CN104250297A - Pair of polypeptides for specifically identifying sheep KRT25 gene, as well as encoding gene and application thereof - Google Patents
Pair of polypeptides for specifically identifying sheep KRT25 gene, as well as encoding gene and application thereof Download PDFInfo
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- CN104250297A CN104250297A CN201410471728.2A CN201410471728A CN104250297A CN 104250297 A CN104250297 A CN 104250297A CN 201410471728 A CN201410471728 A CN 201410471728A CN 104250297 A CN104250297 A CN 104250297A
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Abstract
The invention discloses a pair of polypeptides for specifically identifying a sheep KRT25 gene, as well as an encoding gene and application thereof. The pair of polypeptides comprises polypeptide A and polypeptide B, wherein the polypeptide A consists of 16 TAL nucleic acid recognizing units; each TAL nucleic acid recognizing unit has a double-chain amino acid; the polypeptide B consists of 15 TAL nucleic acid recognizing units; each TAL nucleic acid recognizing unit has a double-chain amino acid. The sheep KRT25 gene can be knocked out or modified in a cellular level or an individual level so as to resolve functions of the sheep KRT25 gene, establish a mutant library of the sheep KRT25 gene or obtain a relevant disease model, therefore, the sheep breeding and pharmaceutical research and development are promoted.
Description
Technical field
The invention belongs to gene engineering technology field, relate to the polypeptide of a pair specific recognition sheep KRT25 gene and encoding gene thereof and application.
Background technology
Keratin sulfate is one of important factor of determination of hair follicle and skin biological function, determines the quality and yield of wool to a certain extent.Internal root sheath keratin gene is generally divided into I acid type keratin gene (KRT25, KRT26, KRT27, KRT28) with in II neutral or alkaline type keratin gene (KRT71, KRT72, KRT73, KRT74), they are all specific expressed at hair follicle internal root sheath.Coded protein participates in the morphogenetic process of hair follicle.Rogers (2004) studies proof I type internal root sheath keratin gene (Rogers GE.Hair follicle differentiation and regulation.International Journal of Developmental Biology, 2004,48:163-170) specific expressed in the internal root sheath of sheep.As can be seen here, KRT25 gene can be used as an important candidate gene of sheep breeding for disease resistance and fineness of wool breeding.Cell or individual level knocking out sheep KRT25 gene or modify, can resolve the function of sheep KRT25 gene, is Sheep Breeding and medicine research and development service.
Transcriptional activation increment effector nuclease (transcription activator-like effector nucleases, TALENs) technology is an efficient gene targeting new tool.There is a kind of albumen (TALEs) can identify, in conjunction with DNA in transcription factor activation effector family.TALE and DNA sequence dna specific binding are mainly mediated by 34 constant aminoacid sequences in TAL structure.The cutting territory of TALEs and FokI endonuclease is connected, forms TALENs, thus the accurate modification to genomic dna sequence can be realized.TALENs is a kind of heterodimer molecule (the TALE DNA binding domains of two units is fused to the catalytic domain of a unit), can cut two nearer sequences of being separated by, thus specificity is strengthened.
Summary of the invention
The object of this invention is to provide the polypeptide of a pair specific recognition sheep KRT25 gene and encoding gene thereof and application.The present invention utilizes this transcriptional activation increment effector (TALE) polypeptide being built to a pair restructuring obtained can identify sheep KRT25 gene specifically.The present invention also utilizes this transcriptional activation increment effector to be built to a pair transcriptional activation increment effector nuclease (TALENs) obtained, and can carry out targeting modification accurately and efficiently to sheep KRT25 gene.
