1hGH was integrated into Bacmid by site-specific transposition, and an expression vector, rBacmid-delta 1hGH DNA was acquired. By transfecting the cultured Sf9 cells with the recombinant expression vector DNA, pure recombinant virus, rAcV-Bac-delta 1hGH was obtained, and hGH gene was expressed. Immuno-blot and Chemiluminescent assay revealed that the expressed hGH had normal immunological activity, the amount of hGH expression level in Sf9 cell supernatant infected with rAcV-Bac-delta 1hGH containing the truncated 5'UTR was four to five times higher than that infected with rAcV-Bac-hGH.
