CN109609535A - A kind of prokaryotic expression carrier of E. coli-galactosidase receptor - Google Patents
A kind of prokaryotic expression carrier of E. coli-galactosidase receptor Download PDFInfo
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- G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
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Abstract
This application involves a kind of prokaryotic expression carriers of E. coli-galactosidase receptor.This application provides beta galactosidase receptors shown in a kind of nucleotide of encoding ss-galactosidase receptor shown in SEQ ID NO:1 and SEQ ID NO:2.According to E. coli-galactosidase structure and function and the complementarity principle of enzyme donor and enzyme acceptor, the enzyme acceptor for deleting the 13rd to 33 amino acids is devised；The coding nucleotide of enzyme acceptor is connect with carrier, and converts to Escherichia coli and constructs recombinant expression host cell；Pass through the great expression enzyme acceptor that ferments；Enzyme acceptor is purified by affinity chromatography technology, the enzyme acceptor purity after desalination is 90% or more.Compared with conventional enzyme acceptor, the enzyme acceptor yield of the application is higher, complementary activity is stronger, can be applied to the development of CEDIA biochemical diagnosis reagent.
Description
The application is application No. is 201610841474.8, and the applying date is on September 22nd, 2016, entitled " a kind of
The divisional application of the Chinese patent application of E. coli-galactosidase receptor and preparation method thereof ".
Technical field
This application involves field of biotechnology, relate more specifically to cloned enzyme donor immuno analytical method.The application provides
A kind of enzyme acceptor of E. coli-galactosidase, code nucleic acid, expression vector, host cell and preparation method.
Background technique
E. coli-galactosidase (GenBank:EDU66561.1) can pass through hydrogen bromide CNBr2Cracking or base
Because recombinant technique obtains two peptide fragments: the large fragment of the missing a small amount of amino acid of N-terminal, referred to as enzyme acceptor (Enzyme acceptor,
EA): lacking the small fragment of most of C-terminal amino acid, referred to as enzyme donor (Enzyme donor, ED).The two individualism does not have
Enzymatic activity can polymerize both when existing simultaneously, and form the holoenzyme with enzymatic activity, this phenomenon is defined as α-complementary.
This characteristic of E. coli-galactosidase has been applied to molecular biology and cloned enzyme donor immunoassay
In technology (Cloned Enzyme Donor Immunoassay, CEDIA).CEDIA technology can be examined in clinical biochemical diagnosis
Survey small molecule compound.
CEDIA is a kind of immunoassay technology, combines the special of beta galactosidase α complementarity principle and antigen-antibody
Property binding property.Small molecule antigens (Ag) and ED are coupled, form ED-Ag conjugate, this conjugate can be with the spontaneous combination of EA, shape
At active beta galactosidase.But if the antibody (Ab) for being directed to the small molecule antigens is added in the reaction system, resist
The specific binding of original antibody will form ED-Ag-Ab compound.At this point, due to the influence of antibody steric hindrance, ED-Ag-Ab is multiple
Active beta galactosidase cannot be formed in conjunction with EA again by closing object.If containing small molecule antigens to be detected in sample,
At this time containing there are two types of the antigens of form in reaction system: one is the free Ag in sample；One is the ED-Ag in kit.
Both antigens all have the ability in conjunction with antibody specificity.Obviously, the antigen of both forms can emulative combination it is anti-
Answer the Ab in system, the Ag that dissociates in sample is more, and the Ab combined is more, so as to cause there are more residue ED-Ag to be coupled
Object (part not in conjunction with antibody) can form active beta galactosidase, and the height of enzymatic activity with the spontaneous combination of EA
It is directly proportional to the content of free antigen in sample.
E. coli-galactosidase EA can be applied to the development of CEDIA technology clinical biochemical diagnostic reagent, be a kind of
Important dhdps enzyme has larger application value.
There are many EA the EA mutant being genetically engineered reported at present, for example, in Langley KE and
(Biochemistry.1976Nov 2 in the paper that Zabin I is delivered；15 (22): 4866-75) refer to a kind of β galactoside
Enzyme is compared with wild type EA, the amino acid residue (referred to as M15) of missing the 11st to 41.
