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Insect-derived promoters for foreign proteins expression in insect cells

A protein and promoter technology, applied in the biological field

Inactive Publication Date: 2011-09-28
替代基因表达有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regulatory sequences similar to those constituting these novel promoters have been identified in Lepidoptera larvae but have not been used in the baculovirus expression system before

Method used

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  • Insect-derived promoters for foreign proteins expression in insect cells
  • Insect-derived promoters for foreign proteins expression in insect cells
  • Insect-derived promoters for foreign proteins expression in insect cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: express green fluorescent protein (GFP) by insect promoter of the present invention

[0050] Clone GFP

[0051] GFP was extracted from pFBpLGFP plasmid by Sphl restriction enzyme and cloned into Sphl restriction site of pFBpB1 or pFBpB2 plasmid. The obtained donor-expression plasmids were named pFBpBIGFP and pFBpB2GFP, respectively. In all cases, the pL sequence was removed, so GFP expression was only due to the activity of the different insect promoters themselves ( image 3 ).

[0052] On the other hand, the pB2 promoter flanked by Not I and PstI restriction sites was ligated to pFBpLGFP which was also opened by the same restriction enzymes. The obtained donor-expression plasmid was named pFBpLpB2GFP, and contained GFP-encoding gene under the control of dual promoters pL and pB2 ( image 3 ).

[0053] Construction of GFP-baculovirus expression vector

[0054] The resulting plasmids were identified by automated sequencing and used to produce recombi...

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Abstract

Insect-derived promoters for foreign proteins expression in insect cells. Regulatory polynucleotide sequences which drive the expression of major insect proteins (hexamerins) at specific evolution stages of larva have been isolated from insects (Trichoplusia ni) in the present invention. Said regulatory polynucleotide sequences promote stronger foreign gene expression in the baculovirus system than the conventional polyhedrin promoter. Additionally, the combination of the new larva-derived promoters of the invention with the pL promoter increased baculovirus expression levels up to 61%-375% (depending on the time of infection) with respect to conventional baculo viruses used in the biotechnology industry. Promoter pB2 also drives gene expression at earlier times than polyhedrin promoter, being this feature a great advantage for correct protein folding and posttranslational modification.

Description

technical field [0001] The invention may relate to the field of biotechnology. The present invention relates to regulatory polynucleotide sequences (such as promoters) derived from hexamerin family genes in insects, and their use for expression of foreign proteins in insect cells and insect larvae, for example using baculovirus expression vectors. Background technique [0002] The baculovirus-insect cell expression system is versatile and widely used to produce heterologous (native and recombinant) proteins. This system is generally accepted as one of the most powerful and versatile systems to produce recombinant proteins of any size. It is based on two main properties: high levels of protein expression and proper protein folding to become biologically active, along with important post-translational modifications in mammalian cells. In addition, the development time of this system is short, and the genetic stability of the recombinant virus is very high. The baculovirus g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/12C12Q1/68C12N15/866A01K67/033C12N15/85
CPCA01K67/033C12N2710/14143C12N15/85A01K2227/706C12N2800/105C12N15/86C12N15/866
Inventor 西尔维娅·戈麦斯萨巴斯蒂安贾维尔·洛佩兹维达尔伊斯梅尔·桑切斯拉莫斯科瓦东加·阿隆索马蒂何塞·安赫尔·马丁内斯埃斯克里瓦诺
Owner 替代基因表达有限公司
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