Repeated module gene-splicing method

A gene splicing and gene technology, applied in the field of repetitive module gene splicing

Inactive Publication Date: 2010-08-18
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 dis

Method used

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  • Repeated module gene-splicing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Take the spliced ​​flagellar silk gene repeat framework as an example;

[0073] (1) Amplify the target gene by using the following PCR primers for the 696bp target repetitive gene;

[0074] Upstream primers:

[0075] 5'-ggt CATATG ttgg GGTCTC g

[0076] Nde I Bsa I

[0077] Downstream primers:

[0078] 5'-ggt CTCGAG ccaa ACCTGC TTCCAG 3'

[0079] Xho I BspM I

[0080] Among them, the underlined part is the enzyme cutting site, and the shaded part is the sequence matching part;

[0081] The 696bp gene sequence of the spider silk flagellar order:

[0082] CTGGTG CTGGTGTTTC TGGCGGTGTT

[0083] GGCCCTGGTG GCCTGGGTGG CCCTGGTGGT TTTGGCGGTC CGGGTGGCCC AGGTGGCCCT

[0084] GGTGGCCCGG GTGCACCTGG TGGCGAAGCG GGTGGTCTTT ATGGTCCGGG CGGTGCGGGT

[0085] GGCCTGTATG GCCCTGGCGG CGCGGGCGGT CTGTATGGTC CAGGCGGTGC GGGTGCACCG

[0086] GGTGGTCCAG GTGGTCCTGG TGGCTTTGGT GGCCCTGGCG GCCTGGGTGG TCCAGGTGGC

[0087] TTTGGCGGTG CAAGCGGTGC AGGCGCCGGT GGCGTTGGTC CGGGT...

Embodiment 2

[0112] Adding a repeat module to the 3' end of the silk repeat gene

[0113] The 819bp repeated spidroin gene was amplified by PCR;

[0114] Upstream primers:

[0115] 5'-ggc catatg aacc ggtctct -3';

[0116] Nde I Bsa I

[0117] Downstream primers:

[0118] 5'-ggc ctcgagc caa acctgcg ctcGGCG

[0119] Xho I BspM I

[0120] Among them, the underlined part is the enzyme cutting site, and the shaded part is the sequence matching part;

[0121] 819bp repeat spidroin protein target gene, the sequence is as follows:

[0122] CTGGGGTCAACGTGGTCCTCGCTCTCAAGGTCCTGGTTCTGGCGGTCAGCAGGGTCCGGGTGGTC

[0123] AGGGTCCTTATGGTCCTAGCGCGGCTGCAGCAGC

[0124] GGTCTGTCTCTGGAAGCAAAAACTAACGCTATC

[0125] GCTTCCGCACTGAGCGCGGCCTTCCTGGAAACCACGGGTTACGTTAATCA

[0126] GCAGTTTGTAAACGAAATCAAAACGCTGATCTTTATGATCGCACAGGCTT

[0127] CCTCCAACGAAATCTCTGGTTCTGCTGCAGCTGCGGGTGGTTCCAGCGGC

[0128] GGTGGTGGTGGCTCCGGTCAGGGTGGCTATGGTCAGGGCGCCTACGCCTC

[0129] TGCCAGCGCGGCGGCTGCCTA...

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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 cut by 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...

Claims

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Application Information

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IPC IPC(8): C12N15/66
Inventor 孟清李梦梦施长华
Owner DONGHUA UNIV
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