CN109609535B - Prokaryotic expression vector of escherichia coli beta galactosidase receptor - Google Patents
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Abstract
The application relates to a prokaryotic expression vector of an escherichia coli beta galactosidase receptor. The present application provides a polypeptide of SEQ ID NO: 1, and the nucleotide sequence of SEQ ID NO: 2, or a beta galactosidase receptor. Designing an enzyme receptor for deleting amino acids from 13 th to 33 th according to the structure and the function of the escherichia coli beta galactosidase and the complementary principle of an enzyme donor and an enzyme receptor; connecting the coding nucleotide of the enzyme receptor with a vector, and converting the coding nucleotide into escherichia coli to construct a recombinant expression host cell; (ii) mass expression of the enzyme receptor by fermentation; purifying the enzyme receptor by affinity chromatography, wherein the purity of the desalted enzyme receptor is more than 90%. Compared with the conventional enzyme receptor, the enzyme receptor has higher yield and stronger complementary activity, and can be applied to the development of CEDIA biochemical diagnostic reagents.
Description
The application is a divisional application of Chinese patent application with the application number of 201610841474.8, the application date of 2016, 9, and 22, and the invention is named as 'an escherichia coli beta galactosidase receptor and a preparation method thereof'.
Technical Field
The present application relates to the field of biotechnology, and more specifically to clonal enzyme donor immunoassay technology. The application provides an enzyme receptor of escherichia coli beta galactosidase, and a coding nucleic acid, an expression vector, a host cell and a preparation method thereof.
Background
Coli beta galactosidase (GenBank: EDU66561.1) can be purified by hydrogen bromide CNBr2Two peptide fragments are obtained by cracking or gene recombination technology: large fragments lacking a small number of amino acids at the N-terminus, called Enzyme receptors (enzymes)aceptor, EA): a small fragment lacking most of the C-terminal amino acids is called an Enzyme Donor (ED). The phenomenon that the two exist independently without enzyme activity and can be polymerized to form the whole enzyme with enzyme activity when the two exist simultaneously is defined as alpha-complementation.
This property of E.coli beta galactosidase has been applied in molecular biology and Cloned Enzyme Donor Immunoassay (CEDIA). The CEDIA technology allows the detection of small molecule compounds in clinical biochemical diagnostics.
CEDIA is an immunoassay technique that combines the principles of β galactosidase α complementation with the specific binding properties of antigen antibodies. Small molecule antigen (Ag) is conjugated to ED to form an ED-Ag conjugate, which binds spontaneously to EA to form active beta galactosidase. However, if an antibody (Ab) against the small molecule antigen is added to the reaction system, the specific binding of the antigen antibody will form an ED-Ag-Ab complex. At this point, the ED-Ag-Ab complex can no longer bind to EA to form active β -galactosidase due to the steric hindrance of the antibody. If the sample contains the small molecule antigen to be detected, the reaction system contains two forms of antigen: one is free Ag in the sample; one is ED-Ag in the kit. Both antigens have the ability to specifically bind to antibodies. Obviously, the two forms of antigen competitively bind to the Ab in the reaction system, the more free Ag in the sample, the more Ab bound, and thus the more the remaining ED-Ag conjugate (the portion not bound to the antibody) can spontaneously bind to EA to form active β -galactosidase, and the level of the enzyme activity is proportional to the amount of free antigen in the sample.
The Escherichia coli beta galactosidase EA can be applied to the development of clinical biochemical diagnostic reagents of CEDIA technology, is an important raw material enzyme and has a great application value.
There are many genetically engineered EA or EA mutants reported so far, for example, in the papers published by Langley KE and Zabin I (biochemistry.1976Nov 2; 15(22):4866-75) a beta galactosidase is mentioned, which lacks the amino acid residues at positions 11 to 41 (called M15) as compared with wild-type EA.
