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Comparison of conventional plasmid vector and Semliki forest virus-derived vectors in expressing growth hormone releasing hormone (GHRH)

Published online by Cambridge University Press:  03 March 2009

Ren Xiao-Hui ,
Luo Hu-Ying ,
Liu Song-Cai ,
Zhang Ming-Jun ,
Ouyang Song-Ying ,
Li Hong-Yi  and
Zhang Yong-Liang
Ren Xiao-Hui
Affiliation:
College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China College of Ocean, Heibei Agricultural University, Qinhuangdao 066003, China
Luo Hu-Ying
Affiliation:
College of Ocean, Heibei Agricultural University, Qinhuangdao 066003, China
Liu Song-Cai
Affiliation:
College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China
Zhang Ming-Jun
Affiliation:
College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China
Ouyang Song-Ying
Affiliation:
China Peking Union Medical College, Beijing 100730, China
Li Hong-Yi
Affiliation:
College of Animal Science, South China Agricultural University, Guangzhou 510642, China
Zhang Yong-Liang*
Affiliation:
College of Animal Science, South China Agricultural University, Guangzhou 510642, China College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

The elements for the Semliki forest virus (SFV) RNA replicon were obtained from the Alphavirus genome. It was designed to overcome the poor efficacy of some current plasmid vectors. Genes coding for viral replicases are preserved while genes coding for structural proteins are replaced by foreign genes in the RNA replicon. High levels of RNA replication and expression of foreign genes in the cytoplasm are regulated by the replicases. To evaluate the effects of the SFV RNA replicon on the improvement of gene expression, a LacZ gene was inserted into pIRES-neo digested by BamHI and dephosphorylated by alkaline phosphatase to construct pIRES-neo-LacZ. The RNA replicon vector pCMV-rep-LacZ and two conventional cytomegalovirus (CMV) promoter-based vectors (pLNCX-LacZ and pIRES-neo-LacZ) were transfected, using Lipofectin, to prepared 293 cells. Growth hormone releasing hormone (GHRH) expression vectors (pCMV-Rep-GHRH, pCDNA3.1(+)-GHRH and pIRES-neo-GHRH) were also tested using the same procedure. Target gene expression was detected with radioimmunoassay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR). Results showed that the expression level of the RNA replicon vector was 2–3 times higher than with normal plasmid vectors. This result will help to improve the efficiency of gene expression in eukaryotic cells.

Keywords

growth hormone releasing hormone (GHRH)Semliki forest virusRNA replicon
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 5 , Issue 3 , December 2008 , pp. 211 - 216
Copyright
Copyright © China Agricultural University 2008

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Footnotes

First published in Journal of Agricultural Biotechnology 2008, 16(2): 286–291

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