CN102866119A - Biological activity detection method of protein of flagellin protein derivative of salmonella and application thereof - Google Patents

Biological activity detection method of protein of flagellin protein derivative of salmonella and application thereof Download PDF

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CN102866119A
CN102866119A CN2012102327841A CN201210232784A CN102866119A CN 102866119 A CN102866119 A CN 102866119A CN 2012102327841 A CN2012102327841 A CN 2012102327841A CN 201210232784 A CN201210232784 A CN 201210232784A CN 102866119 A CN102866119 A CN 102866119A
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flagellin
derivant
albumen
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salmonella
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CN102866119B (en
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张成岗
李伟光
吴永红
李军怀
高艳
李志慧
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Suzhou Sciscape Bio Pharmaceutical Technology Co ltd
Institute of Radiation Medicine of CAMMS
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Suzhou Sciscape Bio Pharmaceutical Technology Co ltd
Institute of Radiation Medicine of CAMMS
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Abstract

The invention discloses a biological activity detection method of a protein of a flagellin protein derivative of salmonella and an application thereof. The biological activity is represented by antioxidant and radical removing capacity of the protein of the flagellin protein derivative of salmonella, and the antioxidant activity, the radical removing dynamics and the activity of PDDH radical of the protein of the flagellin protein derivative of salmonella are used as the detection indexes. The detection method provided by the invention is stable, and has the advantages of reliable detection result and higher detection data repeatability and the like. The detection result can better reflect the antioxidant and radical removing biological activity of the protein of the flagellin protein derivative of salmonella and can be widely applied to activity tracking of the protein of the flagellin protein derivative of salmonella and biological products containing the protein of the flagellin protein derivative of salmonella, and the application prospect is wide.

Description

Biologically active detection method and the application thereof of salmonella flagellin derivant albumen
Technical field
The invention belongs to biologically active detects and biomedicine field, be specifically related to biologically active detection method and the application thereof of salmonella flagellin derivant albumen, particularly relate to the detection method of the anti-oxidant of salmonella flagellin derivant albumen and removing free radical activity and have anti-oxidant application with removing in the free radical function medicament in preparation.
Background technology
Salmonella flagellin (Flagellin) derivant is the product that the structure of salmonella flagellin is transformed, it is to comprise that the salmonella flagellin holds the fusion of 1-176 amino acids residue and 402-505 amino acids residue from N, can contain or not contain comprise Tat protein transduction peptide wear the film peptide, the salmonella flagellin holds 1-176 amino acids residue can connect by flexible peptide linker with 402-505 amino acids residue from N.U.S. Cleveland BioLabs company takes the lead in preparing ((Burdelya LG, Krivokrysenko VI, et al.An agonist of toll-like receptor5has radioprotective activity in mouse and primate models.Science2008,320 (5873): 226-230), called after CBLB502 albumen, the applicant has carried out the transformation of part-structure on the syntheti c route basis of reference CBLB502 albumen, finished the preparation of salmonella flagellin derivant, called after CZLC331.Through comparing, there are small sequence difference between the two in CBLB502 and CZLC331, mainly be conveniently indivedual amino acid to have been carried out small adjustment in order to improve expression efficiency and purifying when using different prokaryotic expression carriers to prepare, both are consistent basically on function.
At present found that the derivant albumen of salmonella flagellin Flagellin has multiple biologically active; for example CBLB502 albumen has anti-radioactivity and antitumor activity; CBLB502 all shows more significant protective effect to hemopoietic system and gastrointestinal system in the mice with radiation injury model; can prolong the life span of mouse behind high dose (dosage the is 14-17Gy) radiation gamma; improve survival rate (the Burdelya LG of low dosage (dosage is 10-13Gy) radiation gamma mouse; Krivokrysenko VI; et al.An agonist of toll-like receptor5has radioprotective activity in mouse and primate models.Science2008; 320 (5873): 226-230), these bioactive achievements in research show that salmonella flagellin (Flagellin) derivant has important commercial production and is worth.Burdelya etc. also disclose the preparation method of CBLB502 albumen in the document, can obtain by the method the CBLB502 albumen of prokaryotic expression, further lay a good foundation for the commercial production of salmonella flagellin (Flagellin) derivant.Yet, commercial production need to have corresponding quality standard and the support of quality inspection system, and present detection and control to salmonella flagellin Flagellin derivant albumen, rest on the immunocompetence (Western blot detection) of albumen and the detection of protein content, and for the biologically active of salmonella flagellin Flagellin derivant albumen, especially anti-oxidant and remove free radical activity, there is no at present corresponding activity research report and suitable detection method and report.
Free radical is the intermediate product (including but not limited to superoxide anion, hydrogen peroxide, hydroxy radical etc.) of body eubolism.Free radical has very strong oxidation reaction ability in body, but excessive free radical is easy to react with various biomacromolecules in body and cause cell and tissue oxidizing damage simultaneously.Too much free radical is the major reason that numerous disease produces in the body, therefore removes the important way that excessive free radicals has become the control various diseases.Under the physiological status, free radical is in the mobile equilibrium that produces and remove in the human body, and this balance mainly relies on the antioxidant system in the body to keep.Too much free radical is the key factor that produces oxidative stress in the body, it also is the major reason of causing a disease, such as immune system relevant disease, nervous system degenerative disease, tumour etc., therefore remove free radical too much in the body or prevented that the generation of polyradical from having become the important channel of epidemic prevention system, the nervous system disease.At present, also there is not salmonella flagellin Flagellin derivant PROTEIN C BLB502 to have report anti-oxidant and the removing free radical activity.
Thus, the present inventor's biologically active (especially anti-oxidant and removing free radical activity) to salmonella flagellin Flagellin derivant albumen on protein level is carried out the series further investigation, and achievement in research is converted into quality standard and the quality detecting method of salmonella flagellin Flagellin derivant protein production, require significant for the Quality Control in the follow-up commercial production.
