CN103301477B - Application of escherichia coli succinodehydrogenase iron-sulfur protein SdhB - Google Patents
Application of escherichia coli succinodehydrogenase iron-sulfur protein SdhB Download PDFInfo
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- CN103301477B CN103301477B CN201310194610.5A CN201310194610A CN103301477B CN 103301477 B CN103301477 B CN 103301477B CN 201310194610 A CN201310194610 A CN 201310194610A CN 103301477 B CN103301477 B CN 103301477B
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- escherichia coli
- sdhb
- sulfur protein
- succinate dehydrogenase
- iron
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the field of medical technology, and specifically relates to an application of escherichia coli succinodehydrogenase iron-sulfur protein SdhB. According to active immunization and passive immunization tests, the escherichia coli succinodehydrogenase iron-sulfur protein SdhB and the corresponding antibody have an obvious immune protection effect on the infection of clinical pathogenic bacteria of escherichia coli. Therefore, the escherichia coli succinodehydrogenase iron-sulfur protein SdhB and the corresponding antibody can be used for preparing a medicine for preventing and treating diseases caused by the infection of clinical pathogenic bacteria of animal escherichia coli. The medicine does not generate drug resistance and can effectively prevent and treat the diseases caused by the infection of clinical pathogenic bacteria of escherichia coli.
Description
Technical field
The present invention relates to medical art, more specifically, relate to the application of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB.
Background technology
Escherichia coli (
escherichia coli) be commonly referred to escherichia coli, be distributed in nature, great majority are non-pathogenic, mainly grow nonparasitically upon another plant in the intestinal of human or animal, and be normal flora, the escherichia coli of minority have toxicity, can cause disease, comprise the relevant disease of the mankind and domestic animals and fowls.In recent years, the loss that colibacillosis provisions fowl industrial belt is come is increasing, and it is difficult to cure, mortality rate is high, the very easily clinical characters such as recurrence, is just annoying the numerous raisers of basic unit and veterinarian.Such as, the intestine of young pigs infectious disease that escherichia coli can cause, is called colibacillosis of pigs.Common are yellow scour of piglet, piglet pujos blancos and edema disease three kinds, there is enteritis, enterotoxemia for feature.Wherein yellow scour of piglet is the infectious intestinal disease that is a kind of acute, highly lethal occurring in 1 ~ 7 age in days piglet.
Although the disease caused by escherichia coli can adopt antibiotic to control, this pathogen very easily produces drug resistance, causes antibiotic effect invalid.And long-term a large amount of quality using antibiotic not only to affect domestic animals and fowls food, and contaminated environment, affect human body healthy.Therefore, vaccine control is carried out in the selection controlling this type of disease the best.
Develop vaccine and need good protection former, just may exciting human immune system effectively, produce specific immune response, protection body exempts the infection of pathogen.At present, about the vaccine of escherichia coli is developed, but due to escherichia coli serotype more, its effect awaits further raising.Therefore, find that the former development to vaccine of new efficient protection is very important.
Succinate dehydrogenase (Succinatedehydrogenase is called for short SDH) is positioned at mitochondrial matrix, is the complexⅱ of mitochondrial respiratory chain, is made up of A, B, C, D 4 subunits.Wherein the iron-sulfur protein of SDHB coding and the SDHA flavoprotein of encoding form enzyme contact center.
Summary of the invention
Technical problem to be solved by this invention overcomes the deficiencies in the prior art, provides a kind of application of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB gene.
Another object of the present invention is to provide the application of a kind of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB.
Another object of the present invention is to provide the application of the antibody of a kind of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB.
Another object of the present invention is to provide a kind of pharmaceutical composition.
By active immunity and passive immunity test, the present invention finds that the infection of antibody to escherichia coli clinical bacteria of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB and escherichia coli succinate dehydrogenase iron-sulfur protein SdhB has obvious immanoprotection action.Therefore, the antibody of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB and escherichia coli succinate dehydrogenase iron-sulfur protein SdhB can be used for the medicine of the disease prepared caused by the infection of control escherichia coli clinical bacteria.This medicine can not produce drug resistance, effectively can prevent and treat the disease caused by the infection of escherichia coli clinical bacteria.
