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

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...

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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 industrial The cost of production is high; although the fusion expression vector has a relatively high expression level and can be separated and purified through separation / purification tags, etc., the fusion of a foreign protein often affects the activity of the expressed foreign protein and affects the recombinant protein. function of

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