Recombinant vector containing polyhedrosis gene, and method for expressing and purifying protein

Abstract

The invention relates to the technical field of gene engineering, in particular to a recombinant vector containing polyhedrosis genes and a method for expressing and purifying proteins. The invention provides a recombinant plasmid pBacPAKph, a recombinant plasmid pBacPAKph-M and a linear virus recon with a connection number of CGMCC No.2768. Linear virus recon BmBacPAKph-M is adopted to infect a host body and express fusion protein of polyhedrosis protein and target protein, the expressed fusion protein is separated and purified, enzyme digestion is carried out on prolease to obtain the target protein. The purified target protein can be prepared into intravenous medicines or used as an object for study on protein functions. A plurality of proteins which are provided by the invention and hard to be expressed and purified but can be easily expressed and purified by protein expression and purification systems, especially membrane proteins have a broad prospect of application.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant plasmid containing polyhedrin gene, a preparation method thereof, and a protein expression and purification method. Background technique [0002] Insect cell expression system (ie, baculovirus expression system) has unique biological characteristics and has been paid more and more attention by people. During the very late gene expression process of the baculovirus gene, there are two highly expressed proteins, polyhedrin and P10 protein: polyhedrin is the main component of inclusion bodies, and the accumulation in cells at the late stage of infection can be as high as 30% to 50%, forming crystals, which are non-essential components for virus replication; P10 protein is also a non-essential component for virus replication, and can form fibrous substances in cells. Both the polyhedron gene and the P10 gene have been located and cloned now, and the promote...

AI Technical Summary

Problems solved by technology

[0004] When using traditional recombinant insect viruses to screen recombinant viruses with exogenous genes, it is necessary to use a microscope to screen according to the shape of plaques in virus-infected cells. In addition to requiring skilled technology, it is also time-consuming and long-term, and the recombinant target protein obtained is less , not conducive to preservation, and the purification of the target protein is also difficult

In addition, membrane proteins are difficult to purify due to their own hydrophobic properties, and when expressed in expression systems such as Escherichia coli and yeast cells, membrane proteins are generally integrated into the cell membrane, which greatly increases the difficulty of purification

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