The present invention is achieved by the following technical programs:
Escherichia coli succinate dehydrogenase iron-sulfur protein gene SdhB is infected in preparation control the application caused in the medicine of disease by escherichia coli clinical bacteria; Described escherichia coli succinate dehydrogenase iron-sulfur protein gene SdhB has one of following nucleotide sequences;
S1. there is the nucleotide sequence shown in SEQ ID NO:1;
S2. the nucleotide sequence limited with SEQ ID NO:1 in sequence table has the homology of 95%, and identical biological function protein DNA sequence of encoding.
Escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is infected in preparation control the application caused in the medicine of disease by escherichia coli clinical bacteria; Described escherichia coli succinate dehydrogenase iron-sulfur protein SdhB has one of following amino acid sequences;
S1. there is the aminoacid sequence shown in SEQ ID NO:2;
S2 passes through the aminoacid sequence of the derived protein replacement of one or several amino acid residue of aminoacid sequence process shown in SEQ ID NO:2, disappearance or interpolation produced, and described derived protein has identical biological function with the albumen of SEQ ID NO:2.
The antibody of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is infected in preparation control the application caused in the medicine of disease by escherichia coli clinical bacteria; The aminoacid sequence of described escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is as shown in SEQ ID NO:2.
Preferably, the antibody of described escherichia coli succinate dehydrogenase iron-sulfur protein SdhB comprises monoclonal antibody and polyclonal antibody.
A kind of pharmaceutical composition, comprises the antibody of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB.Preferably, the antibody of described escherichia coli succinate dehydrogenase iron-sulfur protein SdhB comprises monoclonal antibody and polyclonal antibody.
The antibody of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB and escherichia coli succinate dehydrogenase iron-sulfur protein SdhB; to the infection of the escherichia coli clinical bacteria of various kind; all there is obvious immanoprotection action; described escherichia coli clinical bacteria is all escherichia coli pathogenic bacterium, comprises intestinal and produces malicious type escherichia coli, intestinal pathological form escherichia coli, intestinal invasion and attack type escherichia coli, prolapse of rectum blood group escherichia coli and/or intestinal adhesive type escherichia coli.Although the mechanism of action of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB and antibody thereof is that the present invention creatively finds that it can detect on the surface at bacterial outer membrane because previously everybody thinks that succinate dehydrogenase iron-sulfur protein SdhB is only positioned endochylema.
The present invention has following beneficial effect: the antibody of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB of the present invention and escherichia coli succinate dehydrogenase iron-sulfur protein SdhB infects the escherichia coli clinical bacteria of most kind and all has obvious immanoprotection action; therefore can be used as vaccine component or bacterial-infection resisting preparation, infecting for preventing and treating the clinical bacterium of escherichia coli the disease caused.And described medicine can not make pathogenic bacterium produce drug resistance, the disease caused by the infection of escherichia coli clinical bacteria effectively can be prevented and treated.
Accompanying drawing explanation
Fig. 1. the agarose gel electrophoresis result of escherichia coli SdhB gene pcr amplification product; Wherein M is DNA molecular amount standard.
Fig. 2. the pET-32a recombiant plasmid double digestion qualification result containing escherichia coli SdhB gene; Wherein M is protein molecular weight standard.
Fig. 3. the polyacrylamide gel electrophoresis testing result of escherichia coli SdhB protein expression.
Fig. 4. the polyacrylamide gel electrophoresis testing result of the SdhB albumen that purification obtains; Wherein M is protein molecular weight standard.
Fig. 5. observe the Survival that 14 days respectively organize mice in active immunity test.
Fig. 6. observe the Survival that 14 days respectively organize mice in passive immunity test.
Fig. 7. clinical bacteria strain outer membrane face exists the situation of SdhB.
Detailed description of the invention
The present invention is set forth further below in conjunction with drawings and Examples.Should be understood that these embodiments are only not used in for further illustrating the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the such as condition described in Sambrook equimolecular cloning experimentation room handbook (2001 by Cold Spring Harbor Laboratory Press), or according to the condition that manufacturer advises.
Embodiment 1: the preparation of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB and purification
S1. the amplification of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB gene.
