CN1238505C - Cyclic amp receptor protein specifically combined with beta-acceptor excitomotor group pharmaceutical, coding gene and application thereof - Google Patents
Cyclic amp receptor protein specifically combined with beta-acceptor excitomotor group pharmaceutical, coding gene and application thereof Download PDFInfo
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- CN1238505C CN1238505C CN 02153850 CN02153850A CN1238505C CN 1238505 C CN1238505 C CN 1238505C CN 02153850 CN02153850 CN 02153850 CN 02153850 A CN02153850 A CN 02153850A CN 1238505 C CN1238505 C CN 1238505C
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Abstract
The present invention discloses receptor protein which is rummily combined with beta-receptor agonist medicine, and an encoding gene and the application of the beta-receptor agonist class medicine. The present invention aims to provide the receptor protein which can be rummily combined with the beta-receptor agonist class medicine and the encoding gene of the receptor protein. The encoding gene of the receptor protein which can be rummily combined with the beta-receptor agonist class medicine provided by the present invention is one of the following nucleotide sequences: 1) SEQ ID No. 1 in a sequence list; 2) polynucleotide of the protein sequence of SEQ ID No. 2 in a coding sequence list; 3) a DNA sequence which has more than 90% of homology with the DNA sequence limited by the SEQ ID No. 1 in the sequence list, and encodes the same functional protein. The receptor protein which can be rummily combined with the beta-receptor agonist class medicine is an amino acid residue sequence of the SEQ ID No. 2 in the sequence list, or protein which is formed by that replacement, deletion or addition are carried out on the amino acid residue sequence of the SEQ ID No. 2 through one or a plurality of amino acid residues and has the same activity with the SEQ ID No. 2.
Description
Technical field
The present invention relates to a kind of receptor protein and encoding gene thereof and application, particularly relate to receptor protein and encoding gene and application with beta-receptor stimulant medicine specific combination.
Background technology
The beta-receptor agonist is a kind of objectionable impurities, if contain such material in animal-feed, can directly have influence on the quality of animal meat product, thereby has influence on human life and health.Intravital some acceptor of animal can combine with beta-receptor agonist specificity, and receptor protein has been proved to be the regulatory factor of a stress response simultaneously, can start Expression of Related Genes to reach the effect that promotes growth of animal.
Under extraneous environmental stimulus, the variation of physio-biochemical characteristics can take place in the animal body.Animal experienced the variation of external environment before this by number of ways, and extracellular signal transferred to cell interior, through a series of phosphorylation level chain reaction signal being passed to some then transcribes or regulatory factor, transcribe or regulatory factor again by its specific function albumen and beta-receptor agonist specificity binding interactions, the expression that starts the goal gene that generation is replied to environment improves the stress reaction ability of animal.
Livestock product are as the important source of human edible material from soybeans, and supervision detects the objectionable constituent in the animal-feed, guarantee that the quality of product is one of vital task of feed testing person.
Summary of the invention
The purpose of this invention is to provide a kind of can with the receptor protein and the encoding gene thereof of beta-receptor stimulant medicine specific combination.
Provided by the present inventionly can derive from hamster (Golden Hamster), called after Gh β AR with the encoding gene of beta-receptor stimulant medicine specific combination receptor protein.
Can with the encoding gene of beta-receptor stimulant medicine specific combination receptor protein, it is one of following nucleotide sequences:
1) the SEQ ID № in the sequence table: 1;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.
The dna sequence dna of sequence 1 is by 792 based compositions in the sequence table, and the reading frame of this gene is from 5 ' end the 1st to the 792nd bit base.
Can with the receptor protein of beta-receptor stimulant medicine specific combination, it is SEQ ID № in the sequence table: 2 amino acid residue sequence or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 is identical active by SEQ ID №: 2 deutero-protein.
The protein that sequence 2 amino acid residue sequences are made up of 254 amino-acid residues in the sequence table.