Summary of the invention
The purpose of this invention is to provide the higher method from protein level direct-detection salmonella flagellin Flagellin derivant protein biological activity of a kind of sensitivity.
The biologically active detection method of salmonella flagellin Flagellin derivant albumen provided by the present invention, be by the anti-oxidant of salmonella flagellin Flagellin derivant albumen and remove the free radical ability and embody, with the total antioxidation activity of salmonella flagellin Flagellin derivant albumen, the activity removing ABTS free radical dynamics and remove the DPPH free radical as detecting index.
Particularly, the biologically active detection method of salmonella flagellin Flagellin derivant albumen mainly is to detect for the total antioxidation activity of this albumen, uses the ABTS method to detect, and can may further comprise the steps:
The biologically active detection method of salmonella flagellin Flagellin derivant albumen, to embody by the anti-oxidant of salmonella flagellin Flagellin derivant albumen and removing free radical ability, with the total antioxidation activity of salmonella flagellin Flagellin derivant albumen, remove free radical dynamics and PDDH free radical activity as detecting index, can may further comprise the steps:
1) salmonella flagellin Flagellin derivant albumen to be checked is mixed with the sample solution of setting concentration;
2) the anti-oxidant ability with removing free radical of test sample solution;
3) calculate detect data and with the biologically active normal data of salmonella flagellin Flagellin derivant albumen relatively, draw testing result;
Described salmonella flagellin Flagellin derivant albumen includes but not limited to the albumen of called after CBLB502 and CZLC331.
In the described detection method, detect for the total antioxidation activity of salmonella flagellin Flagellin derivant albumen, detect with the ABTS method, may further comprise the steps:
1) production standard curve: make ABTS concentration and absorbance A according to the ABTS detection method 735Typical curve;
2) salmonella flagellin Flagellin derivant protein sample to be measured is made into the sample solution of 1mg/mL;
3) according to typical curve, according to the A of sample 735Value calculation sample and Trolox standard solution etc. volumetric molar concentration, calculate the TAC of sample.
Specifically, described ABTS method detects and can comprise following process:
1) sample detection
1. 10mM Trolox standard solution is diluted to 0,0.05,0.1,0.2,0.4,0.6,0.8 and 1.0mM; With distilled water testing sample salmonella flagellin Flagellin derivant albumen is mixed with 1mg/mL solution;
2. get 200 μ l ABTS working fluid (ABTS and oxygenant K 2S 2O 8Press the preparation of 7mM:28mM final concentration), blank is 10 μ l distilled water or PBS, typical curve detects the Trolox standard solution that adds respectively 10 each concentration of μ l, sample detection adds 10 μ l salmonella flagellin Flagellin derivant protein solutions, mixing was measured A after incubated at room 2-6 minute gently 735
3. according to the blank of measuring and the A of standard solution 735Value, drawing out horizontal ordinate is that solution concentration, ordinate are A 735The typical curve of value is according to the A of typical curve and sample 735Value calculation sample and Trolox standard solution etc. volumetric molar concentration, and then calculate the total antioxidant activity of sample with following method;
2) the total antioxidation activity of calculation sample
The expression mode of total antioxidant activity: when adopting Trolox to carry out the oxidation resistance detection as standard items, the oxidation resistance of the sample Trolox-Equivalent Antioxidant Capacity (oxidation resistance of Trolox equivalence, TEAC) represent, computing formula is TEAC=Trolox equivalence volumetric molar concentration/sample concentration (mol/g), and the TEAC value is total antioxidation activity.
Set the total antioxidation activity of described salmonella flagellin Flagellin derivant albumen for＞0.05Torlox mmol/g is that salmonella flagellin Flagellin derivant albumen has the standard of total antioxidation activity.
In the described detection method, detect except ABTS free radical kinetic activity for salmonella flagellin Flagellin derivant albumin, can may further comprise the steps:
1) testing sample solution is prepared: the sample solution that salmonella flagellin Flagellin derivant protein sample to be measured is made into 0.1mM;
2) salmonella flagellin Flagellin derivant albumen is dynamically removed the detection of ABTS free radical activity
(1) reagent preparation
1. with ABTS and oxygenant K 2S 2O 8After being mixed with ABTS work mother liquor by the 7mM:28mM final concentration, the room temperature lucifuge was deposited 12-16 hour;
2. the mother liquor of will working dilutes 50,62.5,125,250 with PBS or 80% ethanol, and 375 times become the ABTS working fluids;
(2) absorption peak of working sample
The ABTS working fluid 0.98mL and 0.02mL albumen (0.1mM) solution that add respectively different extension rates in the Eppendorf pipe, add the 0.02mL distilled water in the blank group, then measure rapidly the absorbance of 340nm and 415nm with the Cary50UV-VIS of U.S. Varian company, minute is 0s (before not adding sample) and 30s, records the light absorption value (A of each time point in the ABTS working fluid of different extension rates 340And A 415);
(3) calculate the dynamic (dynamical) maximum reaction rate of removing free radical
By light absorption value A 340(or A 415) normalized form (M=A that converts with amount of substance concentration M 340(or A 415)/ε 340(or ε 415)) calculate reacted amount of substance concentration M (mol/L), wherein, ε 340=4.8 * 10 4(mol/L) -1Cm -1, ε 415=3.6 * 10 4(mol/L) -1Cm -1).This albumin is maximum reaction rate Vmax except the kinetic parameter of ABTS free radical, and ABTS working fluid concentration M of free radical after reaction that can calculate by general Hill equation different extension rates obtains.