S11. design of primers: according to the whole genome sequence of the escherichia coli that NCBI announces, design pair of primers: nucleotide sequence 5'-CCGGAATTCATGAGACTCGAGTTTTCA-3' as shown in SEQ ID NO 3 of forward primer F; The nucleotide sequence of reverse primer R is as shown in SEQ ID NO 4: 5'-CCGAAGCTTTTACGCATTACGTTGCAACA-3'.For ease of the structure of cloning and expressing carrier, primer is introduced restriction endonuclease sites EcoR I and Hind III respectively.Primer is synthesized by prompt base (Shanghai) trade Co., Ltd in the English Weihe River.
S12. gene amplification: the complete genome DNA extracting escherichia coli, the extraction of complete genome DNA adopts the bacterial genomes DNA Mini Kit of Beijing Pu Boxin biotechnology Co., Ltd; With the complete genome DNA of the escherichia coli obtained for template, carry out PCR reaction, reaction system is as follows: PCR reaction buffer 2.5mL, 10mmol/L dNTP is 1L altogether, 10mol/L forward primer F and each 1L of reverse primer R, template DNA 1L, is supplemented to 25L with ultra-pure water by reaction system.PCR reaction cycle parameter is as follows: the first step: 94 DEG C of 2min; Second step: 94 DEG C of degeneration 60s, 55 DEG C of annealing 60s, 72 DEG C extend 60s, 30 circulations.Continue subsequently to extend 10 min at 72 DEG C, and preserve at 4 DEG C.Reclaim DNA fragmentation by the length (see figure 1) of agarose gel electrophoresis identification of dna fragment.
S13. the clone of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB gene, screening and identification.
Use restricted enzyme
ecoRi and
hindiII DNA fragmentation that obtains of enzyme action step S12 and pET-32a(Novagen respectively) expression vector, DNA ligation kit operation instructions with reference to Takara company carry out coupled reaction, reaction system is as follows: ligase solution 5L, through the DNA fragmentation 1L of enzyme action, through the pET-32a expression vector 0.5L of enzyme action, with ultra-pure water, reaction system is supplemented to 10L, in about 16 DEG C insulations 14 ~ 16 hours.Recombinant vector is transformed escherichia coli DH5 α competence, after conversion, thalline is applied on the LB solid plate containing ampicillin (50 ug/mL), cultivate 12 ~ 16 hours in 37 DEG C.Random picking, containing the recon on flat board, is inoculated on the LB fluid medium containing ampicillin (50 ug/mL), extracts plasmid respectively carry out double digestion qualification (see figure 2) with alkaline lysis.Deliver to Shenzhen Huada Genetic Technology Co., Ltd by there being the recon of insertion and carry out sequencing.Sequencing result shows: escherichia coli succinate dehydrogenase iron-sulfur protein SdhB gene coded sequence is as shown in SEQ ID NO:1, and the aminoacid sequence of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is as shown in SEQ ID NO:2.By the escherichia coli succinate dehydrogenase iron-sulfur protein SdhB gene sequence alignment reported in the escherichia coli succinate dehydrogenase iron-sulfur protein SdhB gene sequence obtained and NCBI, result shows the homology having 100% with the escherichia coli succinate dehydrogenase iron-sulfur protein SdhB that reports in NCBI, illustrates and obtains correct restructuring SdhB gene.
S14. the expression of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB gene
The recombinant vector containing SdhB gene obtained by step S13 transforms Escherichia coli BL21 with heat shock method and expresses bacterium, cultivates 12 ~ 16 hours in 37 DEG C.By single colony inoculation in the LB fluid medium containing ampicillin (50 ug/mL), Simultaneous vaccination is containing being the Escherichia coli BL21 of free pET-32a plasmid in contrast, and being cultured to OD value in 37 DEG C is 0.6, adds 0.5mmol/L IPTG, in 35 DEG C of inductions 3 hours, collected by centrifugation thalline.Detected the molecular size range of restructuring SdhB gene whether expressing protein and expressing protein by SDS-PAGE, result as shown in Figure 3.As seen from the figure, the molecular weight of restructuring SdhB gene expressing protein is consistent with expectation.
S15. the purification of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB.