Contain expression carrier of the present invention and clone and all belong to protection scope of the present invention, utilize existing molecular biological method can obtain different expression vectors, as pGEX-4T-1 β AR (collection of illustrative plates as shown in Figure 1).
Receptor protein of the present invention can combine with beta-receptor stimulant medicine specificity, detecting the beta-receptor stimulant medicine, particularly detect in the beta-receptor stimulant medicine in animal-feed and the livestock product and will be used widely, have important theory and practice significance.
Description of drawings
Fig. 1 is the gene mapping of Gh β AR amalgamation and expression genophore.
Fig. 2 is the gel electrophoresis figure of different treatment.
Embodiment
Clone and the sequential structure analysis of embodiment 1, hamster Gh β AR cDNA
Be collected in after the hamster lung channel is cleaned in the gold of growing 5 months and be ground to fine degree in the liquid nitrogen, place the 4mol/L guanidinium isothiocyanate, use acid phenol/chloroform extracting mixture again, get supernatant liquor,, repeat above-mentioned steps more once with the total RNA of isopropanol precipitating, RNA is dissolved in DEPC water, be stored in-80 ℃ (Davis etc. compile 1994, molecular biological basic skills: [Basic Methods in Molecular Biology], pp.777).Get the total RNA of 1 μ g and carry out separately an independently step reverse transcription chain type amplified reaction (RT-PCR) (Takara kit DR2409A) by a pair of specificity degenerated primers.A pair of forward primer (Forward primers): MaARF:5 '-CAAAGAATTCATGGGGCCACCCGGGAACGAC-3 ' and the reverse primer of holding corresponding to C-(Reverse Primer): MaARR1:5 '-GCAACTCGAGGAACTTGGAGGACCTTCGGAG-3 ' corresponding to the conservative territory N-end of hamster beta-receptor albumen in the gold, the PCR condition is: 4 ℃, and 3 minutes; 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1.5 minutes-30 the circulation; 72 ℃ 10 minutes; 4 ℃ of preservations.The PCR product is connected to and is used for order-checking on the pMD18-T carrier.The result shows that this gene and external packaging sequence are in full accord, and cDNA function fragment length is 792bp, called after Gh β AR, the nucleotide sequence with sequence 1 in the sequence table.
264 amino acid of this gene fragment coding, the amino acid residue sequence with sequence 2 in the sequence table.This protein contains the conservative territory of a hydrophobic amino acid structure, and this conserved regions has core sequence and the binding mechanism with beta-receptor agonist specific combination, can judge the existence of purpose compound thus.
The structure of embodiment 2, amalgamation and expression gene pGEX-4T-1 β AR
Utilize hamster lung flesh tissue in the gold, extract total RNA, and according to a pair of Auele Specific Primer of sequence 1 design, by RT-PCR amplification obtain a length be 792bp comprise the conservative territory of hydrophobic structure cDNA fragment, then by EcoRI and this gene fragment of XhoI digestion with restriction enzyme, be melted in water after purified and be used for connecting.Get 1 μ gcDNA fragment and cut postdigestive pGEX-4T-1 carrier, utilize dna ligase, under 16 ℃ of temperature, connect more than 12 hours through EcoRI and XhoI enzyme.Connect after product and transform,, extract plasmid, carry out enzyme again and cut and check order and determine the size and the constitutional features of this gene fragment through 37 ℃ of cultivations in intestinal bacteria.PGEX-4T-1 β AR (as shown in Figure 1) after the empirical tests transforms in intestinal bacteria, in order to purified fusion protein.
The above-mentioned fusion expression vector that builds is transformed behind intestinal bacteria, cultivate down at 37 ℃, induce in intestinal bacteria through IPTG, SDS-PAGE detects and shows that this gene can carry out external accurate translation in intestinal bacteria.And do not have protein band to occur without the inductive bacterial strain, show this amalgamation and expression gene can purifying after as the existence that detects feed and hold beta-receptor agonist in the product whether, the result as shown in Figure 2, wherein the M road is the standard protein molecular weight, 1 road is not derivative intestinal bacteria, 2 roads are through inductive empty carrier (GST), and 3 is without the inductive fusion vector, and 4 is through inductive fusion vector (expressing the fusion molecule amount is 54.8kD).