Set described salmonella flagellin Flagellin derivant albumen and have that to remove the dynamic (dynamical) standard of ABTS free radical be that the salmonella flagellin Flagellin derivant albumin of 2 μ M is except the maximum reaction rate of ABTS free radical 0.3 μ Ms -1
In the described detection method, detect for the activity of salmonella flagellin Flagellin derivant albumin except the DPPH free radical, can may further comprise the steps:
1) salmonella flagellin Flagellin derivant protein sample to be measured is made into the sample solution of 10,25,50,75,100,125 μ g/mL; With the methyl alcohol of analyzing pure level just DPPH be dissolved into the DPPH free-atom aqueous solution of 0.1mM;
2) six centrifuge tube difference labels, the DPPH free-atom aqueous solution that adds equal-volume 470 μ l, then the salmonella flagellin Flagellin derivant protein solution that adds successively respectively above-mentioned variable concentrations, measure its absorbance in the 517nm place behind the reaction 5min under the room temperature, its value is respectively blank A 0, and sample A 1
3) calculate removing hydroxy radical activity: be calculated as follows the DPPH free radical scavenging activity:
DPPH removes ratio=(1-A 1/ A 0) * 100%.
Set described salmonella flagellin Flagellin derivant albumen to the DPPH free radical scavenging activity〉1.5% have the standard of removing the hydroxy radical activity for salmonella flagellin Flagellin derivant albumen.
The present invention is to provide described method to the tracking of salmonella flagellin Flagellin derivant albumen at protein biological activity on the other hand, the activity of salmonella flagellin Flagellin derivant albumen is confirmed in the preparation of salmonella flagellin Flagellin derivant protein production, storage, the transportation, and the application of the activity that contains the biological products of salmonella flagellin Flagellin derivant albumen in detecting.
The present invention also is to provide the salmonella flagellin Flagellin derivant albumen that comprises CBLB502 albumen and CZLC331 albumen to have anti-oxidant application with removing in the free radical function medicament in preparation on the one hand, described have a drug candidate that medicine anti-oxidant and that remove the free radical function comprises the panimmunity systemic disease that the treatment oxidative stress is induced, medicine in the disease of immune system that the treatment oxidative stress is induced, the panimmunity systemic disease that the treatment oxidative stress is induced etc. causes the medicine of the relevant disease that the body free radical increases, and also comprises their medical product.
The present invention proposes biologically active detection method of the flagellin Flagellin derivant albumen that comprises CBLB502, CZLC331 etc. and uses thereof, this detection method comprises: 1) flagellin Flagellin derivant albumen to be checked is mixed with the sample solution of setting concentration; 2) test sample solution is anti-oxidant and remove the ability of free radical, with the total antioxidation activity of this albumen, this albumin except free radical dynamics and this albumen to the clearance rate of DPPH free radical as detecting index; 3) calculate detect data and with the biologically active normal data of this albumen relatively, draw testing result.Detection method of the present invention is stable, have measurement result reliable, detect Data duplication than advantages of higher, the result who measures can reflect preferably the anti-oxidant of flagellin Flagellin derivant albumen and remove the biologically active of free radical, and flagellin Flagellin derivant itself and active track side's mask of containing the biological products of flagellin Flagellin derivant are had extensive use, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is 296 amino acid sequences of salmonella flagellin Flagellin derivant CZLC331 albumen and the sequence alignment result of 331 amino acid sequences
Fig. 2 is the testing result of the total antioxidation activity of salmonella flagellin Flagellin derivant CZLC331 albumen
Fig. 3 is that salmonella flagellin Flagellin derivant CZLC331 albumen is to the dynamics of ABTS radicals scavenging
Fig. 4 is that salmonella flagellin Flagellin derivant CZLC331 albumen is to DPPH radicals scavenging result
Embodiment
The invention provides a kind of sensitivity higher from the bioactive method of protein level direct-detection salmonella flagellin Flagellin derivant.
Among the present invention, salmonella flagellin Flagellin derivant comprises the albumen such as CBLB502 of existing document record, the CZLC331 albumen of applicant's preparation and the albumen of other similar structures, hereinafter general designation " albumen " or " this albumen ".
The biologically active detection method of salmonella flagellin Flagellin derivant albumen provided by the present invention, that dynamic characteristic anti-oxidant, that remove free radical and remove free radical by this albumen embodies,, can may further comprise the steps as detecting index with the total antioxidation activity of this albumen, the activity removing ABTS free radical dynamics and remove the DPPH free radical:
1) albumen to be checked is mixed with the sample solution of setting concentration;
2) the anti-oxidant ability with removing free radical of test sample solution;
3) calculate detect data and with the biologically active normal data of this albumen relatively, draw testing result.
First aspect in the biologically active detection method of albumen, detects for its total antioxidation activity, detects with the ABTS method.
The ABTS method is measured the principle of TAC:
(2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, Chinese are 2,2-connection nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts) is oxidized to green ABTS to ABTS under suitable oxygenant effect +, ABTS when polyphenoils exists +The generation meeting suppressed, measure ABTS at 735nm +Absorbance can measure and calculate the TAC of sample.Trolox(quino dimethylacrylate) is a kind of analog of vitamin E, has the oxidation resistance close with vitamin E, as the reference of other polyphenoils TAC.For example, the TAC of Trolox is 1, and in the same concentrations situation, the multiple that the oxidation resistance of other material is compared with Trolox with its oxidation resistance represents.
Therefore detect and can may further comprise the steps with the ABTS method for the total antioxidation activity of this albumen:
1) production standard curve: make ABTS concentration and absorbance A according to the ABTS detection method 735Typical curve;
2) the testing protein sample is made into the sample solution of 1mg/mL;
3) according to typical curve, according to the A of sample 735Value calculation sample and Trolox standard solution etc. volumetric molar concentration, and then calculate the TAC of sample.
