[Enhancing hGH expression level in insect cells by shortening the 5'-UTR of hGH cDNA].
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.
Geng
Zhao-Hui
ZH
Department of Biochemistry & Molecular Biology, Nankai University, Tianjin 300071, China.
Liu
Ying
Y
Gao
Peng
P
Zhao
Dong-Ming
DM
Li
Shu
S
Yu
Xin-Da
XD
Zhang
Bao-Zhu
BZ
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D019382:Human Growth Hormone / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007313:Insecta / Q000166:cytology / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D011994:Recombinant Proteins; D019382:Human Growth Hormone