The vivoexpression product ligand affinity of the pGEX-4T-1 β AR gene of embodiment 4, amalgamation and expression in a big bacillus identified
The above-mentioned fusion expression vector that builds is transformed behind intestinal bacteria, cultivate down at 37 ℃.Induce in intestinal bacteria through IPTG, make this gene can in intestinal bacteria, carry out external accurate translation.Its product after protein mentions that slightly affinity column separate to be purified, with
3The Clenbuterol hydrochloride of H mark (typical β
2-receptor stimulant, CBL, clenbuterol hydrochloride) check and part bonded avidity, and compare with unaltered Ma β AR.Unit volume Gh β AR bonded
3H CBL mmole number is 142% of Ma β AR, and the time that reaches maximum combined concentration, the former was the latter's 51%.
Sequence table
<160>2
<210>1
<211>792
<212>DNA
＜213〉hamster (Mesocricetus auratus) in the Cricetus gold in
<400>1
atggggccac?ccgggaacga?cagtgacttc?ttgctgacaa?ccaacggaag?ccatgtgcca 60
gaccacgatg?tcactgagga?acgggacgaa?gcatgggtgg?taggcatggc?catccttatg 120
tcggttatcg?tcctggccat?cgtgtttggc?aacgtgctgg?tcatcacagc?cattgccaag 180
ttcgagaggc?tacagactgt?caccaactac?ttcataacct?ccttggcgtg?tgctgatcta 240
gtcatgggcc?tagcggtggt?gccgtttggg?gccagtcaca?tccttatgaa?aatgtggaat 300
tttggcaact?tctggtgcga?gttctggact?tccattgatg?tgttatgcgt?cacagccagc 360
attgagaccc?tgtgcgtgat?agcagtggat?cgctacattg?ctatcacatc?gccattcaag 420
taccagagcc?tgctgaccaa?gaataaggcc?cgaatggtca?tcctaatggt?gtggattgta 480
tccggcctta?cctccttctt?gcccattcag?atgcactggt?accgtgccac?ccaccagaaa 540
gccatcgact?gctatcacaa?ggagacttgc?tgcgacttct?tcacgaacca?ggcctacgcc 600
attgcttcct?ccattgtatc?tttctacgtg?cctctagtgg?tcatggtctt?tgtctattcc 660
agggtcttcc?aggtggccaa?aaggcagctc?cagaagatag?acaaatctga?gggaagattc 720
cactccccaa?acctcggcca?ggtggagcag?gatgggcgga?gtgggcacgg?actccgaagg 780
tcctccaagt?tc 792
<210>2
<211>264
<212>PRT
＜213〉hamster (Mesocricetus auratus) in the Cricetus gold in
<400>2
Met?Gly?Pro?Pro?Gly?Asn?Asp?Ser?Asp?Phe?Leu?Leu?Thr?Thr?Asn
1 5 10 15
Gly?Ser?His?Val?Pro?Asp?His?Asp?Val?Thr?Glu?Glu?Arg?Asp?Glu
20 25 30
Ala?Trp?Val?Val?Gly?Met?Ala?Ile?Leu?Met?Ser?Val?Ile?Val?Leu
35 40 45
Ala?Ile?Val?Phe?Gly?Asn?Val?Leu?Val?Ile?Thr?Ala?Ile?Ala?Lys
50 55 60
Phe?Glu?Arg?Leu?Gln?Thr?Val?Thr?Asn?Tyr?Phe?Ile?Thr?Ser?Leu
65 70 75
Ala?Cys?Ala?Asp?Leu?Val?Met?Gly?Leu?Ala?Val?Val?Pro?Phe?Gly
80 85 90
Ala?Ser?His?Ile?Leu?Met?Lys?Met?Trp?Asn?Phe?Gly?Asn?Phe?Trp
95 100 105
Cys?Glu?Phe?Trp?Thr?Ser?Ile?Asp?Val?Leu?Cys?Val?Thr?Ala?Ser
110 115 120
Ile?Glu?Thr?Leu?Cys?Val?Ile?Ala?Val?Asp?Arg?Tyr?Ile?Ala?Ile
125 130 135
Thr?Ser?Pro?Phe?Lys?Tyr?Gln?Ser?Leu?Leu?Thr?Lys?Asn?Lys?Ala
140 145 150
Arg?Met?Val?Ile?Leu?Met?Val?Trp?Ile?Val?Ser?Gly?Leu?Thr?Ser
155 160 165
Phe?Leu?Pro?Ile?Gln?Met?His?Trp?Tyr?Arg?Ala?Thr?His?Gln?Lys
170 175 180
Ala?Ile?Asp?Cys?Tyr?His?Lys?Glu?Thr?Cys?Cys?Asp?Phe?Phe?Thr
185 190 195