Wherein, step 1) and 2) be used for sample tests, can be by following process operation:
1. 10mM Trolox standard solution is diluted to 0,0.05,0.1,0.2,0.4,0.6,0.8 and 1.0mM; With distilled water testing sample albumen is mixed with 1mg/mL solution;
2. get 200 μ l ABTS working fluids (prescription: with ABTS and oxygenant K 2S 2O 8After being mixed with ABTS work mother liquor by the 7mM:28mM final concentration, the room temperature lucifuge was deposited 12-16 hour, the mother liquor of will working dilutes 125 times with PBS or 80% ethanol and becomes the ABTS working fluid), blank is 10 μ l distilled water or PBS, typical curve detects the Trolox standard solution that adds respectively 10 each concentration of μ l, sample detection adds 10 μ l protein solutions, and mixing was measured A after incubated at room 2-6 minute gently 735
3. according to the blank of measuring and the A of standard solution 735Value, drawing out horizontal ordinate is that solution concentration, ordinate are A 735The typical curve of value is according to the A of typical curve and sample 735Value calculation sample and Trolox standard solution etc. volumetric molar concentration.
And then go out the total antioxidant activity of sample with the following process computation of step 3):
1. the expression mode of total antioxidant activity: adopting Trolox to carry out oxidation resistance when detecting as standard items, the oxidation resistance of sample can be used the Trolox-Equivalent Antioxidant Capacity (oxidation resistance of Trolox equivalence, TEAC) represent, for the albumen with antioxidation activity or antioxidant sample, concentration such as sample is 0.15mg/mL, measure the volumetric molar concentration that obtains identical with 0.3mM Trolox, then the antioxidation activity of sample is 0.3mM/0.15mg/mL=2mmol/g, and namely computing formula is: TEAC=Trolox equivalence volumetric molar concentration/sample concentration (mol/g);
2. calculate by above-mentioned formula, the TEAC value is the total antioxidation activity of sample.
Second aspect in the biologically active detection method of this albumen, for its dynamics to ABTS radicals scavenging, can may further comprise the steps:
1) testing sample solution is prepared: the sample solution that protein sample to be measured is made into 0.1mM;
2) albumen is dynamically removed the detection of ABTS free radical activity
(1) reagent preparation
1. with ABTS and oxygenant K 2S 2O 8After being mixed with ABTS work mother liquor by the 7mM:28mM final concentration, the room temperature lucifuge was deposited 12-16 hour;
2. the mother liquor of will working dilutes 50,62.5,125,250,375 times with PBS or 80% ethanol and becomes the ABTS working fluid;
(2) absorption peak of working sample
Manage ABTS working fluid 0.98mL and 0.02mL albumen (0.1mM) solution that adds respectively different extension rates in (microcentrifugal tube) to Eppendorf, add the 0.02mL distilled water in the blank group, then measure rapidly the absorbance of 340nm and 415nm with the Cary50UV-VIS of U.S. Varian company, minute is 0s (before not adding sample) and 30s, records the light absorption value (A of each time point in the ABTS working fluid of different extension rates 340Be reaction product absorption peak and A 415Be the free radical absorption peak);
(3) calculate the dynamic (dynamical) maximum reaction rate of removing free radical
Normalized form ε by light absorption value and material concentration conversion 340=4.8 * 10 4M -1Cm -1And ε 415=3.6 * 10 4M -1Cm -1Calculate reacted concentration (concentration=A 340(or A 415)/ε 340(or ε 415)), in the formula, ε 340And ε 415The absorbance at expression 340nm and 415nm place, M represents amount of substance concentration (mol/L), cm represents centimetre.This albumin is except the kinetic parameter (maximum reaction rate Vmax is gone out by ABTS working fluid concentration of free radical after reaction that the Hill equation calculates different extension rates) of ABTS free radical, and the reaction rate that calculates is the numerical value that testing sample is removed ABTS free radical kinetic activity.
The third aspect in the biologically active detection method of this albumen, detects for its activity of removing the DPPH free radical, can may further comprise the steps:
1) testing sample solution is prepared: protein sample to be measured is made into the sample solution of 10,25,50,75,100,125 μ g/mL, with the methyl alcohol of pure grade of analysis just DPPH be dissolved into the DPPH free-atom aqueous solution of 0.1mM;
2) six centrifuge tubes are distinguished labels, add the DPPH free-atom aqueous solution of equal-volume 470 μ l, then add successively respectively the protein solution of above-mentioned variable concentrations, measure its absorbance in the 517nm place behind the reaction 5min under the room temperature, and its value is respectively blank A 0With sample A 1
3) calculate removing hydroxy radical activity: be calculated as follows the DPPH free radical scavenging activity:
DPPH removes ratio=(1-A 1/ A 0) * 100%
In above three kinds of detection methods, the antioxidation activity that this albumen is total〉0.05Trolox mmol/g has the index of total antioxidation activity as product; This albumin with 2 μ M removes the maximum reaction rate of ABTS free radical〉0.3 μ Ms -1Remove the index of ABTS free radical kinetic activity as product; This albumin of 3 μ g/mL is except the ratio of DPPH free radical〉1.5% have the index of DPPH free radical scavenging activity as product.For detected protein product, can select wherein any one as detecting index, as long as being arranged, a detection index product up to standard can think that it has the removing free radical activity, as specification product, and can be further used for preparing the active component with medicine of removing the free radical function.
The biologically active detection method of above-mentioned salmonella flagellin Flagellin derivant CZLC331 albumen is in the tracking to this protein biological activity; activity to this albumen in protein production preparation, storage, the transportation is confirmed; the activity that contains the biological products of this albumen detects, and the application in setting up this albumen quality standard and quality inspection system also belongs to protection scope of the present invention.