For another example, the M112 of 23 to 31 amino acids is lacked；Lack the EA5 of 35 to 52 amino acids；Missing 30 to 37
The EA14 of amino acid；Lack the EA11 of 35 to 54 amino acids；Lack the EA17 of 21 to 53 amino acids；Lack 13 to 45 ammonia
The EA18 of base acid；Lack the EA20 of 26 to 45 amino acids；Lack the EA22 of 13 to 40 amino acids；Lack 16 to 35 bit aminos
The EA23 of acid；Lack 22 to 35 amino acids EA24 (Daniel R.Henderson, Clinical Chemistry,
Vol.32,No.9,1986,1637-1641).The domestic β-for reporting while lacking 1 to 5 and 14 to 40 amino acids there are also scholar
Galactosidase EA (the expression of Escherichia coli beta galactosidase EA, ED fusion protein；Chinese microbiology and immunology are miscellaneous
Will, 2003,23, No 2:113-114).
Table 1. it has been reported that EA mutant summarize
However, enzyme acceptor in the prior art still has the detection higher phenomenon of background.In consideration of it, this field needs one
Kind novel beta galactosidase, with higher yield, lower background, and the complementation between enzyme acceptor and enzyme donor
Activity is stronger.
Summary of the invention
According to some embodiment there is provided a kind of beta galactosidase receptor, amino acid sequence is SEQ ID No.2.
It is compared according to the beta galactosidase receptor of the application with wild type, lacks the 13rd to 33 amino acids residue.
The present inventor is former according to the biological structure and function and EA of E. coli-galactosidase and the complementation of ED
Reason, fixed point delete the 13rd to 33 amino acids of beta galactosidase, obtain the beta galactosidase receptor of the application.Inventor goes out
Expect ground discovery, by the recombination beta galactosidase receptor that this genetic engineering is transformed, there is low-down detection background,
Be conducive to expression and the raising of complementary activity simultaneously.
According to some embodiment there is provided a kind of polynucleotides, the beta galactosidase receptor of the application is encoded.?
In some embodiments, polynucleotides are DNA；In other embodiments, polynucleotides are RNA.In specific embodiment party
In formula, polynucleotides are DNA.In a particular embodiment, the nucleotide sequence of polynucleotides is SEQ ID No.1, SEQ
The complementary series of ID No.1.
According to some embodiment there is provided a kind of expression vector, the beta galactosidase receptor of the application is expressed.?
In some embodiments, the expression vector of the application includes above-mentioned polynucleotides.In some embodiments, the expression of the application
Carrier includes polynucleotides shown in SEQ ID No.1.In a particular embodiment, multicore glycosides shown in SEQ ID No.1
Acid is operably coupled in expression vector.In some embodiments, expression vector is prokaryotic expression carrier.In some implementations
In mode, expression vector is selected from pET41a, pET28a, pET20a and combinations thereof.In a particular embodiment, expression vector is
pET41a。
Embodiment there is provided a kind of host cells according to some, and it includes the expression vectors of the application.In some implementations
In mode, the host cell of the application is prokaryotic host cell.In a particular embodiment, the prokaryotic host cell is big
Enterobacteria.In a particular embodiment, Escherichia coli be selected from Rosetta-gami2 (DE3) pLysS, BL21, Rosetta and
A combination thereof.
Technical staff knows, other than expressing target product using host cell, can also use cell-free expression
The beta galactosidase receptor of system expression the application.
Embodiment there is provided a kind of reagents according to some, and it includes the beta galactosidase receptors of the application.Some
In embodiment, the reagent is cloned enzyme donor immunological assay reagents.In some embodiments, the reagent includes one
A or two or three or more autonomous containers.It in some embodiments, in a vessel include the β of the application
Galactoside enzyme acceptor；It include beta galactosidase donor in another container.In other embodiments, β galactoside
Enzyme donor can be free；Or beta galactosidase donor can be coupled antigen, such as the lesser antigen of molecular weight or
Person's haptens.In some embodiments, antigen is selected from: peptide, polysaccharide, lipid, nucleic acid, drug.In some embodiments, originally
The beta galactosidase receptor and beta galactosidase donor of application are in different containers.In some embodiments, β galactolipin
Glycosides enzyme donor can be beta galactosidase donor any known in the art or following.
According to some purposes embodiment there is provided any one selected from the following or combinations thereof in reagent preparation: this
The beta galactosidase receptor of application, the polynucleotides of the application, the expression vector of the application, the application host cell.One
In a little embodiments, the reagent is cloned enzyme donor immunological assay reagents.