Large scale cultivating transforms thalline, and carry out supersound process after centrifugal, collected by centrifugation supernatant Ni-NTA His Bind affinity column carries out purification.With dcq buffer liquid Buffer E(8M carbamide, 0.1M NaH
2pO
4, 10mm Tris-HCl, by HCl adjust ph to 6.3) washing 5 times, each consumption is 1mL; With elution buffer Buffer F(8M carbamide, 0.1MNaH
2pO
4, 10mm Tris-HCl, by HCl adjust ph to 5.9) eluting destination protein 4 times, each consumption is 1mL, collects eluent A; With elution buffer Buffer G (8M carbamide, 0.1M NaH
2pO
4, 10mm Tris-HCl, by HCl adjust ph to 4.5) eluting is not in conjunction with the albumen 4 times of Ni-NTA His Bind affinity column, each consumption is 1mL, collects eluent B.The eluent A and the eluent B that get collection carry out SDS-PAGE analysis, and purification result as shown in Figure 4.By escherichia coli succinate dehydrogenase iron-sulfur protein SdhB subpackage good for purification, after lyophilization powdered, frozen in-80 DEG C of refrigerators.
Embodiment 2: the sero-fast preparation of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB and purification.
S1. the sero-fast preparation of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB:
By the escherichia coli succinate dehydrogenase iron-sulfur protein SdhB of embodiment 1 purification immunity Kunming mouse, obtain SdhB antiserum.Concrete steps are as follows: be dissolved in appropriate ultra-pure water by escherichia coli succinate dehydrogenase iron-sulfur protein SdhB lyophilized powder, then with isopyknic Freund's complete adjuvant (lanoline: paraffin oil=1:4 v/v, bacillus calmette-guerin vaccine is 30 mg/mL) mix homogeneously, wherein protein content is about 100 ug, then supersound process 2 min, namely obtained SdhB antigen, adopts lumbar injection; After 14 days, mixed with isopyknic incomplete Freund's adjuvant (lanoline: paraffin oil=1:4 v/v) by escherichia coli succinate dehydrogenase iron-sulfur protein SdhB, inject to Kunming mouse, wherein protein content is about 50 ug; Inject again once after 14 days.After 10 days, eye gets blood, by the blood hold over night in 4 DEG C of refrigerators obtained, serum is separated out naturally; 4 DEG C of 3000 r/min, centrifugal 30 minutes, gets supernatant and namely obtains SdhB antiserum.Having carried out titration by Wethern blotting method, is 1:16000.
S2. the sero-fast purification of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB
Saturated ammonium sulfate method preliminary purification antiserum: the antiserum of collection adds the mixing of isopyknic normal saline, and then dropwise add saturated ammonium sulfate two parts, limit edged stirs, and prevents precipitation, and ammonium sulfate saturation reaches 50%, leaves standstill 4 DEG C of refrigerator overnight after mixing.Centrifugal (12000 g, 10min), discarded by supernatant, taking precipitate is dissolved in a small amount of normal saline.By above-mentioned physiological saline solution liquid 33% saturated ammonium sulphate, centrifugal (12000 g, 10min), collecting precipitation, is dissolved in the PBS of pH=7.4.Extract is loaded bag filter, dialyses in normal saline, removing ammonium sulfate.Take out centrifugal, the antiserum that namely sucking-off supernatant is slightly carried.
The sepharose 4B post affinity purification antiserum of CNBr activation: by protein-coupled for the SdhB of the about 4mg purification sepharose 4B post to activating through CNBr, make the affinity column of the anti-SdhB antibody of purification.Be added on post after again the antiserum that about 5mg slightly carries being mixed with 10mL PBS, 4 DEG C of placements are spent the night, with PBS solution, 2mol/L NaCI/PBS solution and PBS(pH4.5) after solution washes each about 10 bed volumes of post successively, carry out eluting with the glycine (pH2.4) of 0.1mol/L and collect albumen, often pipe adds appropriate 1mol/L Tris-HCI(pH8.8) neutralize.
S3. escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is quantitatively sero-fast: carry out quantitatively (concrete grammar can refer to) purified antibodies content by Brandford method.
Embodiment 3: the sero-fast passive immune protection effect of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB.
Adopt the Kunming mouse of about about 3 weeks as subjects, the escherichia coli succinate dehydrogenase iron-sulfur protein SdhB antiserum that intramuscular injection 40 microgram embodiment 2 prepares, the serum of the non-immunizing rabbit of matched group injection equivalent, with escherichia coli clinical drug-resistant pathogenic strain Y17(1.5 × 10 be separated in clinical disease human body after 1 hour
8cfu/ is only) infect.Observe the Survival of each group Kunming mouse after 72 hours, and the relative immunity protective rate (table 2) of Computation immunity Kunming mouse.Result shows, with the Kunming mouse of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB antiserum passive immunity, its mortality rate obviously declines, and relative protection ratio is 93.75%.
Table 2 escherichia coli succinate dehydrogenase iron-sulfur protein SdhB antiserum is to the protected effect of Kunming mouse
Embodiment 4: escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is to the active immunity protective effect of pathogen.
The escherichia coli succinate dehydrogenase iron-sulfur protein SdhB lyophilized powder of the purification obtained by embodiment 1 is dissolved in appropriate ultra-pure water, then with isopyknic Freund's complete adjuvant (lanoline: paraffin oil=1:4 v/v, bacillus calmette-guerin vaccine is 30mg/mL) mix homogeneously, wherein protein content is about 100 ug, then supersound process 2 minutes, i.e. obtained SdhB antigen, intramuscular injection experiment Kunming mouse; After 10 days, mixed with isopyknic incomplete Freund's adjuvant (lanoline: paraffin oil=1:4 v/v) by escherichia coli succinate dehydrogenase iron-sulfur protein SdhB, wherein protein content is about 100 ug, intramuscular injection experiment Kunming mouse.Matched group Kunming mouse corresponding intramuscular injection equal volume normal saline.After 2nd immunity about 7 days, counteracting toxic substances protective effect is carried out to mice.Counteracting toxic substances bacterium adopts the escherichia coli clinical drug-resistant pathogenic strain Y17 be separated in clinical disease the human body respectively and escherichia coli clinical drug-resistant pathogenic strain HY obtained in the sick pig body in pig farm.
The challenge test of clinical patient fastbacteria
Adopt clinical patient fastbacteria Y17, counteracting toxic substances dosage is 1.5 × 10
8cfu/ only.Then the Survival that 14 days respectively organize mice is observed, and the relative immunity protective rate of Computation immunity mice.Fig. 5 be in 14 days every day mice Survival, as can be seen from Fig., namely matched group does not have 19 mices of immune group after pathogen counteracting toxic substances, its death mainly occurs in the 1st day, up to 15, the 2nd day dead 1, from the 3rd day until the 14th day, there is no dead mouse.And immune SdhB group mice is from the 1st day until the 14th day, without dead mouse.Table 3 is for after clinical patient fastbacteria counteracting toxic substances, succinate dehydrogenase iron-sulfur protein SdhB is to the protected effect of Kunming mouse, and its relative protection ratio is 100%.
The protected effect that table 3 escherichia coli succinate dehydrogenase iron-sulfur protein SdhB active immunity infects Y17
The challenge test of pig source pathogenic bacterium
Adopt pig source pathogenic bacterium HY, counteracting toxic substances dosage is 1.25 × 10
9cfu/ only.Then the Survival that 14 days respectively organize mice is observed, and the relative immunity protective rate of Computation immunity mice.Fig. 6 be in 14 days every day mice Survival, as can be seen from Fig., namely matched group does not have 20 mices of immune group after pathogen counteracting toxic substances, and its death mainly occurs in 2 days, up to 18, from the 3rd day until the 14th day, does not have dead mouse.And immune SdhB group mice only the 1st day dead 3.Table 4 is for after the counteracting toxic substances with pig source pathogenic bacterium, succinate dehydrogenase iron-sulfur protein SdhB is to the protected effect of Kunming mouse, and its relative protection ratio is 83.33%.
The protected effect that table 4 escherichia coli succinate dehydrogenase iron-sulfur protein SdhB active immunity infects HY
In sum, escherichia coli succinate dehydrogenase iron-sulfur protein SdhB has obvious immanoprotection action for clinical bacteria, can be used as vaccine component or bacterial-infection resisting preparation, infects for preventing and treating the clinical bacterium of escherichia coli the disease caused.
