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Silkworm Bmlp3 gene promoter and use thereof

A promoter, pmd19-bmlp3 technology, applied in the direction of using vectors to introduce foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effect of simple separation and purification, efficient synthesis and storage capacity, and low cost of large-scale production

Active Publication Date: 2011-09-21
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These features provide theoretical support for the use of the silkworm Bmlp3 gene promoter to express foreign proteins in the fat body, and in the research report on the use of the piggBac-derived transposon-mediated silkworm transgenesis, the use of the silkworm 30K protein gene promoter in fat body The method of expressing foreign protein in vivo has not been reported

Method used

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  • Silkworm Bmlp3 gene promoter and use thereof
  • Silkworm Bmlp3 gene promoter and use thereof
  • Silkworm Bmlp3 gene promoter and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Obtaining of Bombyx mori Bmlp3 Gene Promoter

[0038] The PCR amplification of the promoter element is based on the genome of the five-instar-three-day silkworm variety P50 as a template, and is amplified according to the designed upstream and downstream primers of the lp3 promoter. The specific construction is as follows:

[0039] The first is the acquisition of the promoter element: the Bmlp3 promoter is composed of the 5'-upstream sequence of the lp3 gene, exon 1, intron 1 and exon 2 of 17bp, it does not contain the signal peptide sequence of the lp3 gene, and is composed of the above The downstream primers were amplified from the five-instar and three-day-old genome of silkworm variety P50 as a template. The upstream primer P-f contained an EcoR I site: 5′-ggaattcCAGTATAGTTACAACGGCTGCCCC-3′, and the downstream primer P-r contained a BamH I site: 5′-cgggatccCGCGTCGAGTCCTGCAATATGT -3′, the amplification conditions were pre-denaturation at 94°C for 4 minutes, d...

Embodiment 2

[0061] Example 2 Obtaining of the recombinant vector containing the silkworm Bmlp3 gene promoter

[0062] The pBac[3xP3-EGFPafm] injection transposon vector used in the present invention is a vector derived from piggBac. The microinjection vector pBac[Bmlp3-DsRed-SV40, 3xP3EGFP] was constructed with reference to Horn & Wimmer (2000) using the pSLfa1180fa cloning shuttle vector using a two-step cloning method: obtained in the examples, as shown in SEQ ID NO:1 The recombinant expression vector obtained after TA cloning of the silkworm Bmlp3 gene promoter and the vector pMD19-T simple is pMD19-Bmlp3; then a complete expression frame is constructed on the shuttle vector pSLfa1180fa, as shown in SEQ ID NO: 4, specifically : The recombinant expression vector obtained in step A was digested with EcoR I and BamHI respectively to obtain the pMD19-Bmlp3 plasmid, the Bmlp3 gene promoter of the silkworm was recovered, and the pMD19-Bmlp3 plasmid was digested with EcoR I and BamHI respecti...

Embodiment 3

[0100] Example 3 Application of Bombyx mori Bmlp3 Gene Promoter to Express Foreign Protein in Fat Body

[0101] pHA3PIG (Tamura et al., 2000) was used as an auxiliary plasmid to produce transposase, and the pBac[BmLSP-DsRed-SV40,3xP3EGFP] and pHA4PIG plasmids were extracted with the QIAGEN Plasimd Mini Kit (Qiagen) plasmid extraction kit at a ratio of 1: After mixing at 1 mole ratio, microinject into 357 Dazao early embryos (G0 generation) that have been released from diapause (2-5 h after oviposition). The injected silkworm eggs are sealed with non-toxic dehydration, and accelerated to hatch at 25°C. The hatched larvae (G0 generation) were reared with artificial feed, and then self-crossed or backcrossed to produce seeds after adulthood. The obtained G1-generation silkworm eggs (7th day) were detected under a macroscopic stereofluorescence microscope (Olypus MVX10, Japan). Green fluorescence observation uses excitation light with a wavelength of 460-490 nm to screen out trans...

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Abstract

The invention relates to the technical field of biology, in particular to a silkworm Bmlp3 gene promoter represented by SEQ ID No.1. The promoter can be used for expressing an extrinsic protein in a fat body. The invention also comprises a preparation method of a microscopic injection carrier for expressing the extrinsic protein in the fat body of silkworms, which comprises: constructing a recombinant expression vector containing a silkworm Bmlp3 gene promoter and an expression cassette; and preparing the microscopic injection carrier. The Bmlp3 gene promoter can realize the expression of the extrinsic protein in a development period in which the fat body is the most developed. The method can realize the controllability of an expression period; the transgenosis-mediated by a transposons derived from pggBac has characteristics of high stability, high heritability and the like; and through continuous rearing, colony can be enlarged quickly, and a quantitative guarantee for purification of the EQ ID No.1. The promoter can be used for expressing an extrinsic protein is provided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the promoter of the silkworm Bmlp3 gene and its application. Background technique [0002] The silkworm is an important economic insect, which has made extremely important contributions to the cultural, economic and social development of the Chinese nation. At the same time, its genetic background is clear, its genetic resources are abundant, and it is an internationally recognized model insect of Lepidoptera. One of the important traits of the silkworm is that the fat body of the last instar larva has the ability to efficiently synthesize proteins. During the period from the late fifth instar to pupation, the fat body of silkworms is very developed, accounting for about 30% of the body weight. Based on this feature, Developing it into a new type of bioreactor can use transgenic silkworms to produce more types of useful proteins, broaden the application fields of silkworms, improve t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/66C12N15/85
Inventor 夏庆友邓党军徐汉福马三垣
Owner SOUTHWEST UNIV
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