Embodiment there is provided a kind of beta galactosidases according to some, and it includes beta galactosidase donor and this Shens
Beta galactosidase receptor please.In some embodiments, the beta galactosidase receptor of beta galactosidase donor and the application
Interaction, forms active beta galactosidase.In some embodiments, the β of beta galactosidase donor and the application
Galactoside enzyme acceptor Non-covalent binding.In some embodiments, beta galactosidase donor can also be coupled antigen, example
Such as the lesser antigen of molecular weight or haptens.In some embodiments, antigen is selected from: peptide, polysaccharide, lipid, nucleic acid, medicine
Object.In some embodiments, beta galactosidase donor can be β galactolipin any known in the art or following
Glycosides enzyme donor.
Embodiment there is provided a kind of methods for preparing beta galactosidase receptor according to some, comprising steps of
1) under conditions of allowing beta galactosidase receptor to be expressed, the host cell of the application is cultivated；With
2) it purifies and collects the beta galactosidase receptor.
In some embodiments, the culture is carried out by fermentation.In some embodiments, fermentation is according to choosing
From mode below: batch fermentation continuously ferments, fed-batch fermentation and combinations thereof.In some embodiments, the culture
It carries out in a reservoir.In a particular embodiment, the container is selected from: shaking flask, culture dish and fermentor.
In some embodiments, it is trained in 28 to 40 DEG C, 30 to 40 DEG C, 35 to 38 DEG C, preferably 37 DEG C of temperature
It supports.
In some embodiments, under aerobic conditions, it is cultivated.In a particular embodiment, it is greater than in dissolved oxygen
It is carried out under conditions of 40%.
In some embodiments, it is cultivated under conditions of pH value is 6.8 to 7.2.
In some embodiments, the expression is constitutive expression or inducible expression；This depends on expression vector
Type.
In some embodiments, induction is further included the steps that between step 1) and step 2).In specific embodiment party
In formula, inducer is added into the culture of step 1) to induce the expression of beta galactosidase receptor.In specific embodiment party
In formula, inducer is IPTG.
In some embodiments, the purifying refers to affinity purification.The method of purifying depends on the type of expression vector.
Such as, but not limited to, when in expression vector including His label, using Ni-NTA affinity chromatography technology.
In a particular embodiment, a kind of method for preparing beta galactosidase receptor is provided, comprising steps of
1) 1.5 × 10 are pressed8The host cell of the application is inoculated in antibiotic first by the density of/L in the fermenter
In beginning culture medium；
2) host cell is cultivated in 37 DEG C, until the OD of host cell600Up to 3 to 5；
3) temperature of culture is reduced to 25 DEG C, the first feed supplement is added, IPTG to final concentration of 1mM is added later；
4) it is added the second feed-batch culture 8 to 12 hours by the speed of 10ml/h/L；
5) culture is collected；
6) pass through Ni-NTA affinitive layer purification and collect beta galactosidase receptor；
7) desalination optionally, is carried out to beta galactosidase receptor.
In a particular embodiment, antibiotic initial medium includes: 10g/L tryptone, 5g/L yeast mention
Take object, 0.5 to 2g/L magnesium sulfate, 7 to 10g/L dipotassium hydrogen phosphate, 3 to 6g/L sodium dihydrogen phosphate, 10ml/L microelement mother liquor,
50mg/L kanamycins, 20% to 25% glucose.
In a particular embodiment, the first feed supplement includes 100 ± 20g/L tryptone, the extraction of 250 ± 50g/L yeast
Object, 10 ± 2g/L lactose.
In a particular embodiment, the second feed supplement includes 400 ± 50g/L glycerol.
Detailed description of the invention
Fig. 1: the expression of beta galactosidase receptor.Left road: molecular weight of albumen Marker；Right road: Escherichia coli β galactolipin
The band of glycosides enzyme acceptor.
