Method for purifying fusion protein based on bombyx mori baculovirus polyhedron dissolving characteristic

A purification method and polyhedron gene technology, applied in the field of high-efficiency fusion expression vectors, can solve the problems of foreign protein separation and purification, affect the activity of foreign proteins, and affect the function of recombinant proteins, etc., and achieve easy purification, high expression, and instrumentation. simple effect

Inactive Publication Date: 2012-06-27
TIANJIN YAOYU BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Generally speaking, although the non-fusion expression vector can better maintain the primary structure of the foreign protein, its expression level is low, the separation and purification of the foreign protein is difficult, the recovery rate is low, the separation and purification process is complicated, and large-scale indus...

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  • Method for purifying fusion protein based on bombyx mori baculovirus polyhedron dissolving characteristic
  • Method for purifying fusion protein based on bombyx mori baculovirus polyhedron dissolving characteristic
  • Method for purifying fusion protein based on bombyx mori baculovirus polyhedron dissolving characteristic

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specific Embodiment 1

[0035] 1. Cloning experiment design

[0036] The present invention takes enhanced green fluorescent protein (EGFP) as an example, inserts the Polh gene between the BamH I and EcoR I restriction sites of the pET-28a vector; adds TEV before the start codon ATG when designing the upstream primer of EGFP Enzyme sequence, the TEV-EGFP sequence is inserted between the EcoR I and Xho I restriction sites of the pET-28a vector connected with the Polh gene to obtain the recombinant plasmid pET-28a-Polh-EGFP.

[0037] 2. Vector construction of pET-28a-Polh-EGFP

[0038] The upstream primer P1 and the downstream primer P2 were designed according to the ORF sequence of the Bombyx mori Polh gene, respectively containing the BamH I restriction site and the EcoR I restriction site. The specific sequence is as follows:

[0039] P1: 5'-GCG ATGCCGAATTATTCATAC-3'

[0040] (The box is the BamH I restriction site)

[0041] P2: 5'-GC ATACGCCGGACCAGTGAAC-3'

[0042] (EcoR I restriction site ...

specific Embodiment 2

[0057] 1. Cloning experiment design

[0058] The present invention takes anti-apoptotic factor-1 (BI-1) as an example, inserts the Polh gene between the BamH I and EcoR I restriction sites of the pFastBac HTb carrier; TEV enzyme sequence was added before ATG, and TEV-BI-1 sequence was inserted between EcoR I and Xho I restriction sites of pFastBac HTb vector connected with Polh gene to obtain recombinant plasmid pFastBac HTb-Polh-BI-1.

[0059] 2. Vector construction of pFastBac HTb-Polh-BI-1

[0060] The upstream primer P1 and the downstream primer P2 were designed according to the ORF sequence of the Bombyx mori Polh gene, respectively containing the BamH I restriction site and the EcoR I restriction site. The specific sequence is as follows:

[0061] P1: 5'-GCG ATGCCGAATTATTCATAC-3'

[0062] (The box is the BamH I restriction site)

[0063] P2: 5'-GC ATACGCCGGACCAGTGAAC-3'

[0064] (EcoR I restriction site is in the box)

[0065] The wild silkworm baculovirus geno...

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Abstract

The invention relates to a method for expressing and purifying fusion protein based on bombyx mori baculovirus polyhedron dissolving characteristic and a high-efficiency fusion expression vector for implementing the fusion protein expression and purification method. The method comprises the following steps of: constructing a bombyx mori polyhedrin gene (Po1h) and a target protein gene into a prokaryotic expression vector and introducing a protease enzyme digestion site between the bombyx mori polyhedrin gene (Po1h) and the target protein gene so as to perform fusion expression in a prokaryotic expression bacterium; due to the solubility characteristics that the polyhedrin is dissolved under the alkaline condition and precipitated under the neutral condition, washing the precipitate, which is obtained after ultrasonic induction of the thallus, by using buffer solutions with different pH values; dissolving the precipitate collected finally by using a buffer solution with high pH value; centrifuging, adjusting the pH value of the collected supernate to be neutral; centrifugally collecting the precipitate which is the fusion protein obtained through separation and purification; performing enzyme digestion on specific protease and performing purification, performing nickel column purification on the polyhedrin tag of the fusion protein to obtain the target protein, so that the fusion protein purification method based on bombyx mori baculovirus polyhedron dissolving characteristic and prokaryotic expression is formed.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for expressing and purifying a fusion protein based on the polyhedron dissolution properties of the silkworm baculovirus, and also relates to a high-efficiency fusion expression vector for implementing the above method for expressing and purifying the fusion protein. Background technique [0002] In the process of life science research and production of biological products, the use of expression vectors to prepare recombinant proteins is one of the most important technologies. Obtaining a large amount of active recombinant proteins is a necessary condition for the research and production of biological products. Constructing an effective expression vector is a basic requirement for expressing a target gene, and it is also an important factor affecting gene expression level and protein activity. The existing expression vectors mainly include non-fusion expression ve...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/74C12N15/79C12N1/15C12N1/19C12N1/21C12N5/10C07K19/00C07K1/14
Inventor 张耀洲舒特俊陈剑清白舒婉陈昊
Owner TIANJIN YAOYU BIOLOGICAL TECH
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