Embodiment 5: clinical bacteria strain detects universality outer membrane face existing SdhB
Adopt the escherichia coli pathogenic strain of clinical separation to carry out SdhB sero-fast ELISA experiment, if result is positive, illustrate to there is SdhB on the outer membrane face of antibacterial.Detailed process is as follows:
Picking 27 strain be separated escherichia coli pathogenic bacterium (Y1-Y20, EPEL, E3-H, E4-H, O157,100108d, HY2011, G × 2011) monoclonal respectively in 5mLLB test tube, 37 DEG C of 200rpm incubated overnight.Transfer in 100mL fresh LB by 1:100, centrifugal receipts bacterium when 37 DEG C of 200rpm are cultured to OD600=1.0, after brine 2 times, add the formaldehyde normal saline 20mL of 1%, 80 DEG C of deactivation 90min.After brine residues of formaldehyde, then adjust OD600=0.2 with normal saline, often pipe 1mL subpackage is used for follow-up experiment.
By centrifugal for often kind of antibacterial remove supernatant after, add 200 μ L SdhB(5% milk PBS and dilute, 1:500) antiserum, room temperature shake is hatched.Simultaneously with normal saline and BSA for contrast.Centrifugal segregation serum after effect 90min, then wash 2 times with PBS.Add anti-(5% milk PBS dilutes, 1:3000) room temperature vibrations of 200 μ L bis-and cultivate 1h, centrifugal segregation supernatant, washs 3 times with PBS.
Add after precipitation mixes by 20 μ LPBS and be transferred in ELISA Plate, then add 100 μ L nitrite ions (50 μ L hydrogen peroxide+50 μ LTMD), 37 DEG C of lucifuge colour developing 10min, add stop buffer (2M H
2sO
4), in 450nm reading, Fig. 7 is measurement result.SdhB albumen is all detected as can be seen from Fig. on the surface of this 27 strain multi-drug resistant bacteria.
SEQUENCE LISTING
<110> Zhongshan University
The application of <120> escherichia coli succinate dehydrogenase iron-sulfur protein SdhB
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 717
<212> DNA
<213> escherichia coli succinate dehydrogenase iron-sulfur protein SdhB gene
<400> 1
atgagactcg agttttcaat ttatcgctat aacccggatg ttgatgatgc tccgcgtatg 60
caggattaca ccctggaagc ggatgaaggt cgcgacatga tgctgctgga tgcgcttatc 120
cagctaaaag agaaagatcc cagcctgtcg ttccgccgct cctgccgtga aggtgtgtgc 180
ggttccgacg gtctgaacat gaacggcaag aatggtctgg cctgtattac cccgatttcg 240
gcactcaacc agccgggcaa gaagattgtg attcgcccgc tgccaggttt accggtgatc 300
cgcgatttgg tggtagacat gggacaattc tatgcgcaat atgagaaaat taagccttac 360
ctgttgaata atggacaaaa tccgccagct cgcgagcatt tacagatgcc agagcagcgc 420
gaaaaactcg acgggctgta tgaatgtatt ctctgcgcat gttgttcaac ctcttgtccg 480
tctttctggt ggaatcccga taagtttatc ggcccggcag gcttgttagc ggcatatcgt 540
ttcctgattg atagccgtga taccgagact gacagccgcc tcgacggttt gagtgatgca 600
ttcagcgtat tccgctgtca cagcatcatg aactgcgtca gtgtatgtcc gaaggggctg 660
aacccgacgc gcgccatcgg ccatatcaag tcgatgttgt tgcaacgtaa tgcgtaa 717
<210> 2
<211> 238
<212> PRT
<213> escherichia coli succinate dehydrogenase iron-sulfur protein SdhB
<400> 2
Met Arg Leu Glu Phe Ser Ile Tyr Arg Tyr Asn Pro Asp Val Asp Asp
1 5 10 15
Ala Pro Arg Met Gln Asp Tyr Thr Leu Glu Ala Asp Glu Gly Arg Asp
20 25 30
Met Met Leu Leu Asp Ala Leu Ile Gln Leu Lys Glu Lys Asp Pro Ser
35 40 45
Leu Ser Phe Arg Arg Ser Cys Arg Glu Gly Val Cys Gly Ser Asp Gly
50 55 60
Leu Asn Met Asn Gly Lys Asn Gly Leu Ala Cys Ile Thr Pro Ile Ser
65 70 75 80
Ala Leu Asn Gln Pro Gly Lys Lys Ile Val Ile Arg Pro Leu Pro Gly
85 90 95
Leu Pro Val Ile Arg Asp Leu Val Val Asp Met Gly Gln Phe Tyr Ala
100 105 110
Gln Tyr Glu Lys Ile Lys Pro Tyr Leu Leu Asn Asn Gly Gln Asn Pro
115 120 125