Specific embodiment
The building of the expression vector of 1. E. coli-galactosidase EA of embodiment
According to the E. coli-galactosidase nucleotide sequence that the website NCBI is announced, wild type EA gene order is determined；
It deletes the 13rd to 33 amino acids residue and obtains sequence shown in SEQ ID No.2；It is public that Radix Polygalae biotechnology is won in commission Beijing three
Department carries out full genome synthesis, and adds two restriction enzyme sites (such as Nco I, Xho I) respectively at sequence both ends, by synthesis
Gene order is inserted on plasmid；
Above-mentioned plasmid is subjected to double digestion, electrophoresis recycles E. coli-galactosidase EA genetic fragment；Vector plasmid
PET41a equally carries out double digestion processing, the plasmid after Purified in electrophoresis digestion；
PET41a plasmid and E. coli-galactosidase EA gene after digestion, obtain after connecting under the conditions of 16 DEG C
Expression plasmid, conversion to bacillus coli DH 5 alpha, the culture medium flat plate for being coated with Kan resistance are screened；Screen positive gram obtained
Confirmation is sequenced after grand extraction expression plasmid.
The acquisition of 2. expression host cell of embodiment
The resulting expression plasmid of embodiment 1 is transformed into host cell E. coli Rosetta-gami2 (DE3) pLysS,
Obtain expression host cell.
Embodiment 3. prepares beta galactosidase EA
The resulting expression host cell of embodiment 2 is inoculated in seed culture medium and (weighs 10g tryptone, 5g yeast mentions
Object, 10g NaCl is taken to be dissolved in 900ml deionized water, 10M NaOH adjusts pH to 7.0, and it adds deionized water and is settled to 1L, 121
DEG C high pressure sterilization 20min, it is cooling after room temperature preservation, kanamycins is added to its final concentration of 50mg/L), 37 DEG C of cultures in when use
To OD600Reach 3 to 5；
By 1.5 × 108Host cell is inoculated in fermentation initial medium (tryptone 10g, yeast extraction by the density of/L
Object 5g, magnesium sulfate 0.5 to 2g, dipotassium hydrogen phosphate 7 to 10g, sodium dihydrogen phosphate 3 to 6g, 10ml microelement mother liquor (10g
FeSO4·7H2O、0.1g NH46Mo7O24、0.5g MnSO4·5H2O、2.25g ZnSO4·7H2O、0.23g Na2B4O7·
10H2O、1g CuSO4·7H2O、2g CaCl2Be dissolved in 5M hydrochloric acid), be added 900ml deionized water dissolving after, adjust pH to 6.8 to
7.2, it adds after deionized water is settled to 1L, high pressure sterilization 20min is used after cooling.It is dense to end that kanamycins is added when use
Degree is 50mg/L, and the glucose of sterilizing is added to final concentration of 20% to 25%), ventilatory capacity is adjusted, in dissolved oxygen greater than 40%
Under the conditions of make its growth, and adjust the pH value of culture medium 6.8 to 7.2 by the way that acid or alkali is added dropwise；
It gradually exhausts to the ingredient in initial medium, when the oxygen dissolving value of culture medium is down to 40%, cultivation temperature is down to
25 DEG C, while feed supplement 1 (100 ± 20g/L tryptone, 250 ± 50g/L yeast extract, 10 are added with the ratio of 100ml/L
± 2g/L lactose, room temperature preservation after 121 DEG C of high-temperature sterilization 20min).After the completion of feed supplement 1 is added, IPTG is added to final concentration of
1mM starts to induce；
And (400 ± 50g/L glycerol, room temperature is protected after 121 DEG C of high-temperature sterilization 20min by the speed of 10ml/h/L addition feed supplement 2
Deposit), while the pH value of culture medium is adjusted 6.8 to 7.2 with ammonium hydroxide or sulfuric acid；After inducing about 10h under 25 DEG C of cryogenic conditions, fermentation
Culture terminates；
Resulting cell is collected by centrifugation with tube centrifuge, high pressure homogenizer is cooled to 4 DEG C of temperature or less in advance；
Cell is resuspended, with high pressure homogenizer 850bar high-pressure homogeneous 3 times, makes clasmatosis, discharges Escherichia coli β galactolipin
Glycosides enzyme EA；
Cell pyrolysis liquid is centrifuged 30min, 0.22 μm of membrane filtration of gained supernatant in 4 DEG C of 18000rpm；
Filtered supernatant is loaded onto Ni affinity column；It removes not in conjunction with column or the lower impurity of binding force；Elute mesh
Albumen Escherichia coli beta galactosidase EA, be collected simultaneously protein peak；
It is loaded onto desalting column and carries out desalination, collect protein peak.
