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Method for preparing tandem repeat dna and related biological materials and applications

A tandem repeat, DNA molecule technology, applied in the biological field, can solve the problems of low spinning strength, low toughness, high price, etc.

Active Publication Date: 2021-12-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2002, some scholars used mammalian cells to express a 60kDa recombinant spidroin protein, whose spinning strength was 4.2 times lower than that of natural spider silk.
Liraglutide (lir for short) is a kind of active small peptide, which is a highly effective drug for treating diabetes and is expensive

Method used

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  • Method for preparing tandem repeat dna and related biological materials and applications
  • Method for preparing tandem repeat dna and related biological materials and applications
  • Method for preparing tandem repeat dna and related biological materials and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1, preparation of tandem repeat MaSp1

[0057] In this example, an expression vector named pMaSp1 containing a single-copy DNA expression cassette was prepared. The nucleotide sequence of pMaSp1 is a double-stranded DNA of sequence 1 (SEQ ID No.1) in the sequence listing. In Sequence 1, positions 1500-2312 are an apramycin resistance gene; positions 2537-3073 are DNA molecules named single-copy DNA expression cassettes, hereinafter referred to as MaSp1 RNA expression cassettes. The structure of the MaSp1 RNA expression cassette is as follows figure 1 Shown, by the T7 promoter (the nucleotide sequence is the 2537-2556th nucleotide of sequence 1), the intron (the nucleotide sequence is the sequence 2557-2790 nucleotides of 1, of which 2557-2785 is the 3'ss sequence, and 2786-2790 is the 3' end splicing site), and the name connected to the 3' intron is single-copy DNA The target DNA (hereinafter referred to as the MaSp1 gene, the nucleotide sequence is the 2791-2...

Embodiment 2

[0096] Embodiment 2, preparation lir

[0097] In this example, an expression vector named plir containing a single-copy DNA expression cassette was prepared. The nucleotide sequence of plir is a double-stranded DNA of sequence 3 (SEQ ID No.3) in the sequence listing. In sequence 3, positions 1500-2312 are the apramycin resistance gene; positions 2537-3079 are DNA molecules named single-copy DNA expression cassettes, hereinafter referred to as lirRNA expression cassettes. The structure of the lirRNA expression cassette is as follows Figure 7 Shown, by the T7 promoter (the nucleotide sequence is the 2537-2556th nucleotide of sequence 3), the intron (the nucleotide sequence is the sequence 2557-2790 nucleotides of 3, wherein the 2557-2785th is the 3'ss sequence, and the 2786-2790th is the 3' end splicing site), and the name connected to the 3' intron is single-copy DNA Target DNA (wherein, No. 2791-2883 of sequence 3 is the lir gene, and No. 2884-2904 is the TEV enzyme recogni...

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Abstract

The invention provides a method for preparing tandem repeated DNA molecules and related products and applications. The method for preparing tandem repeat DNA molecules of the present invention comprises introducing an expression vector containing a double-stranded DNA molecule named a single-copy DNA expression cassette into a recipient cell to obtain a recombinant cell, extracting the total RNA of the recombinant cell, and reversing the total RNA Recorded into cDNA to obtain tandem repeat DNA molecules, which greatly reduces the time for preparing long tandem repeat sequences. Experiments have shown that the tandem repeat MaSp1 with 10 repeats can be obtained in only 4 days, which greatly shortens the time from MaSp1 to MaSp8 by traditional methods. Through the implementation of the present invention, the lir tandem repeat fragment with a copy number of 7 can be quickly obtained, and then degraded into lir small peptides. The method of the invention has the characteristics of short experiment period, time and cost saving, high efficiency and the like.

Description

technical field [0001] The invention relates to a method for preparing tandem repeat DNA, related biological materials and applications in the field of biotechnology. Background technique [0002] Tandem repeat DNA is a segment of DNA that is concatenated end to end into multiple copies. Tandem repeat DNA can be used for the expression of tandem repeat proteins. At present, there are two methods used in the construction of tandem repeat DNA, the asymmetric cohesive end complementation method and the homologous enzyme method. The copy number generated by the asymmetric cohesive end complementation method is relatively random, and multiple enzymes are required for enzyme cleavage and ligation. The homologous enzyme method is also relatively cumbersome, requiring repeated enzyme digestion and ligation. Spider silk has high strength and good plasticity, and is widely used in many fields. In the industrial field, such as the preparation of parachutes, protective clothing, com...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/70C07K19/00C12N1/21C12R1/19
CPCC12N15/1096C12N15/70C07K14/43518C07K14/605C07K2319/00
Inventor 毕昌昊张学礼刘丽赵东东李斯微
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI