Pair of polypeptides specifically identifying porcine NFkappaBp65 gene, and coding gene and application thereof

A pair of coding technology, applied in the field of genetic engineering, can solve the problem of low gene knockout efficiency

Inactive Publication Date: 2014-02-05
青岛市畜牧兽医研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The efficiency of traditional gene knockout technology is very low, so it is ver

Method used

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  • Pair of polypeptides specifically identifying porcine NFkappaBp65 gene, and coding gene and application thereof
  • Pair of polypeptides specifically identifying porcine NFkappaBp65 gene, and coding gene and application thereof
  • Pair of polypeptides specifically identifying porcine NFkappaBp65 gene, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, the construction of TALE target recognition module

[0039] 1. Description of the four modules and the mechanism of combining modules

[0040] The nucleic acid recognition unit of TAL is a double-linked amino acid (that is, two adjacent amino acids) 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 ...

Embodiment 2

[0055] Embodiment 2, the construction of reporter plasmid

[0056] 1. Synthesize the double-stranded DNA molecule (target fragment) shown in sequence 6 of the sequence listing.

[0057] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of NFκB p65F and NFκB p65R to obtain a PCR amplification product.

[0058] NFκB p65F: 5'-ACTTATGTTA CCCGGG CGGATTGAGGAGAAACGCAAAAGG-3';

[0059] NFκB p65R: 5'-ATACATGTAT CCCGGG AGAAACAGGAGCCCAACAGAGGG-3'.

[0060] 3. Digest the PCR amplified product in step 2 with restriction endonuclease XmaI, and recover the digested product.

[0061] 4. Digest the pGL4-SSA vector with restriction endonuclease XmaI, and recover the vector backbone of about 5.7 kb.

[0062] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain recombinant plasmid pGL4-SSA-p65 (reporter plasmid). According to the sequencing results, the structure of the reco...

Embodiment 3

[0063] Example 3, verifying the TALEN activity of the plasmid constructed in Example 1 by the luciferase reporter gene method

[0064] Carry out the following 2 groups of experimental treatments respectively (each experimental treatment carries out three repeated experiments, each repeated experiment is set 3 repeated treatments, the average value of three repeated treatments is taken as the result; the transfection reagents are all DNA Fect Transfection Reagent DNA transfection reagents, CWBIO, Cat No.CW0860, the amount of transfection reagent added in each treatment is 6 μl, and the operation is carried out according to the instructions):

[0065] Group 1: 0.4 μg pRL-TK plasmid (reference control plasmid), 2.0 μg recombinant plasmid pGL4-SSA-p65, 4.0 μg plasmid vector pCS2-Fok I (PEAS type) and 4.0 μg plasmid vector pCS2-Fok I (PERR type) co-transfection 1×10 6 293T cells;

[0066] Group 2: 0.4 μg pRL-TK plasmid (reference control plasmid), 2.0 μg recombinant plasmid pGL4-...

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Abstract

The invention discloses a pair of polypeptides specifically identifying a porcine NFkappaBp65 gene, and a coding gene and an application thereof. The polypeptides comprise a polypeptide A and a polypeptide B; double-strand amino acids in the polypeptide A sequentially comprise amino acid residues having site numbers of 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 in a sequence 3; and double-strand amino acids in the polypeptide B sequentially comprise amino acid residues having site numbers of 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 in a sequence 5. The polypeptides can specifically identify the porcine NFkappaBp65 gene, and can be used for the knockout or reconstruction of the porcine NFkappaBp65 gene to obtain porcine disease resistance breeding materials, xenograft donors and human disease animal models.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a pair of polypeptides specifically recognizing porcine NFκB p65 gene, its encoding gene and application. Background technique [0002] p65 is the most important subunit of NFκB transcription factor. Porcine NFκB p65 is significantly associated with the antibody levels of PRRSV, CSFV and Pseudorabies virus in piglets (Li et al., 2011), and the change of a single amino acid can cause the infection ability of domestic pigs to African swine fever virus (ASFV). Significant changes (Palgrave et al., 2011). It can be seen that the porcine NFκB p65 gene can be used as an important candidate gene for pig disease resistance breeding. [0003] As a major subunit of NFκB transcription factor, p65 plays an important role in mammalian immune response. Of the three TAD-containing subunits, p65 is the only one expressed systemically (Doleschall et al., 2007). Deletion of the p65 s...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11C12N15/63C12N15/10C40B50/06
CPCC07K14/00C07K14/47C07K2319/80C12N9/22C12N15/85
Inventor 李和刚赵金山阮进学李培培张宝珣刘明团代永联侯乐乐郝小静江科吴海港
Owner 青岛市畜牧兽医研究所
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