Test case
The measurement of test case 1. purity of protein and yield
By 3 gained albumen of embodiment with PAGE gel electroresis appraisal purity of protein (Fig. 1), and egg is measured with BCA method
White concentration calculates EA yield.EA purity made from the application is 90%.
2. yield of table
2. Activity determination of test case
The resulting E. coli-galactosidase EA of embodiment 3 is subjected to Activity determination.
E. coli-galactosidase EA can form beta galactosidase with ED complementation.Beta galactosidase can will be adjacent
Nitrobenzene β-D- synthesis (ONPG) hydrolysis, generates the o-nitrophenol (ONP) of galactolipin and yellow.Measure ONP's
Amount can determine the enzyme activity of galactosidase, so that it is determined that the complementary activity of EA.
(1) EA is diluted to 0.2mg/ml with PB buffer (100mM PB, 150mM NaCl, 0.1%NaN3, pH 7.3),
ED is diluted to 1 μ g/ml with PB buffer.Complementary activity is measured with Siemens's Biochemical Analyzer 1800:
100 μ l EA solution are as R1；
100 μ l ONPG substrate solution (5mg/ml ONPG, 100mM PB, 150mM NaCl, 10mM MgCl2,0.1%
NaN3, pH7.3) it is used as R2；
10 μ l enzyme donor solution are as sample S, for measuring the complementary activity of EA.
10 μ l PB buffers are as blank control sample, for measuring the background activity of EA；
EA-ED program is run, determines reaction rate,
(2) it is control with M15, carries out aforesaid operations in parallel.
(3) result:
The EA complementary activity ratio M15 high 11.2% of the application, and background activity low 21%.
3. complementary activity of table
| EA | OD571 | % |
| M15 | 0.21874 | 100 |
| The EA of the application | 0.24332 | 111.2% |
4. background activity of table
| EA | OD571 | % |
| M15 | 0.00167 | 100 |
| The EA of the application | 0.00132 | 79 |
Sequence table
<110>Beijing Strong Biotechnologies, Inc.
<120>a kind of prokaryotic expression carrier of E. coli-galactosidase receptor
<130> 390002CG-360019
<150> 201610841474.8
<151> 2016-09-22
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 3006
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<213> Escherichia coli
<400> 1
accatgatta cggattcact ggccgtcgtt ttacaagcca gctggcgtaa tagcgaagag 60
gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgctttgcc 120
tggtttccgg caccagaagc ggtgccggaa agctggctgg agtgcgatct tcctgaggcc 180
gatactgtcg tcgtcccctc aaactggcag atgcacggtt acgatgcgcc catctacacc 240
aacgtgacct atcccattac ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt 300
tgttactcgc tcacatttaa tgttgatgaa agctggctac aggaaggcca gacgcgaatt 360
atttttgatg gcgttaactc ggcgtttcat ctgtggtgca acgggcgctg ggtcggttac 420
ggccaggaca gtcgtttgcc gtctgaattt gacctgagcg catttttacg cgccggagaa 480
aaccgcctcg cggtgatggt gctgcgctgg agtgacggca gttatctgga agatcaggat 540
atgtggcgga tgagcggcat tttccgtgac gtctcgttgc tgcataaacc gactacacaa 600
atcagcgatt tccatgttgc cactcgcttt aatgatgatt tcagccgcgc tgtactggag 660
gctgaagttc agatgtgcgg cgagttgcgt gactacctac gggtaacagt ttctttatgg 720
cagggtgaaa cgcaggtcgc cagcggcacc gcgcctttcg gcggtgaaat tatcgatgag 780
cgtggtggtt atgccgatcg cgtcacacta cgtctgaacg tcgaaaaccc gaaactgtgg 840
agcgccgaaa tcccgaatct ctatcgtgcg gtggttgaac tgcacaccgc cgacggcacg 900
ctgattgaag cagaagcctg cgatgtcggt ttccgcgagg tgcggattga aaatggtctg 960
ctgctgctga acggcaagcc gttgctgatt cgaggcgtta accgtcacga gcatcatcct 1020
ctgcatggtc aggtcatgga tgagcagacg atggtgcagg atatcctgct gatgaagcag 1080
aacaacttta acgccgtgcg ctgttcgcat tatccgaacc atccgctgtg gtacacgctg 1140
tgcgaccgct acggcctgta tgtggtggat gaagccaata ttgaaaccca cggcatggtg 1200
ccaatgaatc gtctgaccga tgatccgcgc tggctaccgg cgatgagcga acgcgtaacg 1260
cgaatggtgc agcgcgatcg taatcacccg agtgtgatca tctggtcgct ggggaatgaa 1320