Confirm through the present invention, salmonella flagellin Flagellin derivant albumen has anti-oxidant and removes the free radical function, thereby can it have anti-oxidant medicine with removing the free radical function for the active ingredient preparation.Described salmonella flagellin Flagellin derivant albumen is preferably the CZLC331 albumen that obtains by the bioengineering fermentation.
Described have a drug candidate that medicine anti-oxidant and that remove the free radical function comprises the panimmunity systemic disease that the treatment oxidative stress is induced, medicine in the disease of immune system that the treatment oxidative stress is induced, the panimmunity systemic disease that the treatment oxidative stress is induced etc. causes the medicine of the relevant disease that the body free radical increases, and also comprises their medical product.
The present invention comes further from removing free radical ability angle salmonella flagellin Flagellin derivant protein biological activity to be detected to illustrate the present invention with embodiment; following examples describe as an example of the CZLC331 albumen of inventor preparation example, but protection scope of the present invention is not limited to following embodiment.
Method therefor is conventional method if no special instructions among the following embodiment.
The preparation of embodiment 1, salmonella flagellin Flagellin derivant CZLC331 albumen
1, makes up the prokaryotic expression carrier pET28b-Tat-CZLC331 of salmonella flagellin derivant CZLC331 albumen
1) salmonella flagellin derivant CZLC331 protein coding gene is artificial synthetic: the encoding gene of salmonella flagellin derivant CZLC331 albumen is by flexible arm the coded sequence of salmonella flagellin to be held the 1-528 bit base and is connected 5 ' (corresponding salmonella flagellin held 1-176 amino acids residue and 402-505 amino acids residue from N respectively) of holding the connection of 1206-1515 bit base to obtain from 5 ', its nucleotide sequence is shown in sequence in the sequence table 1, length is that 891bp(is the core protein sequence of coding CZLC331 albumen), protein sequence (is comprised of 296 amino acid shown in sequence in the sequence table 2, core protein sequence for CZLC331 albumen), synthetic by the rich company of Deco skill Development Co., Ltd that steps in Beijing.2% agarose gel electrophoresis testing result of synthetic gene shows the genes of interest that has obtained 891bp, conforms to expected results.
2) structure of recombinant expression carrier pET28b-Tat-CZLC331
A) pcr amplification salmonella flagellin derivant CZLC331 protein coding gene
Adopt the method for conventional PCR to amplify the salmonella flagellin derivant CZLC331 protein coding gene of 891bp, 50 μ l PCR reaction systems are: plasmid template (carries the cloning vector pGH-CZLC331 of salmonella flagellin derivant CZLC331 protein coding gene, construction method is that the CZLC331 protein coding gene that chemical method is synthetic inserts cloning vector pGH(available from the rich Deco skill Development Co., Ltd that steps in Beijing) the SmaI restriction enzyme site obtain) 0.5 μ l, 10 * dNTP5 μ l, 10 * Ex Taq damping fluid, 5 μ l, on, downstream primer (the upstream primer sequence is:
5'-CGCGGGATCCATGGCTCAGGTTATCA-3', the downstream primer sequence is:
5'-CCGCTCGAGTGCGTCGTCTCTGTCTTG-3') each 0.5 μ l, Ex Taq enzyme 0.25 μ l, ddH2O38.25 μ l; The PCR reaction conditions is: first 95 ℃ 4 minutes; Then 95 ℃ 45 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds, totally 30 circulations; Last 72 ℃ 7 minutes.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, testing result shows the dna fragmentation that has obtained 891bp through amplification, conforms to expected results, reclaims and this purpose fragment of purifying.
B) restriction enzyme respectively enzyme cut genes of interest CZLC331 and pET28b-Tat carrier
Respectively to salmonella flagellin derivant CZLC331 protein coding gene and pET28b-Tat carrier (construction method see below) with restriction enzyme BamH I and Xho I double digestion, then adopt the T4DNA ligases to connect to spend the night and be transformed among the competent escherichia coli cell DH5 α at 16 ℃.The pET28b-Tat carrier construction method is: at first the method by chemosynthesis synthetic to contain restriction enzyme site be the NcoI(upstream) and the NdeI(downstream) double-stranded TAT core sequence, then adopt restriction enzyme NcoI and NdeI double digestion TAT core fragment and carrier pET28b, reclaim the purpose fragment and cut evaluation by the connection of T4DNA ligase and enzyme, send at last the prokaryotic expression carrier pET28b-Tat that Ying Jun company direct Sequencing conclusive evidence contains the TAT core fragment to make up correct, its nucleotide sequence is shown in sequence in the sequence table 3, length is 996bp, (sequence 3 length are the coding protein sequence of the dna sequence dna of 996bp to coding protein sequence shown in sequence in the sequence table 4, formed by 331 amino acid, it is the amino acid sequence of CZLC331 albumen, CZLC331 is name therefore), do not contain the CZLC331 protein sequence (sequence 2) of TAT core fragment and contain the TAT core fragment CZLC331 protein sequence (sequence 4) the sequence alignment result as shown in Figure 1, for sequence 2, the aminoterminal of sequence 4 has increased TAT and has worn the film peptide sequence, and c-terminus has increased by 6 histidines and indivedual amino acid.
C) identify
The clone who grows after cultivating is identified by digestion with restriction enzyme and order-checking respectively, obtain all correct prokaryotic expression carriers of sequence and insertion position, called after pET28b-Tat-CZLC331.
2, transform the recovery of host e. coli and thalline
Step 1 is made up correct prokaryotic expression carrier pET28b-Tat-CZLC331 transform e. coli bl21 (DE3) and also be applied to the LB plate, clone to be grown, each is organized respectively clone of picking and is inoculated into 5mL and contains Kana +In the LB nutrient culture media of (final concentration 100 μ g/mL), 37 ℃, 220rpm shook bacterium approximately 16 hours, treated that thalline recovers fully.
