Enhancer Hr3

A technology of promoter and terminator, which can be used in DNA preparation, DNA/RNA fragments, introduction of foreign genetic material using vectors, etc., which can solve the problems of reduced expression and complexity of foreign genes.

Active Publication Date: 2012-06-13
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The expression of foreign genes in eukaryotic cells and tissues is regulated by a series of regulation such as transcription level, post-transcriptional regulation (including mRNA transport and stability regulation), translation efficiency, post-translational regulation, and protein secretion. The regulatory region, the cis-acting elements in the ORF of the foreign gene and the trans-regulatory factors in the host coop

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Establishment and Optimization of a Cell Expression System Based on the Bombyx mori Act4 Promoter

[0043] 1 Expression vector construction

[0044] A. Cloning of the promoter of the silkworm actin 4 gene ( B. mori Action4 ): Using the silkworm dimorphic variety Dazao genome as a template, an upstream primer containing a Sal I site was designed according to the silkworm A4 promoter sequence published by NCBI (GenBank accession number GI: 1794195): 5'GCgtcgacTCATCTTGTCACACCCTA 3' and a downstream primer containing A BamH I site: 5'cccggatccCTTGAATTAGTTTTTAATTAAATAGTTTCTATTATAA 3, amplified by PCR, the amplification conditions are: 94°C pre-denaturation for 4 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 30 seconds, a total of 30 cycles ;Final extension at 72°C for 10 min and stored at 4°C. The recovered A4 promoter fragment was named BmAct4-57, its nucleotide sequence is shown in SEQ ID NO: 2, and connected to...

Embodiment 3

[0085] Example 3. Functional identification and application of three key gene termination signals for silk gland silk protein synthesis in Bombyx mori

[0086] 1 Cell expression vector construction

[0087] The 3'UTR upstream primer FibH PolyA-F: 5' was designed based on the cDNA of the fifth instar third tencel gland cDNA of the dimorphic silkworm variety Dazao as a template, and based on the silkworm FibH gene sequence published by NCBI (GenBank accession number GI: 8572060) ttgcggccgcTTTTAATATAAAATAACCCTTGTT 3'contains a Not I site and downstream primer FibH PolyA R: 5'ccaagcttCTTAGTTGAGAAGGCATACTTG 3'contains a Hind III site; the 3'is designed based on the silkworm FibL gene sequence published by NCBI (GenBank accession number GI:112984493) UTR upstream primer FibL PolyA-F: 5'ttgcggccgcATAAGAACTGTAAATAATGTATATA 3' contains a Not I site and downstream primer FibL PolyA-R: 5'ccaagcttATCTGGAAAACTGGATACATCT 3' contains a Hind III site; according to the silkworm Sericin1 gene...

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Abstract

The invention relates to enhancer Hr3 adopting the nucleotide sequence shown in the SEQIDNO: 1. The enhancer Hr3 can be used for preparing and recombining heterologous protein by bombyx mori. The invention further relates to a recombination carrier containing the enhancer Hr3, and the recombination carrier is prepared in such a manner that the enhancer Hr3 is connected with a Nco1 restriction site at the upstream of a promoter of a carrier containing no enhancer through the Nco1 restriction site of the enhancer Hr3. In the invention, functional elements such as the first grade enhancer, activating transcription factor IE1, untranslated region sequences (5' UTR and 3' UTR) and the like which are expressed by intensifier are identified in the cellular level, and an efficient and stable sericin I expression system is built by integrating optimum elements on the basis, so that efficient expression recombination heterologous protein can be made of middle silk gland of transgenic bombyx mori.

Description

technical field [0001] The invention relates to biological genetic engineering, in particular to the field of transgenic silkworm. Background technique [0002] With the rapid development of bioengineering and genetic engineering in the 21st century, people have an increasing demand for functional proteins for various purposes such as medical, medicinal, edible, beauty, and health care, which cannot be met by relying on protein extraction and production from natural sources. Rapidly growing market demand. Establishing and improving various high-efficiency prokaryotic and eukaryotic expression systems, and using strains, cells, and insects as host bioreactors is an effective and sustainable way to achieve low-cost, large-scale production of recombinant foreign proteins with biological activity. method, and has become a research hotspot in the world today. Using Chinese hamster ovary cells as a bioreactor to produce foreign proteins is currently the most standard expression ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/63C12N15/85A01K67/04
Inventor 夏庆友王峰徐汉福马三垣赵萍向仲怀
Owner SOUTHWEST UNIVERSITY
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