tcaggccacg gcgctaatca cgacgcgctg tatcgctgga tcaaatctgt cgatccttcc 1380
cgcccggtgc agtatgaagg cggcggagcc gacaccacgg ccaccgatat tatttgcccg 1440
atgtacgcgc gcgtggatga agaccagccc ttcccggctg tgccgaaatg gtccatcaaa 1500
aaatggcttt cgctacctgg agagacgcgc ccgctgatcc tttgcgaata cgcccacgcg 1560
atgggtaaca gtcttggcgg tttcgctaaa tactggcagg cgtttcgtca gtatccccgt 1620
ttacagggcg gcttcgtctg ggactgggtg gatcagtcgc tgattaaata tgatgaaaac 1680
ggcaacccgt ggtcggctta cggcggtgat tttggcgata cgccgaacga tcgccagttc 1740
tgtatgaacg gtctggtctt tgccgaccgc acgccgcatc cagcgctgac ggaagcaaaa 1800
caccagcagc agtttttcca gttccgttta tccgggcaaa ccatcgaagt gaccagcgaa 1860
tacctgttcc gtcatagcga taacgagctc ctgcactgga tggtggcgct ggatggtaag 1920
ccgctggcaa gcggtgaagt gcctctggat gtcgctccac aaggtaaaca gttgattgaa 1980
ctgcctgaac taccgcagcc ggagagcgcc gggcaactct ggctcacagt acgcgtagtg 2040
caaccgaacg cgaccgcatg gtcagaagcc ggacacatca gcgcctggca gcagtggcgt 2100
ctggctgaaa acctcagcgt gacactcccc gccgcgtccc acgccatccc gcatctgacc 2160
accagcgaaa tggatttttg catcgagctg ggtaataagc gttggcaatt taaccgccag 2220
tcaggctttc tttcacagat gtggattggc gataaaaaac aactgctgac gccgctgcgc 2280
gatcagttca cccgtgcacc gctggataac gacattggcg taagtgaagc gacccgcatt 2340
gaccctaacg cctgggtcga acgctggaag gcggcgggcc attaccaggc cgaagcagcg 2400
ttgttgcagt gcacggcaga tacacttgct gatgcggtgc tgattacgac cgctcacgcg 2460
tggcagcatc aggggaaaac cttatttatc agccggaaaa cctaccggat tgatggtagt 2520
ggtcaaatgg cgattaccgt tgatgttgaa gtggcgagcg atacaccgca tccggcgcgg 2580
attggcctga actgccagct ggcgcaggta gcagagcggg taaactggct cggattaggg 2640
ccgcaagaaa actatcccga ccgccttact gccgcctgtt ttgaccgctg ggatctgcca 2700
ttgtcagaca tgtatacccc gtacgtcttc ccgagcgaaa acggtctgcg ctgcgggacg 2760
cgcgaattga attatggccc acaccagtgg cgcggcgact tccagttcaa catcagccgc 2820
tacagtcaac agcaactgat ggaaaccagc catcgccatc tgctgcacgc ggaagaaggc 2880
acatggctga atatcgacgg tttccatatg gggattggtg gcgacgactc ctggagcccg 2940
tcagtatcgg cggaattcca gctgagcgcc ggtcgctacc attaccagtt ggtctggtgt 3000
caaaaa 3006
<210> 2
<211> 1002
<212> PRT
<213> Escherichia coli
<400> 2
Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln Ala Ser Trp Arg
1 5 10 15
Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg Ser
20 25 30
Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe Pro Ala Pro Glu Ala Val
35 40 45
Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro Glu Ala Asp Thr Val Val
50 55 60
Val Pro Ser Asn Trp Gln Met His Gly Tyr Asp Ala Pro Ile Tyr Thr
65 70 75 80
Asn Val Thr Tyr Pro Ile Thr Val Asn Pro Pro Phe Val Pro Thr Glu
85 90 95
Asn Pro Thr Gly Cys Tyr Ser Leu Thr Phe Asn Val Asp Glu Ser Trp
100 105 110
Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe Asp Gly Val Asn Ser Ala
115 120 125
Phe His Leu Trp Cys Asn Gly Arg Trp Val Gly Tyr Gly Gln Asp Ser
130 135 140
Arg Leu Pro Ser Glu Phe Asp Leu Ser Ala Phe Leu Arg Ala Gly Glu
145 150 155 160
Asn Arg Leu Ala Val Met Val Leu Arg Trp Ser Asp Gly Ser Tyr Leu
165 170 175
Glu Asp Gln Asp Met Trp Arg Met Ser Gly Ile Phe Arg Asp Val Ser
180 185 190
Leu Leu His Lys Pro Thr Thr Gln Ile Ser Asp Phe His Val Ala Thr
195 200 205
Arg Phe Asn Asp Asp Phe Ser Arg Ala Val Leu Glu Ala Glu Val Gln
210 215 220
Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg Val Thr Val Ser Leu Trp
225 230 235 240
Gln Gly Glu Thr Gln Val Ala Ser Gly Thr Ala Pro Phe Gly Gly Glu
245 250 255
Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu
260 265 270
Asn Val Glu Asn Pro Lys Leu Trp Ser Ala Glu Ile Pro Asn Leu Tyr
275 280 285
Arg Ala Val Val Glu Leu His Thr Ala Asp Gly Thr Leu Ile Glu Ala
290 295 300
Glu Ala Cys Asp Val Gly Phe Arg Glu Val Arg Ile Glu Asn Gly Leu
305 310 315 320
Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile Arg Gly Val Asn Arg His
325 330 335
Glu His His Pro Leu His Gly Gln Val Met Asp Glu Gln Thr Met Val
340 345 350
Gln Asp Ile Leu Leu Met Lys Gln Asn Asn Phe Asn Ala Val Arg Cys
355 360 365
Ser His Tyr Pro Asn His Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr
370 375 380
Gly Leu Tyr Val Val Asp Glu Ala Asn Ile Glu Thr His Gly Met Val
385 390 395 400
Pro Met Asn Arg Leu Thr Asp Asp Pro Arg Trp Leu Pro Ala Met Ser
405 410 415
Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg Asn His Pro Ser Val
420 425 430
Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His Gly Ala Asn His Asp
435 440 445
Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp Pro Ser Arg Pro Val Gln
450 455 460
Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala Thr Asp Ile Ile Cys Pro
465 470 475 480
Met Tyr Ala Arg Val Asp Glu Asp Gln Pro Phe Pro Ala Val Pro Lys
485 490 495
Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu
500 505 510
Ile Leu Cys Glu Tyr Ala His Ala Met Gly Asn Ser Leu Gly Gly Phe
515 520 525
Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr Pro Arg Leu Gln Gly Gly
530 535 540
Phe Val Trp Asp Trp Val Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn
545 550 555 560
Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe Gly Asp Thr Pro Asn
565 570 575
Asp Arg Gln Phe Cys Met Asn Gly Leu Val Phe Ala Asp Arg Thr Pro
580 585 590
His Pro Ala Leu Thr Glu Ala Lys His Gln Gln Gln Phe Phe Gln Phe
595 600 605
Arg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser Glu Tyr Leu Phe Arg
610 615 620
His Ser Asp Asn Glu Leu Leu His Trp Met Val Ala Leu Asp Gly Lys
625 630 635 640
Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val Ala Pro Gln Gly Lys
645 650 655
Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro Glu Ser Ala Gly Gln
660 665 670
Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn Ala Thr Ala Trp Ser
675 680 685
Glu Ala Gly His Ile Ser Ala Trp Gln Gln Trp Arg Leu Ala Glu Asn
690 695 700
Leu Ser Val Thr Leu Pro Ala Ala Ser His Ala Ile Pro His Leu Thr
705 710 715 720
Thr Ser Glu Met Asp Phe Cys Ile Glu Leu Gly Asn Lys Arg Trp Gln
725 730 735
Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln Met Trp Ile Gly Asp Lys
740 745 750
Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe Thr Arg Ala Pro Leu
755 760 765
Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg Ile Asp Pro Asn Ala
770 775 780
Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr Gln Ala Glu Ala Ala
785 790 795 800
Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp Ala Val Leu Ile Thr
805 810 815
Thr Ala His Ala Trp Gln His Gln Gly Lys Thr Leu Phe Ile Ser Arg
820 825 830
Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln Met Ala Ile Thr Val Asp
835 840 845
Val Glu Val Ala Ser Asp Thr Pro His Pro Ala Arg Ile Gly Leu Asn
850 855 860
Cys Gln Leu Ala Gln Val Ala Glu Arg Val Asn Trp Leu Gly Leu Gly
865 870 875 880
Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp Arg
885 890 895
Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro Ser
900 905 910
Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu Asn Tyr Gly Pro His
915 920 925
Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser Arg Tyr Ser Gln Gln
930 935 940
Gln Leu Met Glu Thr Ser His Arg His Leu Leu His Ala Glu Glu Gly
945 950 955 960
Thr Trp Leu Asn Ile Asp Gly Phe His Met Gly Ile Gly Gly Asp Asp
965 970 975
Ser Trp Ser Pro Ser Val Ser Ala Glu Phe Gln Leu Ser Ala Gly Arg
980 985 990
Tyr His Tyr Gln Leu Val Trp Cys Gln Lys
995 1000
Claims (2)
1. a kind of prokaryotic expression carrier, it includes the polynucleotides of encoding ss-galactosidase receptor；
The beta galactosidase receptor is shown in SEQ ID No.2.