Particularly, the object of the present invention is achieved like this:
The polypeptide of a pair specific recognition sheep KRT25 gene (called after specific polypeptides to), is made up of polypeptide I and polypeptide II; Described polypeptide I is made up of 16 TAL nucleic acid recognizing unit, has a doubly-linked amino acid in each TAL nucleic acid recognizing unit; Described polypeptide II is made up of 15 TAL nucleic acid recognizing unit, has a doubly-linked amino acid in each TAL nucleic acid recognizing unit;
16 doubly-linked amino acid in described polypeptide I are as follows successively: the sequence 3 of sequence table is from N-terminal 12-13 amino acids residue, 46-47 amino acids residue, 80-81 amino acids residue, 114-115 amino acids residue, 148-149 amino acids residue, 182-183 amino acids residue, 216-217 amino acids residue, 250-251 amino acids residue, 284-285 amino acids residue, 318-319 amino acids residue, 352-353 amino acids residue, 386-387 amino acids residue, 420-421 amino acids residue, 454-455 amino acids residue, 488-489 amino acids residue and 522-523 amino acids residue.
15 doubly-linked amino acid in described polypeptide II are as follows successively: the sequence 4 of sequence table is from N-terminal 12-13 amino acids residue, 46-47 amino acids residue, 80-81 amino acids residue, 114-115 amino acids residue, 148-149 amino acids residue, 182-183 amino acids residue, 216-217 amino acids residue, 250-251 amino acids residue, 284-285 amino acids residue, 318-319 amino acids residue, 352-353 amino acids residue, 386-387 amino acids residue, 420-421 amino acids residue, 454-455 amino acids residue and 488-489 amino acids residue.
Described polypeptide I specifically can as shown in the sequence 2 of sequence table.
Described polypeptide II specifically can as shown in the sequence 4 of sequence table.
The present invention also protects a pair DNA molecular (called after specific DNA molecular to), is made up of the DNA molecular first of coding said polypeptide first and the DNA molecular second of coding said polypeptide second.
In described DNA molecular first, 16 amino acid whose Nucleotide of doubly-linked of coding said polypeptide first are as follows successively: the sequence 4 of sequence table is from 5 ' end 34-39 position Nucleotide, 136-141 position Nucleotide, 238-243 position Nucleotide, 340-345 position Nucleotide, 442-447 position Nucleotide, 544-549 position Nucleotide, 646-651 position Nucleotide, 748-753 position Nucleotide, 850-855 position Nucleotide, 952-957 position Nucleotide, 1054-1059 position Nucleotide, 1156-1161 position Nucleotide, 1258-1263 position Nucleotide, 1360-1365 position Nucleotide, 1462-1467 position Nucleotide and 1564-1569 position Nucleotide.
In described DNA molecular second, 15 amino acid whose Nucleotide of doubly-linked of coding said polypeptide second are as follows successively: the sequence 4 of sequence table is from 5 ' end 34-39 position Nucleotide, 136-141 position Nucleotide, 238-243 position Nucleotide, 340-345 position Nucleotide, 442-447 position Nucleotide, 544-549 position Nucleotide, 646-651 position Nucleotide, 748-753 position Nucleotide, 850-855 position Nucleotide, 952-957 position Nucleotide, 1054-1059 position Nucleotide, 1156-1161 position Nucleotide, 1258-1263 position Nucleotide, 1360-1365 position Nucleotide and 1462-1467 position Nucleotide.
Described DNA molecular first specifically can as shown in the sequence 1 of sequence table.
Described DNA molecular second specifically can as shown in the sequence 3 of sequence table.
The present invention also protects described specific polypeptides to the application in specific recognition and targeting modification sheep KRT25 gene.Described sheep KRT25 gene, its NCBI GI is 57619200.
The present invention also protects described specific polypeptides to building the application in sheep KRT25 transgenation storehouse.Described sheep KRT25 gene, its NCBI GI is 57619200.
Compared with prior art, the invention provides the polypeptide pair of specific recognition sheep KRT25 gene, and provide the right encoding gene of this polypeptide.Present invention can be implemented in cell or individual level and knock out sheep KRT25 gene or modify, to resolve function, the structure sheep KRT25 transgenation storehouse of sheep KRT25 gene or to obtain relative disease model, is Sheep Breeding and medicine research and development service.