3, the abduction delivering of prokaryotic expression carrier pET28b-Tat-CZLC331
The bacterium liquid of recovery is diluted to OD600=0.8, then drawing 5mL bacterium liquid is inoculated in the LB nutrient culture media that 150mL contains Kana+ (final concentration 100 μ g/mL), 37 ℃, 220rpm shake bacterium and approximately measure OD600 after 4-5 hour, add rapidly derivant IPTG (final concentration 1mM) when treating OD600=0.6-1.0, and in 30 ℃, 220rpm abduction delivering 8 hours.
4, the preparation of salmonella flagellin derivant CZLC331 protein sample
Above-mentioned abduction delivering bacterium liquid to collect thalline, adopts ultrasonication behind the 20mM sodium phosphate buffer dilute sample in centrifugal 10 minutes of 4 ℃, 12000rpm, and preparation CZLC331 protein sample is left and taken supernatant purifying to be separated.
5. separation, the purifying of salmonella flagellin derivant CZLC331 albumen
The supernatant of step 4 preparation is adopted initial buffer liquid (20mM Na3PO4+0.5M NaCl, pH7.4) after the balance directly loading to the good HisTrap HP5mL post (available from GE company) of balance, then adopt the above-mentioned damping fluid flushing pillar of 4-5 column volume to the eluting peak baseline, adopt at last elution buffer (20mM Na 3PO 4+ 0.5M NaCl+0.5M imidazoles, pH7.4) eluted protein sample obtains salmonella flagellin derivant after the desalination, called after CZLC331 albumen, purity reaches more than 95%.Through identifying, its amino acid sequence holds 1-176 amino acids residue identical with 402-505 amino acids residue with the salmonella flagellin from N.
Embodiment 2, the total antioxidation activity of salmonella flagellin Flagellin derivant CZLC331 albumen detect
Detect with the ABTS method, can may further comprise the steps:
1, reagent preparation
(1) CZLC331 protein sample solution preparation: on the CZLC331 protein sample basis that has prepared by embodiment 1 method, with distilled water CZLC331 albumen is mixed with the 1mg/mL sample solution.
(2) preparation of reference substance vitamin C (Vc), reduced glutathione (GSH) and N-acetylcystein (NAC) solution: prepare with CZLC331 albumen, take by weighing reference substance 10mg, with distilled water it is mixed with the reference substance solution of 1mg/mL.
2, total antioxidation activity detects
1. with ABTS and oxygenant K 2S 2O 8After being mixed with ABTS work mother liquor by the 7mM:28mM final concentration, the room temperature lucifuge was deposited 12-16 hour;
2. the mother liquor of will working dilutes 125 times with PBS or 80% ethanol and becomes the ABTS working fluid;
3. 10mM Trolox standard solution is diluted to 0,0.05,0.1,0.2,0.4,0.6,0.8 and 1.0mM; With distilled water testing sample CZLC331 albumen, Vc, GSH and NAC are mixed with 1mg/mL solution;
4. get 200 μ l ABTS working fluids;
5. add 10 μ l distilled water or PBS in the blank, typical curve detects the Trolox standard solution that adds the various concentration of 10 μ l, and sample detection adds the sample of the various concentration of 10 μ l, gently mixing.
6. measure A after incubated at room 2-6 minute 735
7. the A that obtains according to bioassay standard solution 735Value, drawing out horizontal ordinate is that solution concentration, ordinate are A 735The typical curve of value;
8. according to typical curve, according to the A of sample 735Value calculation sample and Trolox standard solution etc. volumetric molar concentration, and then calculate the TAC of sample.
9. the expression mode of TAC: adopting Trolox to carry out oxidation resistance when detecting as standard items, the oxidation resistance of sample can represent with Trolox-Equivalent Antioxidant Capacity (TEAC).For the albumen with antioxidation activity or antioxidant, concentration such as sample is 0.15mg/mL, measure the volumetric molar concentration that obtains identical with 0.3mM Trolox, then the oxidation resistance of sample is 0.3mM/0.15mg/mL=2mmol/g, and namely computing formula is: TEAC=Trolox equivalence volumetric molar concentration/sample concentration (mol/g).
10. carry out detection and the calculating of reference substance by same method.
Testing result is referring to Fig. 2, and the result shows, CZLC331 albumen has certain antioxidation activity, the antioxidation activity that the 1mg/mL sample solution is total〉0.0951Trolox mmol/g.
Embodiment 3, salmonella flagellin Flagellin derivant CZLC331 albumen are to the dynamics of ABTS radicals scavenging
Measure CZLC331 albumen to the dynamics of ABTS radicals scavenging, concrete grammar can may further comprise the steps:
1) testing sample solution is prepared: the sample solution that CZLC331 protein sample to be measured is made into 0.1mM;
2) CZLC331 albumen is dynamically removed the detection of ABTS free radical activity
(1) reagent preparation
1. with ABTS and oxygenant K 2S 2O 8After being mixed with ABTS work mother liquor by the 7mM:28mM final concentration, the room temperature lucifuge was deposited 12-16 hour;
2. the mother liquor of will working dilutes 50,62.5,125,250,375 times with PBS or 80% ethanol and becomes the ABTS working fluid;
(2) absorption peak of working sample
Manage ABTS working fluid 0.98mL and 0.02mL albumen (0.1mM) solution that adds respectively different extension rates in (microcentrifugal tube) to Eppendorf, add the 0.02mL distilled water in the blank group, then measure rapidly the absorbance of 340nm and 415nm with the Cary50UV-VIS of U.S. Varian company, minute is 0s (before not adding sample) and 30s, records the light absorption value (A of each time point in the ABTS working fluid of different extension rates 340And A 415);
(3) calculate the dynamic (dynamical) maximum reaction rate of removing free radical
By light absorption value A 340(or A 415) normalized form (M=A that converts with amount of substance concentration M 340(or A 415)/ε 340(or ε 415)) calculate reacted amount of substance concentration M (mol/L), wherein, ε 340=4.8 * 10 4(mol/L) -1Cm -1, ε 415=3.6 * 10 4(mol/L) -1Cm -1).This albumin is maximum reaction rate Vmax except the kinetic parameter of ABTS free radical, and ABTS working fluid concentration M of free radical after reaction that can calculate by general Hill equation different extension rates obtains.
