Repeated module gene-splicing method

Abstract

The invention relates to a repeated module gene-splicing method, which includes the following steps: (1) two PCR primers are utilized to amplify target gene; (2) the PCR primers and plasmid pET-LIC are cut by double restriction enzymes, segments are connected with carrier, and after competent cells are transformed, plasmid pET-TS is obtained; (3) the plasmid pET-TS is cut by double restriction enzymes, and plasmid segments are recovered, and are then connected with the target gene segments to transform, so that plasmid pET-TS2 is obtained; (4) the plasmid pET-TS2 is cut by double restriction enzymes, and plasmid segments are recovered, and are then connected with the target gene segments to transform, so that plasmid pET-TS3 is obtained; (5) the plasmid and target gene PCR product are cutby double restriction enzymes, and the same method is utilized to continuously insert repeated segments. The invention repetitively splices a plurality of repeated module genes, moreover, no restriction enzyme cutting site residues are left between the modules, thereby the seamless connection between the repeated modules is truly realized, and moreover, splicing is directional.

Description

technical field [0001] The invention belongs to the field of gene splicing, and in particular relates to a gene splicing method of repeat modules. Background technique [0002] With the development of genetic engineering technology, protein biosynthesis has become a practical technology, providing a steady stream of raw material protein. The application of protein materials has a long history, but many scientific studies are limited to the synthesis of identical, random polymers (α-amino acids), or the reconstruction of natural proteins. The main advantage of protein biosynthesis is that it can directly produce high molecular weight polypeptides with accurate molecular weight and defined primary structure. Compared with traditional polymer synthesis methods, peptide biosynthesis is closer to the complete macromolecular structure, such as size, composition, sequence, topology and stereochemistry. Biosynthetic polymers can be viewed as a single modular polymer and may have u...

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Problems solved by technology

[0004] Although the above method has been successfully applied to the synthesis of some multimeric protein polymers, the method still has many disadvantages

The most significant disadvantage relies on a pool of restriction endonucleases that recognize non-rhythmic cleavage sites and are required for the formation of head-to-tail self-ligations of DNA monomers

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