Asn?Gln?Ala?Tyr?Ala?Ile?Ala?Ser?Ser?Ile?Val?Ser?Phe?Tyr?Val
200 205 210
Pro?Leu?Val?Val?Met?Val?Phe?Val?Tyr?Ser?Arg?Val?Phe?Gln?Val
215 220 225
Ala?Lys?Arg?Gln?Leu?Gln?Lys?Ile?Asp?Lys?Ser?Glu?Gly?Arg?Phe
230 235 240
His?Ser?Pro?Asn?Leu?Gly?Gln?Val?Glu?Gln?Asp?Gly?Arg?Ser?Gly
245 250 255
His?Gly?Leu?Arg?Arg?Ser?Ser?Lys?Phe
260 264
Claims (9)
1, can with the encoding gene of beta-receptor stimulant medicine specific combination receptor protein, it is one of following nucleotide sequences:
1) the SEQ ID № in the sequence table: 1;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.
2, according to the described gene of claim 1, it is characterized in that: the SEQ ID № in the sequence table of described gene tool: 1.
3, can with the receptor protein of beta-receptor stimulant medicine specific combination, it is to have SEQ ID № in the sequence table: 2 amino acid residue sequence or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 is identical active by SEQ ID №: 2 deutero-protein.
4, according to the described albumen of claim 3, it is characterized in that: it has SEQ ID № in the sequence table: 2 amino acid residue sequence.
5, contain claim 1 or 2 described expression carrier.
6, expression vector according to claim 5 is characterized in that: described carrier is pGEX-4T-1 β AR.
7, the transgenic cell line that contains claim 1 or 2 described genes.
8, claim 3 or the 4 described receptor proteins application in detecting the beta-receptor stimulant medicine.
9, application according to claim 8 is characterized in that: described application is meant receptor protein application in the beta-receptor stimulant medicine in detecting animal-feed and livestock product.
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|---|---|---|---|
| CN 02153850 CN1238505C (en) | 2002-12-05 | 2002-12-05 | Cyclic amp receptor protein specifically combined with beta-acceptor excitomotor group pharmaceutical, coding gene and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02153850 CN1238505C (en) | 2002-12-05 | 2002-12-05 | Cyclic amp receptor protein specifically combined with beta-acceptor excitomotor group pharmaceutical, coding gene and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1504570A CN1504570A (en) | 2004-06-16 |
| CN1238505C true CN1238505C (en) | 2006-01-25 |
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| CN 02153850 Expired - Fee Related CN1238505C (en) | 2002-12-05 | 2002-12-05 | Cyclic amp receptor protein specifically combined with beta-acceptor excitomotor group pharmaceutical, coding gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2005176988A (en) | 2003-12-17 | 2005-07-07 | Ge Medical Systems Global Technology Co Llc | Data correcting method, and x-ray ct device |
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Granted publication date: 20060125 Termination date: 20121205 |