Testing result shows in the situation that the CZLC331 protein concentration is constant referring to Fig. 3, and along with the ABTS number of free radical constantly increases, ABTS free radical wear rate is also in continuous increase.Utilize the Hill equation to calculate the maximum reaction rate of ABTS radicals scavenging by this diagram data, the result is 0.3665 μ M/s, reflects that CZLC331 albumen has stronger removing ability to free radical in measuring concentration range.
Embodiment 4, salmonella flagellin Flagellin derivant CZLC331 albumen free radical scavenging detect
Adopt the DPPH colourimetry that the CZLC331 albumin is measured except the activity of hydroxy radical, DPPH is dissolved in the methanol solution, can obtain CZLC331 albumen to the scavenging action of DPPH free radical by the concentration that detects the 517nm absorption peak, specifically can may further comprise the steps:
1) CZLC331 protein sample to be measured is made into the sample solution of 10,25,50,75,100,125 μ g/mL, take Vc, NAC(N-acetylcysteine) and the GSH(reduced glutathione) be contrast; With the methyl alcohol of analyzing pure level just DPPH be dissolved into the DPPH free-atom aqueous solution of 0.1mM;
2) six centrifuge tubes are distinguished labels, add the DPPH free-atom aqueous solution of equal-volume 470 μ l, then add successively respectively the CZLC331 protein solution of above-mentioned variable concentrations, measure its absorbance in the 517nm place behind the reaction 5min under the room temperature, and its value is respectively A 0(blank) and A 1(sample);
3) calculate removing hydroxy radical activity: be calculated as follows the DPPH free radical scavenging activity:
DPPH removes ratio=(1-A 1/ A 0) * 100%
Testing result is referring to Fig. 4, and the result shows, under 3 μ g/mL final concentrations, the CZLC331 albumin is 2.35% except the activity of DPPH free radical, but compares with Vc, NAC and GSH, its removing ability slightly a little less than.
The biologically active of embodiment 5, different batches CZLC331 protein product detects
1) preparation of samples
For the CZLC331 protein product of different batches, the CZLC331 protein product is mixed with the concentration of 3mg/mL with distilled water, the follow-up dilution (referring to table 1) of carrying out according to demand variable concentrations;
2) required reagent and instrument
Reagent: agents useful for same is prepared with reference to the method among the embodiment 2-4;
Instrument: have and detect the ultraviolet spectrophotometer that uses wave band, such as Cary50UV-VIS;
3) detect operating process: the concrete operation step with reference to embodiment 2-4 is measured;
4) result and evaluation criterion
The biologically active testing result of table 1 different batches CZLC331 protein product
Figure BDA00001855469800131
Judgment criteria:
A) for the total antioxidation activity of CZLC331 albumen, CZLC331 albumen is in above-mentioned mensuration system, the total antioxidation activity of the CZLC331 albumen of 1mg/mL is 0.095 ± 0.029Trolox mmol/g, has lower total antioxidant activity than conventional antioxidants such as Vc.Therefore can set the CZLC331 albumen total antioxidant activity of 1mg/mL〉0.05Trolox mmol/g is the index that product has total antioxidation activity.
B) remove the dynamic (dynamical) reaction rate of ABTS free radical for the CZLC331 albumin, therefore the clearance rate of CZLC331 albumen (final concentration 2 μ M) is 0.3665 μ M/s, can set the CZLC331 albumin removal rates of 2 μ M〉0.3 μ M/s removes the dynamic (dynamical) index of ABTS free radical as product.
C) for the CZLC331 albumin except the activity of DPPH free radical, CZLC331 albumen is when 3 μ g/mL, clearance rate is 2.35 (%), illustrate that its removing ability slightly a little less than.Therefore can set the CZLC331 albumen clearance rate of 3 μ g/mL〉1.5% have the index of free radical scavenging as product.
For detected CZLC331 protein product, can select wherein any one as detecting index, as long as namely being arranged, a detection index product up to standard can think that it has the removing free radical activity, as specification product, and can be further used for preparing the active component with medicine of removing the free radical function.
The present invention can remove free radical with the detection techniques such as ABTS method and DPPH method proof salmonella flagellin Flagellin derivant CZLC331 albumen, thereby can it have the medicine of removing the free radical function for active ingredient preparation, and set up bioactive detection method for salmonella flagellin Flagellin derivant albumen with this.Those skilled in the art can understand, because the albumen such as CBLB502 are identical with CZLC331 albumen basic structure, function is basically identical, and therefore above embodiment conclusion is equally applicable to the salmonella flagellin Flagellin derivant of other name.Particularly the given biologically active judgment criteria of embodiment 5 is applicable equally to this albuminoid of other name, can be used as the general detection of this albuminoid and quality control standard.
Activity test method provided by the invention include but not limited to for the total antioxidation activity of salmonella flagellin Flagellin derivant albumen, remove the DPPH free radical and to the dynamics of ABTS radicals scavenging etc. as the detection index.Detection method of the present invention is stable, has measurement result reliable, detect Data duplication than advantages of higher, the result who measures can reflect the biologically active for salmonella flagellin Flagellin derivant albumen preferably, and to for salmonella flagellin Flagellin derivant itself and active track side's mask of containing for the biological products of salmonella flagellin Flagell in derivant albumen extensive use being arranged, also can be used for for the follow-up of quality in the salmonella flagellin Flagellin derivant protein production process, provide assurance to setting up for salmonella flagellin Flagellin derivant albumen quality standard and quality inspection system, be fit to promotion and application.