As another example, M112 deleted for amino acids 23 to 31; EA5 deleted for amino acids 35 to 52; EA14 deleted for amino acids 30 to 37; EA11 deleted for amino acids 35 to 54; EA17 deleted for amino acids 21 to 53; EA18 deleted for amino acids 13 to 45; EA20 deleted for amino acids 26 to 45; EA22 deleted for amino acids 13 to 40; EA23 deleted for amino acids 16 to 35; EA24 which lacks amino acids 22 to 35 (Daniel R.Henderson, Clinical Chemistry, Vol.32, No.9,1986, 1637-1641). Yet scholars report beta-galactosidase EA lacking amino acids 1 to 5 and 14 to 40 simultaneously (expression of fusion protein of Escherichia coli beta-galactosidase EA and ED; J. Zhonghua microbiology and immunology 2003,23, No 2: 113-114).
TABLE 1 summary of the EA mutants reported
However, the enzyme receptors in the prior art still have the phenomenon of high detection background. In view of this, there is a need in the art for a novel beta galactosidase enzyme that has higher yield, lower background, and stronger complementary activity between the enzyme acceptor and the enzyme donor.
Disclosure of Invention
According to some embodiments, there is provided a beta galactosidase receptor whose amino acid sequence is SEQ ID No. 2. According to the beta galactosidase receptor of the present application, the amino acid residues at positions 13 to 33 are deleted compared to the wild type.
According to the biological structure and function of the beta galactosidase of Escherichia coli and the complementation principle of EA and ED, the amino acids from 13 th to 33 th positions of the beta galactosidase are deleted in a fixed point manner to obtain the beta galactosidase receptor of the application. The inventors have unexpectedly found that recombinant beta galactosidase receptors engineered in this way have a very low detection background, while facilitating improved expression and complementation activities.
According to some embodiments, there is provided a polynucleotide encoding a beta galactosidase receptor of the present application. In some embodiments, the polynucleotide is DNA; in other embodiments, the polynucleotide is RNA. In a specific embodiment, the polynucleotide is DNA. In a specific embodiment, the nucleotide sequence of the polynucleotide is SEQ ID No.1, the complement of SEQ ID No. 1.
According to some embodiments, an expression vector is provided that expresses the beta galactosidase receptor of the present application. In some embodiments, the expression vector of the present application comprises the polynucleotide described above. In some embodiments, the expression vector of the present application comprises the polynucleotide set forth in SEQ ID No. 1. In a specific embodiment, the polynucleotide shown in SEQ ID No.1 is operably linked to an expression vector. In some embodiments, the expression vector is a prokaryotic expression vector. In some embodiments, the expression vector is selected from the group consisting of pET41a, pET28a, pET20a, and combinations thereof. In a specific embodiment, the expression vector is pET41 a.
According to some embodiments, there is provided a host cell comprising an expression vector of the present application. In some embodiments, the host cell of the present application is a prokaryotic host cell. In a specific embodiment, the prokaryotic host cell is e. In a specific embodiment, the E.coli is selected from the group consisting of Rosetta-gami2(DE3) pLysS, BL21, Rosetta and combinations thereof.
The skilled person will appreciate that, in addition to using a host cell to express the product of interest, a cell-free expression system may be used to express the beta galactosidase receptor of the present application.
According to some embodiments, there is provided an agent comprising a beta galactosidase receptor of the present application. In some embodiments, the reagent is a clonase donor immunoassay reagent. In some embodiments, the reagent comprises one, or two, or three, or more separate containers. In some embodiments, the beta galactosidase receptor of the present application is contained in one container; in another container a beta galactosidase donor is contained. In other embodiments, the beta galactosidase donor can be free; or the beta galactosidase donor may be coupled to an antigen, such as a smaller molecular weight antigen, or a hapten. In some embodiments, the antigen is selected from: peptides, polysaccharides, lipids, nucleic acids, drugs. In some embodiments, the beta galactosidase receptor and the beta galactosidase donor of the present application are in different containers. In some embodiments, the beta galactosidase donor can be any beta galactosidase donor known in the art or of the future.
According to some embodiments, there is provided the use of any one or a combination of the following in the preparation of an agent: beta galactosidase receptor of the present application, polynucleotide of the present application, expression vector of the present application, host cell of the present application. In some embodiments, the reagent is a clonase donor immunoassay reagent.
