Polypeptide pair for specifically recognizing muscle myostatin gene as well as encoding gene and application of gene

A muscle growth inhibition, one-to-one technology, applied in the fields of peptides, depsipeptides, DNA preparation, etc., can solve the problems of inability to guarantee gene knockout, time-consuming, cumbersome ZFN preparation process, etc., and achieve the effect of great application value.

Active Publication Date: 2014-05-07
南宁壮博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] ZFNs have been successfully used in the genetic manipulation of plants, zebrafish, insects and various mammals, but there are still many problems to be solved, mainly including the following aspects: (1) The preparation process of ZFNs is cumbersome, complicated, time-consuming and expensive Expensive (a pair of ZFNs from Sigma is 200,000 RMB, and it usually takes 2 months to prepare a pair of ZFNs); (2) It is not guaranteed that all genes can be knocked out with ZFNs, because the number of genes in the zinc finger library is limited ; (3) The cytotoxicity of ZFN is mainly the side effect caused by non-specific cleavage (off-target cleavage)

Method used

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  • Polypeptide pair for specifically recognizing muscle myostatin gene as well as encoding gene and application of gene
  • Polypeptide pair for specifically recognizing muscle myostatin gene as well as encoding gene and application of gene
  • Polypeptide pair for specifically recognizing muscle myostatin gene as well as encoding gene and application of gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, the construction of TALE target recognition module

[0045] 1. Description of the four modules and the mechanism of combining modules

[0046] The nucleic acid recognition unit of TAL is a double-linked amino acid separated by 32 constant amino acid sequences. Double-linked amino acids have a constant correspondence with A, G, C, and T, that is, NI recognizes A, NG recognizes T, HD recognizes C, and NN recognizes G. The pTALE-A plasmid, pTALE-G plasmid, pTALE-C plasmid, and pTALE-T plasmid are single-module vectors, which are plasmids encoding DNA for the nucleic acid recognition units of the above four TALs respectively, and at the 5' end of the encoding DNA. Spe I restriction recognition sequence, 3' end has continuous Nhe I restriction recognition sequence and Hind III recognition sequence. Through the Spe I, Nhe I, and Hind III restriction sites on the single-module vector, the TAL unit corresponding to the target sequence can be cloned in series, an...

Embodiment 2

[0065] Embodiment 2, the construction of reporter plasmid

[0066] 1. Synthesize the double-stranded DNA molecule (target fragment) shown in sequence 6 of the sequence listing.

[0067] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of mstnssaup and mstnssadn to obtain a PCR amplification product.

[0068] mstnssaup:5'-AGT AGATCT GTGATTCAGGATCTATTGCTAC-3';

[0069] mstnssadn:5'-AGT CTCGAG GCAGTAATTGGCCTTATATCT-3'.

[0070] 3. The PCR amplified product of step 2 was double-digested with restriction endonucleases BglII and XhoI, and the digested product was recovered.

[0071] 4. Digest the pSSA vector with restriction endonucleases BglII and XhoI to recover a vector backbone of about 6500 bp.

[0072] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant plasmid pSSA-MSTN (reporter plasmid). According to the sequencing results, the struct...

Embodiment 3

[0073] Example 3, verifying the TALEN activity of the plasmid constructed in Example 1 by the luciferase reporter gene method

[0074] The following two groups of experimental treatments were carried out respectively (each experimental treatment was repeated three times, and each repeated experiment was set with three repeated treatments, and the average value of the three repeated treatments was taken for the results; the transfection reagents were all DNA Fect Transfection Reagent DNA transfection reagents, CWBIO , Cat No.CW0860, the amount of transfection reagent added in each treatment is 6ul, and the operation is performed according to the instructions):

[0075] Group 1: 0.4ug Renilla luciferase plasmid (reference control plasmid) and 2.0ug recombinant plasmid pSSA-MSTN were co-transfected 1×10 6 293T cells;

[0076] Group 2: Co-transfect 1× with 0.4ug Renilla luciferase plasmid (reference control plasmid), 2.0ug recombinant plasmid pSSA-MSTN, 4ug recombinant plasmid pc...

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Abstract

The invention discloses a polypeptide pair for specifically recognizing a muscle myostatin gene as well as an encoding gene and an application of the gene. The polypeptide pair provided by the invention comprises a polypeptide I and a polypeptide II; double-chain amino acid in the polypeptide I sequentially comprises 2-3, 36-37, 70-71, 104-105, 138-139, 172-173, 206-207, 240-241, 274-275, 308-309, 342-343, 376-377, 410-411, 444-445, 478-479 and 512-513 of the sequence 3; and double-chain amino acid in the polypeptide II sequentially comprises 2-3, 36-37, 70-71, 104-105, 138-139, 172-173, 206-207, 240-241, 274-275, 308-309, 342-343, 376-377, 410-411, 444-445, 478-479, 512-513 and 546-547 of the sequence 5. The polypeptide pair provided by the invention can specifically recognize the muscle myostatin gene, and has important application value in obtaining high-quality pig breeds with high lean meat percentage by knockout or modification of MSTN on pigs.

Description

technical field [0001] The invention relates to a pair of polypeptides specifically recognizing myostatin gene, its encoding gene and application. Background technique [0002] Myostatin (MSTN) is a negative regulator of skeletal muscle growth. After its vitality disappears, animals will show symptoms of muscle overdevelopment, and their muscle fibers will thicken or increase in number, showing symptoms of double muscular buttocks. Because it is one of the regulatory factors that can determine the size of muscle organs, it has good application value and application prospect in animal husbandry production. Many scientists have conducted in-depth research on this gene in pigs, expecting to obtain high-quality and high-lean pig breeds by knocking out or transforming MSTN in pigs. [0003] It is very important to find a technology that can target and modify the MSTN gene. The efficiency of traditional targeting technology is very low, which mainly depends on the random exchang...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/63C12N15/10C40B50/06
Inventor 李奎阮进学吴添文刘楠杨述林牟玉莲
Owner 南宁壮博生物科技有限公司
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