2. a kind of prokaryotic expression carrier, it includes polynucleotides, the polynucleotides are shown in SEQ ID No.1 or SEQ ID
Shown in the complementary series of No.1；
Preferably, the prokaryotic expression carrier is selected from: pET41a, pET28a, pET20a and combinations thereof.
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| CN201610841474.8A CN106399278B (en) | 2016-09-22 | 2016-09-22 | A kind of E. coli-galactosidase receptor and preparation method thereof |
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| CN201910039393.XA Active CN109666619B (en) | 2016-09-22 | 2016-09-22 | Host cell for expressing escherichia coli beta galactosidase receptor |
| CN201910039744.7A Active CN109797143B (en) | 2016-09-22 | 2016-09-22 | Reagent containing escherichia coli beta galactosidase receptor |
| CN201910039492.8A Active CN109613251B (en) | 2016-09-22 | 2016-09-22 | Use of escherichia coli beta galactosidase receptor |
| CN201910039636.XA Active CN109609481B (en) | 2016-09-22 | 2016-09-22 | Preparation method of escherichia coli beta galactosidase receptor |
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| CN102662057A (en) * | 2012-06-07 | 2012-09-12 | 广州易航生物科技有限公司 | Cloned enzyme-donor immunoassay (CEDIA) ImmunoChip drug detecting kit |
| US20160054322A1 (en) * | 2014-08-21 | 2016-02-25 | Discoverx Corporation | Homogenous thermal shift ligand binding assay |
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| US5444161A (en) * | 1989-08-16 | 1995-08-22 | Microgenics Corporation | Substrates for β-galactosidase |
| US20060088886A1 (en) * | 2004-10-25 | 2006-04-27 | Anlong Ouyang | Topiramate analogs |
| AU2007225195B2 (en) * | 2006-03-13 | 2013-10-24 | The Board Of Trustees Of The Leland Stanford Junior University | Detection of molecular interactions using a reduced affinity enzyme complementation reporter system |
| JP2008139245A (en) * | 2006-12-05 | 2008-06-19 | Fujifilm Corp | Immunoassay method of specimen |
| CN105203542A (en) * | 2015-09-14 | 2015-12-30 | 绍兴圣康生物科技有限公司 | Reagent kit for determining 25-hydroxyvitamin D and application method thereof |
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| CN102662057A (en) * | 2012-06-07 | 2012-09-12 | 广州易航生物科技有限公司 | Cloned enzyme-donor immunoassay (CEDIA) ImmunoChip drug detecting kit |
| US20160054322A1 (en) * | 2014-08-21 | 2016-02-25 | Discoverx Corporation | Homogenous thermal shift ligand binding assay |
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| CN109613251B (en) | 2021-01-26 |
| CN109797143A (en) | 2019-05-24 |
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| CN109609481A (en) | 2019-04-12 |
| CN109609481B (en) | 2021-02-12 |
| CN109666619A (en) | 2019-04-23 |
| CN109609535B (en) | 2021-02-12 |
| CN106399278B (en) | 2019-06-18 |
| CN109666619B (en) | 2021-03-26 |
| CN109797143B (en) | 2021-02-12 |
| CN106399278A (en) | 2017-02-15 |
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