Accompanying drawing explanation
Fig. 1 is TALENs shot design result schematic diagram.
Fig. 2 is that TALEN plasmid extracts DNA to (left arm TALEN and right arm TALEN) transfection sheep SEF cell and carries out pcr amplification after 72 hours, the part sequencing result (mutant nucleotide sequence) after PCR primer being cloned.
Fig. 3 is the right principle of work schematic diagram of TALEN plasmid.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.In following examples, if no special instructions, low sugar DMEM culture medium culturing cell is all adopted.
FastTALE
tMtALEN rapid build test kit: Shanghai Si Dansai Bioisystech Co., Ltd, Cat No.1802-030.This test kit comprises two module boards: module board 1 comprises 96 modules, each Module recognition continuous print two bases; Module board 2 comprises 76 modules, wherein 48 Module recognition continuous print, two bases, 28 single bases of Module recognition.In addition, this test kit also comprises other components: solution 1, solution 2, solution 3, solution 4, solution 5, competent cell, SOC substratum, ddH
2o; TALEN skeleton carrier, comprises left arm skeleton and right arm skeleton.
The structure that embodiment 1, TALENs plasmid are right
1, TALENs shot design
Adopt online software (
https: //tale-nt.cac.cornell.edu) design.By in sheep KRT25 gene cDNA sequence (NCBI GI is 57619200) Input Software, find target sequence in 130-177bp region, concrete schematic diagram as shown in Figure 1.TALENs left arm recognition sequence is 5 '-TGCAGCATGCCTGGAA-3 ', and right arm recognition sequence is 5 '-GCTCCCAAAGGCACA-3 '.
2, TALENs module assembled
According to FastTALE
tMtALEN rapid build test kit (hereinafter referred to as " test kit ") specification sheets carries out.Concrete steps are as follows:
1) left and right arms recognition sequence is split as 9 modules respectively, be a module with one or two base composition, mark successively, be labeled as 1 as first, last is labeled as 9, as follows:
Left arm L5:T1 GC2 A3 GC4 AT5 GC6 CT7 GG8 AA9 (using skeleton carrier L15)
Right arm R5:G1 C2 TC3 CC4 AA5 AG6 GC7 CA8 CA9 (using skeleton carrier R11)
2) from test kit, take out 9 modules of corresponding numbering, each module gets 1.5 microlitres, and is added in same PCR pipe.
3) be placed in ice chest by solution 1, solution 2 from-20 DEG C of refrigerators taking-ups, solution 3 is positioned in 37 DEG C of water-baths and dissolves.
4) by following system application of sample, first add corresponding TALEN skeleton carrier (L16 or R12), then solution 3, solution 1, solution 2 are added in the PCR pipe of step 2 successively, finally add water and mend to 20 microlitres, of short duration centrifugal mixing.
Linked system:
5) above-mentioned PCR pipe is put into PCR instrument and carry out ligation (turning off the hot lid temperature of PCR instrument).
Linker is as follows:
6) take out PCR pipe, add in this pipe successively by solution 4 (1 microlitre), solution 5 (0.5 microlitre), mixing, hatches one hour for 37 DEG C in PCR instrument.
7) final product (20 microlitre) of step 6 is added in the EP pipe containing competent cell (reagent constituents), mixing, place 30 minutes on ice, then put into 42 DEG C of water-bath heat shocks 45 seconds, be positioned over rapidly 3 minutes on ice.
8) in Bechtop, in the EP pipe of previous step, add 500 microlitre SOC solution (reagent constituents), be placed in 37 DEG C, the shaking table of 250rpm cultivates 30 minutes.
9) the EP pipe of previous step is taken out, centrifugal 5 minutes of 4000rpm.
10) in Bechtop, discard the most of supernatant in EP pipe, leave about 100 microlitres, and it is even to precipitate piping and druming gently, is spread evenly across in the flat board of kalamycin resistance, is placed in 37 DEG C of incubators and cultivates 12-16 hour.
