Preparation method and application of target protein

Abstract

The invention relates to a preparation and application of target protein. The preparation method comprises the following steps: obtaining a nucleotide sequence fragment of the target protein; constructing a recombinant expression vector of the target protein; inductively expressing and purifying the target protein; cleaving; introducing a single cleavage site to the downstream of the nucleotide sequence fragment of the target protein; synthesizing a coding strand and a template strand; and hybridizing the coding strand and the template strand, thus obtaining the target protein, wherein a recognition site for monomer cleavage is introduced while synthesizing the coding strand and the template strand; and preferably, the target protein can be a human nerve growth factor, thymosin, Huwentoxin-XI and Huwena analgesic peptide. The target protein prepared by the preparation method provided by the invention is high in expression quantity and easy to purify; the gene expression product is the target protein monomer and brings no extra amino acid residue, and thus the high biological activity of the target protein is ensured, and the multi-copy cleavage cost is saved; and the target protein is simple to purify and easy for mass production.

Description

technical field [0001] The present invention relates to genetic engineering technology, in particular to a method for preparing a target protein and its use. Background technique [0002] With the development of genetic engineering technology, some small molecular polypeptides with biological activity, such as thymosin, have been highly valued. Thymosin is a protein and polypeptide hormone produced by the thymus gland. As early as the 1960s, Miller et al. (Lancet, 1961, 2:748-749.) discovered that thymosin plays an important role in the immune system, and isolated a group of peptide mixtures with molecular weights of 1000-15000D, called thymosin. Fraction 5 (thymosin fraction 5, TF5); subsequently Goldstein et al. (Proc. Natl. Acad. Sci. USA, 1966, 56: 1010–1017) isolated an acidic polypeptide α1 group from TF5 of bovine thymus extract (thymosin α1, Tα1), Tα1 is the main active component of TF5, which is abnormally conserved in mammals and is widely distributed in mammalia...

AI Technical Summary

Problems solved by technology

In the first method, although the traditional PCR method is simple and easy, the author did not consider the monomer cleavage problem after the expression of the tandem construct, and only discussed the feasibility of this method from a technical perspective

Although the second method can obtain a relatively high copy tandem construct, each expression cassette independently expresses the polypeptide product and avoids the subsequent polypeptide cleavage step, but the molecular weight of the Tα1 monomer expression product is too small, it is easy to be degraded, and it is difficult to achieve the expression level. promote