According to some embodiments, there is provided a beta galactosidase comprising a beta galactosidase donor, and a beta galactosidase receptor of the present application. In some embodiments, the beta galactosidase donor and the beta galactosidase receptor of the present application interact to form an active beta galactosidase. In some embodiments, the beta galactosidase donor and the beta galactosidase receptor of the present application are non-covalently bound. In some embodiments, the beta galactosidase donor can also be coupled to an antigen, such as a smaller molecular weight antigen, or a hapten. In some embodiments, the antigen is selected from: peptides, polysaccharides, lipids, nucleic acids, drugs. In some embodiments, the beta galactosidase donor can be any beta galactosidase donor known in the art or of the future.
According to some embodiments, there is provided a method of preparing a beta galactosidase receptor, comprising the steps of:
1) culturing a host cell of the present application under conditions that allow expression of the beta galactosidase receptor; and
2) purifying and collecting the beta galactosidase receptor.
In some embodiments, the culturing is by fermentation. In some embodiments, the fermentation is in a manner selected from the group consisting of: batch fermentation, continuous fermentation, fed-batch fermentation, and combinations thereof. In some embodiments, the culturing is performed in a vessel. In particular embodiments, the container is selected from the group consisting of: shake flasks, petri dishes and fermentors.
In some embodiments, the culturing is performed at a temperature of 28 to 40 ℃, 30 to 40 ℃, 35 to 38 ℃, preferably 37 ℃.
In some embodiments, the culturing is performed under aerobic conditions. In a specific embodiment, the process is carried out with dissolved oxygen of greater than 40%.
In some embodiments, the culturing is under conditions of pH between 6.8 and 7.2.
In some embodiments, the expression is constitutive expression or inducible expression; depending on the type of expression vector.
In some embodiments, an induction step is further included between step 1) and step 2). In a specific embodiment, an inducer is added to the culture of step 1) to induce the expression of the beta galactosidase receptor. In a specific embodiment, the inducer is IPTG.
In some embodiments, the purification is affinity purification. The method of purification depends on the type of expression vector. For example, but not limited to, when the expression vector contains a His tag, Ni-NTA affinity chromatography is used.
In a specific embodiment, there is provided a method of preparing a beta galactosidase receptor comprising the steps of:
1) by 1.5X 108(ii) density of/L, inoculating the host cells of the present application in a fermenter in a primary medium containing an antibiotic;
2) culturing the host cell at 37 ℃ until the OD of the host cell600Up to 3 to 5;
3) the temperature of the culture was lowered to 25 ℃ and a first feed was added followed by IPTG to a final concentration of 1 mM;
4) adding a second feeding material at the speed of 10ml/h/L for culturing for 8 to 12 hours;
5) collecting the culture;
6) purifying and collecting beta galactosidase receptor by Ni-NTA affinity chromatography;
7) optionally, desalting is performed on the beta galactosidase receptor.
In a specific embodiment, the antibiotic-containing initial medium comprises: 10g/L tryptone, 5g/L yeast extract, 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 kanamycin, and 20% to 25% glucose.
In a specific embodiment, the first feed comprises 100. + -.20 g/L tryptone, 250. + -.50 g/L yeast extract, 10. + -.2 g/L lactose.
In a specific embodiment, the second feed comprises 400 ± 50g/L glycerol.
Drawings
FIG. 1: expression of beta galactosidase receptor. And (3) left lane: protein molecular weight Marker; and (3) right path: band of E.coli beta galactosidase receptor.
Detailed Description
Example 1 construction of expression vector for E.coli beta-galactosidase EA
Determining a wild type EA gene sequence according to an escherichia coli beta galactosidase nucleotide sequence published by an NCBI website; deleting amino acid residues from 13 th to 33 th to obtain a sequence shown as SEQ ID No. 2; entrusts Beijing Sanbo Polygala biological technology company to carry out whole gene synthesis, and adds two enzyme cutting sites (such as Nco I and Xho I) at two ends of the sequence respectively, inserts the synthesized gene sequence on the plasmid;
carrying out double enzyme digestion on the plasmid, and recovering an escherichia coli beta galactosidase EA gene fragment through electrophoresis; carrying out double enzyme digestion treatment on the vector plasmid pET41a, and carrying out electrophoretic purification on the plasmid subjected to enzyme digestion;
connecting the digested pET41a plasmid with an escherichia coli beta galactosidase EA gene at 16 ℃ to obtain an expression plasmid, transforming the expression plasmid into escherichia coli DH5 alpha, and coating a Kan resistant culture medium plate for screening; screening the obtained positive clones, extracting expression plasmids, and sequencing for confirmation.