11) picking next day 10 mono-clonals, are inoculated in mono-clonal and are equipped with in 15 milliliters of centrifuge tubes of 5 milliliters of LB nutrient solutions (containing kantlex), 37 DEG C, cultivate 16 hours in the shaking table of 250rpm.
12) previous step gained mono-clonal bacterium liquid is served the order-checking of Hai Shenggong biotechnology company limited.Order-checking forward primer is 5 '-CTCCCCTTCAGCTGGACAC-3 ', and order-checking reverse primer is 5 '-AGCTGGGCCACGATTGAC-3 '.Gained a pair plasmid called after left arm TALEN and right arm TALEN.
Embodiment 2, the right activity of TALENs built by sequence verification embodiment 1
Sheep fetal fibroblast cell (SEF): isolation cultivation method is see document: Meng Fanhua, Xiao Hongmei, Zhang Dong, Zhou Huanmin. the Mongolian sheep fetal skin inoblast separation and Culture and specificity analysis. " Chinese animal and veterinary " the 3rd phase in 2012,136-140.
1, by the mode cotransfection 1.5 × 10 of each for recombinant plasmid left arm TALEN and right arm TALEN 5 μ g by electricity conversion
6sEF cell, obtains reconstitution cell.
2, reconstitution cell step 1 obtained 30 DEG C is cultivated 72 hours, then collecting cell.
3, extraction step 2 collect cell genomic dna and as template, with PCRF and PCRR composition primer pair carry out pcr amplification, recovery 346bp pcr amplification product.
PCRF:5'CTCTTCGACTTCCCAGTG?3'；
PCRR:5'TTCTCATACCAGCCCTTG?3'。
4, pcr amplification product step 3 obtained and pMD18-T carrier (precious biological, article No.: D101A) connect, and obtain connecting product.
5, connection product conversion bacillus coli DH 5 alpha competent cell step 4 obtained is (precious biological, D9057S), then the LB solid medium flat board coating amicillin resistance is cultivated, random picking 100 is cloned and checks order, the clone calculating sudden change accounts for the ratio of overall clone's number, thus estimates this efficiency to TALEN plasmid.Found that 7 are cloned in appearance sudden change (referring to Fig. 2) near expection cleavage site, the TALEN plasmid that namely recombinant plasmid left arm TALEN and right arm TALEN forms reaches 7% to the cutting efficiency in SEF cellular genome.
The principle of work of recombinant plasmid left arm TALEN and right arm TALEN is shown in Fig. 3, two kinds of difference of the target TALE nuclease with Fok I functional domain high expression under the control of a cmv promoter, and enter in nucleus, specific binding is there is with corresponding target spot DNA, make Fok I endonuclease form dimer and exercise DNA nicking activity, cut off target gene DNA double chain.
Sequence table
Claims (7)
1. the polypeptide of a pair specific recognition sheep KRT25 gene, is made up of polypeptide I and polypeptide II; Described polypeptide I is made up of 16 TAL nucleic acid recognizing unit, has a doubly-linked amino acid in each TAL nucleic acid recognizing unit; Described polypeptide II is made up of 15 TAL nucleic acid recognizing unit, has a doubly-linked amino acid in each TAL nucleic acid recognizing unit;
16 doubly-linked amino acid in described polypeptide I are as follows successively: the sequence 2 of sequence table is from N-terminal 12-13 amino acids residue, 46-47 amino acids residue, 80-81 amino acids residue, 114-115 amino acids residue, 148-149 amino acids residue, 182-183 amino acids residue, 216-217 amino acids residue, 250-251 amino acids residue, 284-285 amino acids residue, 318-319 amino acids residue, 352-353 amino acids residue, 386-387 amino acids residue, 420-421 amino acids residue, 454-455 amino acids residue, 488-489 amino acids residue and 522-523 amino acids residue,
15 doubly-linked amino acid in described polypeptide II are as follows successively: the sequence 4 of sequence table is from N-terminal 12-13 amino acids residue, 46-47 amino acids residue, 80-81 amino acids residue, 114-115 amino acids residue, 148-149 amino acids residue, 182-183 amino acids residue, 216-217 amino acids residue, 250-251 amino acids residue, 284-285 amino acids residue, 318-319 amino acids residue, 352-353 amino acids residue, 386-387 amino acids residue, 420-421 amino acids residue, 454-455 amino acids residue and 488-489 amino acids residue.