Pro Ala Arg Glu His Leu Gln Met Pro Glu Gln Arg Glu Lys Leu Asp
130 135 140
Gly Leu Tyr Glu Cys Ile Leu Cys Ala Cys Cys Ser Thr Ser Cys Pro
145 150 155 160
Ser Phe Trp Trp Asn Pro Asp Lys Phe Ile Gly Pro Ala Gly Leu Leu
165 170 175
Ala Ala Tyr Arg Phe Leu Ile Asp Ser Arg Asp Thr Glu Thr Asp Ser
180 185 190
Arg Leu Asp Gly Leu Ser Asp Ala Phe Ser Val Phe Arg Cys His Ser
195 200 205
Ile Met Asn Cys Val Ser Val Cys Pro Lys Gly Leu Asn Pro Thr Arg
210 215 220
Ala Ile Gly His Ile Lys Ser Met Leu Leu Gln Arg Asn Ala
225 230 235
<210> 3
<211> 27
<212> DNA
<213> forward primer F
<400> 3
ccggaattca tgagactcga gttttca 27
<210> 4
<211> 29
<212> DNA
<213> reverse primer R
<400> 4
ccgaagcttt tacgcattac gttgcaaca 29
Claims (4)
1. escherichia coli succinate dehydrogenase iron-sulfur protein gene SdhB is infected in preparation control the application caused in the medicine of disease by escherichia coli clinical bacteria; Described escherichia coli succinate dehydrogenase iron-sulfur protein gene SdhB is as shown in SEQ ID NO:1;
Described escherichia coli clinical bacteria is all escherichia coli pathogenic bacterium, comprises intestinal and produces malicious type escherichia coli, intestinal pathological form escherichia coli, intestinal invasion and attack type escherichia coli, prolapse of rectum blood group escherichia coli and/or intestinal adhesive type escherichia coli.
2. escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is infected in preparation control the application caused in the medicine of disease by escherichia coli clinical bacteria; Described escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is as shown in SEQ ID NO:2;
Described escherichia coli clinical bacteria is all escherichia coli pathogenic bacterium, comprises intestinal and produces malicious type escherichia coli, intestinal pathological form escherichia coli, intestinal invasion and attack type escherichia coli, prolapse of rectum blood group escherichia coli and/or intestinal adhesive type escherichia coli.
3. the antibody of escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is infected in preparation control the application caused in the medicine of disease by escherichia coli clinical bacteria; The aminoacid sequence of described escherichia coli succinate dehydrogenase iron-sulfur protein SdhB is as shown in SEQ ID NO:2;
Described escherichia coli clinical bacteria is all escherichia coli pathogenic bacterium, comprises intestinal and produces malicious type escherichia coli, intestinal pathological form escherichia coli, intestinal invasion and attack type escherichia coli, prolapse of rectum blood group escherichia coli and/or intestinal adhesive type escherichia coli.
4. apply according to claim 3, it is characterized in that, the antibody of described escherichia coli succinate dehydrogenase iron-sulfur protein SdhB comprises monoclonal antibody and polyclonal antibody.
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| CN113604493B (en) * | 2021-06-13 | 2023-08-01 | 兰州大学 | LDH, recombinant plasmid for expressing LDH, recombinant probiotics for expressing LDH and application |
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Non-Patent Citations (2)
| Title |
|---|
| "Identification and Partial Characterization of a Novel Bipartite Protein Antigen Associated with the Outer Membrane of Escherichia coli";PETER OWEN 等;《JOURNAL OF BACTERIOLOGY》;19870831;第169卷(第8期);第3770-3777页 * |
| "日本血吸虫琥珀酸脱氢酶铁硫蛋白全长cDNA的克隆、表达及保护性免疫评价";余俊龙 等;《中国科学》;20071231;第37卷(第1期);第58-64页 * |
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