Example 2 obtaining of expression host cells
The expression plasmid obtained in example 1 was transformed into host cells E.coli Rosetta-gami2(DE3) pLysS to give expression host cells.
Example 3 preparation of beta galactosidase EA
The expression host cells obtained in example 2 were inoculated in a seed medium (10g of tryptone, 5g of yeast extract, 10g of NaCl were weighed and dissolved in 900ml of deionized water, pH was adjusted to 7.0 with 10M NaOH, deionized water was added to the solution to a constant volume of 1L, autoclaving was carried out at 121 ℃ for 20min, cooling and storage at room temperature were carried out, kanamycin was added to the solution until the final concentration was 50mg/L during use), and culture was carried out at 37 ℃ until OD was reached600To 3 to 5;
by 1.5X 108(density of L) host cells were inoculated into fermentation initiation medium (tryptone 10g, yeast extract 5g, magnesium sulfate 0.5 to 2g, dipotassium hydrogenphosphate 7 to 10g, sodium dihydrogenphosphate 3 to 6g, 10ml of microelement mother liquor (10g FeSO)4·7H2O、0.1g NH46Mo7O24、0.5g MnSO4·5H2O、2.25g ZnSO4·7H2O、0.23g Na2B4O7·10H2O、1g CuSO4·7H2O、2g CaCl2Dissolved in 5M hydrochloric acid), adding 900ml of deionized water to dissolve, adjusting the pH value to 6.8 to 7.2, adding deionized water to constant volume to 1L, sterilizing at high pressure for 20min, and cooling for use. Adding kanamycin to a final concentration of 50mg/L, adding sterilized glucose to a final concentration of 20% to 25%), adjusting aeration, allowing growth under dissolved oxygen of more than 40%, and adjusting the pH value of the culture medium to 6.8 to 7.2 by dropwise adding acid or alkali;
when the components in the initial culture medium are gradually exhausted and the oxygen solubility value of the culture medium is reduced to 40%, the culture temperature is reduced to 25 ℃, and meanwhile, the supplementary material 1(100 plus or minus 20g/L tryptone, 250 plus or minus 50g/L yeast extract, 10 plus or minus 2g/L lactose, high-temperature sterilization at 121 ℃ for 20min and then room-temperature storage) is supplemented according to the proportion of 100 ml/L. After the supplement 1 is completed, IPTG is added until the final concentration is 1mM, and induction is started;
adding supplementary material 2(400 + -50 g/L glycerol, high temperature sterilization at 121 deg.C for 20min, and storing at room temperature) at a speed of 10ml/h/L, and adjusting pH of the culture medium to 6.8-7.2 with ammonia water or sulfuric acid; after the induction is carried out for about 10 hours at the low temperature of 25 ℃, the fermentation culture is finished;
centrifuging and collecting the obtained cells by using a tubular centrifuge, and precooling the cells to a temperature below 4 ℃ by using a high-pressure homogenizer;
resuspending the cells, homogenizing for 3 times at 850bar high pressure by a high pressure homogenizer to break the cells and release the Escherichia coli beta galactosidase EA;
centrifuging the cell lysate at 18000rpm at 4 deg.C for 30min, and filtering the supernatant with 0.22 μm filter membrane;
loading the filtered supernatant to a Ni affinity column; removing impurities which are not combined with the column or have low combination force; eluting target protein escherichia coli beta-galactosidase EA, and collecting a protein peak at the same time;
loading to desalting column for desalting, and collecting protein peak.
Test example
Test example 1 determination of protein purity and yield
The protein obtained in example 3 was subjected to SDS-PAGE gel electrophoresis to determine the purity of the protein (FIG. 1), and the concentration of the protein was measured by BCA method to calculate the EA production. The purity of the EA produced herein is 90%.
TABLE 2 yields
Test example 2 Activity measurement
The activity of E.coli beta-galactosidase EA obtained in example 3 was examined.
Coli beta galactosidase EA is able to complement ED to form beta galactosidase. Beta galactosidase is capable of hydrolyzing o-nitrophenyl beta-D-galactopyranoside (ONPG) to produce galactose and yellow o-nitrophenol (ONP). The enzymatic activity of galactosidase, and thus the complementary activity of EA, can be determined by measuring the amount of ONP.