2. the polypeptide of a pair specific recognition sheep KRT25 gene as claimed in claim 1, is characterized in that: the aminoacid sequence of described polypeptide I is as shown in the sequence 2 in sequence table; The aminoacid sequence of described polypeptide II is as shown in the sequence 4 in sequence table.
3. a pair DNA molecular, is made up of with the DNA molecular second of the described polypeptide II in coding claim 1 the DNA molecular first of the described polypeptide I in coding claim 1.
4. a pair DNA molecular as claimed in claim 3, is characterized in that:
In described DNA molecular first, 16 amino acid whose Nucleotide of doubly-linked of coding said polypeptide first are as follows successively: the sequence 4 of sequence table is from 5 ' end 34-39 position Nucleotide, 136-141 position Nucleotide, 238-243 position Nucleotide, 340-345 position Nucleotide, 442-447 position Nucleotide, 544-549 position Nucleotide, 646-651 position Nucleotide, 748-753 position Nucleotide, 850-855 position Nucleotide, 952-957 position Nucleotide, 1054-1059 position Nucleotide, 1156-1161 position Nucleotide, 1258-1263 position Nucleotide, 1360-1365 position Nucleotide, 1462-1467 position Nucleotide and 1564-1569 position Nucleotide,
In described DNA molecular second, 15 amino acid whose Nucleotide of doubly-linked of coding said polypeptide second are as follows successively: the sequence 4 of sequence table is from 5 ' end 34-39 position Nucleotide, 136-141 position Nucleotide, 238-243 position Nucleotide, 340-345 position Nucleotide, 442-447 position Nucleotide, 544-549 position Nucleotide, 646-651 position Nucleotide, 748-753 position Nucleotide, 850-855 position Nucleotide, 952-957 position Nucleotide, 1054-1059 position Nucleotide, 1156-1161 position Nucleotide, 1258-1263 position Nucleotide, 1360-1365 position Nucleotide and 1462-1467 position Nucleotide.
5. a pair DNA molecular as claimed in claim 4, is characterized in that: the nucleotide sequence of described DNA molecular first is as shown in the sequence 1 in sequence table, and the nucleotide sequence of described DNA molecular second is as shown in the sequence 3 in sequence table.
6. the application of a pair polypeptide according to claim 1 in specific recognition and targeting modification sheep KRT25 gene.
7. a pair polypeptide according to claim 1 is building the application in sheep KRT25 transgenation storehouse.
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Cited By (2)
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| CN105755155A (en) * | 2016-05-09 | 2016-07-13 | 北京泱深生物信息技术有限公司 | Application of KRT71 gene expression product to preparation of lung squamous carcinoma metastasis diagnosis and treatment products |
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| CN104745552A (en) * | 2015-02-13 | 2015-07-01 | 青岛市畜牧兽医研究所 | Polypeptide for specifically recognizing sheep HSP70.1 gene as well as encoding gene and application thereof |
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| CN105755155A (en) * | 2016-05-09 | 2016-07-13 | 北京泱深生物信息技术有限公司 | Application of KRT71 gene expression product to preparation of lung squamous carcinoma metastasis diagnosis and treatment products |
| CN105755155B (en) * | 2016-05-09 | 2019-08-13 | 北京泱深生物信息技术有限公司 | KRT71 gene expression product is preparing the application in lung squamous cancer transfer diagnosis and treatment product |
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