T7 bacteriophage tail fibrin polypeptide and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and relates to T7 bacteriophage tail fibrin polypeptide and an application thereof. Solubility test indicates that P17 can enable solubility of three different single-chain antibodies in escherichia coli to be improved by 2-8 times. The P17 is connected tosingle-chain antibody amino or carboxyl terminal of prokaryotic expression,so that the solubility of the single-chain antibody can be improved. Through deletion mutation and point mutation, the inventor finds that the amino and carboxyl terminal sequence of the P17 polypeptide and an alpha-helix secondary structure located at the carboxyl terminal are necessary for dissolving promotion effects. The single-chain antibody of the P17 polypeptide is coupled, so that targetantigen can be efficiently combined. The P17 polypeptide can be used for preparing polypeptide or other products which can increase the yield of soluble single-chain antibody expressing combination activity in the escherichia coli.

Description

technical field [0001] The technical field of genetic engineering of the present invention relates to a polypeptide capable of improving the solubility of a prokaryotic-expressed single-chain antibody, and specifically relates to a T7 phage tail fibrin polypeptide (named P17) and its application Background technique [0002] Studies have shown that antibodies (Antibody, Ab) are secreted by plasma cells and can specifically recognize foreign antigens (such as bacteria and viruses, etc.) Non-covalent bonds and disulfide bonds are assembled into a Y-shaped protein; both the antibody heavy chain and light chain variable regions contain complementarity determining regions (Complementary determining regions, CDRs) that can specifically recognize antigenic epitopes; and the heavy chain carboxy-terminal Two or three constant regions form a fragment crystallizable region (Fc), which mediates many immune regulatory functions (such as activation of immune cells and antibody-dependent c...

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Problems solved by technology

[0004] It has been reported that scFv can be expressed in various systems such as prokaryotic cells (such as E. coli), mammalian cells (such as CHO cells), yeast and insect cells, although the molecular weight is significantly smaller than that of mAb, scFv heavy chain and light chain variable regions Each still contains cysteines capable of forming intrachain disulfide bonds, which may be important for proper folding of mAbs and scFvs; the cytoplasm of common E. coli expression bacteria is reducing, which may not be conducive to chain The formation of internal disulfide bonds leads to the formation of inactive inclusion bodies of scFv. These inclusion bodies can only be activated by delicate renaturation methods. To obtain active scFv, a common method is to express scFv with different solubilizing protein tags These fusion proteins with solubilizing effects include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin A (TrxA), small ubiquitin-like modified protein (SUMO) and nitrogen source utilization substance A (NusA), etc., but their solubilizing effect may be related to the scFv sequence, which does not always improve the solubility of scFv; another method is in the oxidative periplasmic space of Escherichia coli Expresses scFv, but in low yield

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