(1) EA was diluted to 0.2mg/ml with PB buffer (100mM PB, 150mM NaCl, 0.1% NaN3, pH7.3) and ED was diluted to 1. mu.g/ml with PB buffer. Complementary activities were measured with siemens biochemical analyzer 1800:
100 μ l EA solution as R1;
as R2, 100. mu.l of an ONPG substrate solution (5mg/ml ONPG, 100mM PB, 150mM NaCl, 10mM MgCl2, 0.1% NaN3, pH 7.3);
mu.l of enzyme donor solution was used as sample S for determining the complementary activity of EA.
10 μ l PB buffer as blank control sample for determination of background activity of EA;
running an EA-ED program, determining the reaction rate,
(2) the above operations were performed in parallel, using M15 as a control.
(3) As a result:
the EA complementation activity of the present application was 11.2% higher than M15, whereas the background activity was 21% lower.
TABLE 3 complementary Activity
| EA | OD571 | ％ |
| M15 | 0.21874 | 100 |
| EA of the present application | 0.24332 | 111.2％ |
TABLE 4 background Activity
| EA | OD571 | ％ |
| M15 | 0.00167 | 100 |
| EA of the present application | 0.00132 | 79 |
Sequence listing
<110> Beijing Jiuqiang Biotechnology Ltd
<120> prokaryotic expression vector of escherichia coli beta galactosidase receptor
<130> 390002CG-360019
<150> 201610841474.8
<151> 2016-09-22
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3006
<212> DNA
<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 (3)
1. A prokaryotic expression vector comprising a polynucleotide encoding a beta galactosidase receptor;
the beta galactosidase receptor is shown as SEQ ID No. 2.
2. A prokaryotic expression vector, which comprises polynucleotide, the polynucleotide is shown in SEQ ID No.1 or shown in the complementary sequence of SEQ ID No. 1.
3. The prokaryotic expression vector of claim 2 selected from the group consisting of: pET41a, pET28a, pET20 a.
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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 |
| CN201910039590.1A CN109609535B (en) | 2016-09-22 | 2016-09-22 | Prokaryotic expression vector of escherichia coli beta galactosidase receptor |
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| 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 |
| CN201610841474.8A Active CN106399278B (en) | 2016-09-22 | 2016-09-22 | A kind of E. coli-galactosidase receptor and preparation method thereof |
| CN201910039590.1A Active CN109609535B (en) | 2016-09-22 | 2016-09-22 | Prokaryotic expression vector of escherichia coli beta galactosidase receptor |
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| CN201910039492.8A Active CN109613251B (en) | 2016-09-22 | 2016-09-22 | Use of escherichia coli beta galactosidase receptor |
| CN201610841474.8A Active CN106399278B (en) | 2016-09-22 | 2016-09-22 | A kind of E. coli-galactosidase receptor and preparation method thereof |
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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 |
| CA2645663C (en) * | 2006-03-13 | 2013-11-05 | 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 |
| EP3183580A4 (en) * | 2014-08-21 | 2018-04-25 | DiscoveRx Corporation | Homogenous thermal shift ligand binding assay |
| 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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| Title |
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| CEDIA,a New Homogeneous Immunoassay System;Danlel R.Henderson;《CLIN.CHEM》;19861231;第32卷(第9期);全文 * |
| 大肠杆菌B-半乳糖苷酶EA、ED融合蛋白的表达;张钧;《中华微生物学和免疫学杂志》;20131231;第23卷(第2期);全文 * |
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| CN109613251A (en) | 2019-04-12 |
| CN109609535A (en) | 2019-04-12 |
| CN106399278A (en) | 2017-02-15 |
| CN109666619A (en) | 2019-04-23 |
| CN106399278B (en) | 2019-06-18 |
| CN109613251B (en) | 2021-01-26 |
| CN109797143A (en) | 2019-05-24 |
| CN109797143B (en) | 2021-02-12 |
| CN109609481B (en) | 2021-02-12 |
| CN109609481A (en) | 2019-04-12 |
| CN109666619B (en) | 2021-03-26 |
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