Figure IDA00001855470300011
Figure IDA00001855470300021
Figure IDA00001855470300031
Figure IDA00001855470300041
Figure IDA00001855470300051

Claims (10)

1. the biologically active detection method of salmonella flagellin Flagellin derivant albumen, to embody by the anti-oxidant of salmonella flagellin Flagellin derivant albumen and removing free radical ability, with the total antioxidation activity of salmonella flagellin Flagellin derivant albumen, remove free radical dynamics and PDDH free radical activity as detecting index, can may further comprise the steps:
1) salmonella flagellin Flagellin derivant albumen to be checked is mixed with the sample solution of setting concentration;
2) the anti-oxidant ability with removing free radical of test sample solution;
3) calculate detect data and with the biologically active normal data of salmonella flagellin Flagellin derivant albumen relatively, draw testing result;
Described salmonella flagellin Flagellin derivant albumen includes but not limited to the albumen of called after CBLB502 and CZLC331.
2. detection method according to claim 1 is characterized in that: wherein, detect for the total antioxidation activity of salmonella flagellin Flagellin derivant albumen, detect with the ABTS method, may further comprise the steps:
1) production standard curve: make ABTS concentration and absorbance A according to the ABTS detection method 735Typical curve;
2) salmonella flagellin Flagellin derivant protein sample to be measured is made into the sample solution of 1mg/mL;
3) according to typical curve, according to the A of sample 735Value calculation sample and Trolox standard solution etc. volumetric molar concentration, calculate the TAC of sample.
3. detection method according to claim 2, it is characterized in that: specifically, described ABTS method detects and can comprise following process:
1) sample detection
1. 10mM Trolox standard solution is diluted to 0,0.05,0.1,0.2,0.4,0.6,0.8 and 1.0mM; With distilled water testing sample salmonella flagellin Flagellin derivant albumen is mixed with 1mg/mL solution;
2. get 200 μ l ABTS working fluid (ABTS and oxygenant K 2S 2O 8Press the preparation of 7mM:28mM final concentration), blank is 10 μ l distilled water or PBS, typical curve detects the Trolox standard solution that adds respectively 10 each concentration of μ l, sample detection adds 10 μ l salmonella flagellin Flagellin derivant protein solutions, mixing was measured A after incubated at room 2-6 minute gently 735
3. according to the blank of measuring and the A of standard solution 735Value, drawing out horizontal ordinate is that solution concentration, ordinate are A 735The typical curve of value is according to the A of typical curve and sample 735Value calculation sample and Trolox standard solution etc. volumetric molar concentration, and then calculate the total antioxidant activity of sample with following method;
2) the total antioxidation activity of calculation sample
The expression mode of total antioxidant activity: when adopting Trolox to carry out the oxidation resistance detection as standard items, the oxidation resistance of the sample Trolox-Equivalent Antioxidant Capacity (oxidation resistance of Trolox equivalence, TEAC) represent, computing formula is TEAC=Trolox equivalence volumetric molar concentration/sample concentration (mol/g), and the TEAC value is total antioxidation activity.
4. it is characterized in that according to claim 2 or 3 described detection methods: the total antioxidation activity of described salmonella flagellin Flagellin derivant albumen for＞0.05Torlox mmol/g is that salmonella flagellin Flagellin derivant albumen has the standard of total antioxidation activity.
5. detection method according to claim 1 is characterized in that: wherein, detect except ABTS free radical kinetic activity for salmonella flagellin Flagellin derivant albumin, can may further comprise the steps:
1) testing sample solution is prepared: the sample solution that salmonella flagellin Flagellin derivant protein sample to be measured is made into 0.1mM;
2) salmonella flagellin Flagellin derivant albumen is dynamically removed the detection of ABTS free radical activity
(1) reagent preparation
1. with ABTS and oxygenant K 2S 2O 8After being mixed with ABTS work mother liquor by the 7mM:28mM final concentration, the room temperature lucifuge was deposited 12-16 hour;
2. the mother liquor of will working dilutes 50,62.5,125,250 with PBS or 80% ethanol, and 375 times become the ABTS working fluids;
(2) absorption peak of working sample
The ABTS working fluid 0.98mL and 0.02mL albumen (0.1mM) solution that add respectively different extension rates in the Eppendorf pipe, add the 0.02mL distilled water in the blank group, then measure rapidly the absorbance of 340nm and 415nm with the Cary50UV-VIS of U.S. Varian company, minute is 0s (before not adding sample) and 30s, records the light absorption value (A of each time point in the ABTS working fluid of different extension rates 340And A 415);
(3) calculate the dynamic (dynamical) maximum reaction rate of removing free radical
By light absorption value A 340(or A 415) normalized form (M=A that converts with amount of substance concentration M 340(or A 415)/ε 340(or ε 415)) calculate reacted amount of substance concentration M (mol/L), wherein, ε 340=4.8 * 10 4(mol/L) -1Cm -1, ε 415=3.6 * 10 4(mol/L) -1Cm -1).This albumin is maximum reaction rate Vmax except the kinetic parameter of ABTS free radical, and ABTS working fluid concentration M of free radical after reaction that can calculate by general Hill equation different extension rates obtains.
6. detection method according to claim 5 is characterized in that: described salmonella flagellin Flagellin derivant albumen has that to remove the dynamic (dynamical) standard of ABTS free radical be that the salmonella flagellin Flagellin derivant albumin of 2 μ M is except the maximum reaction rate of ABTS free radical〉0.3 μ Ms -1
7. detection method according to claim 1 is characterized in that: wherein, detect for the activity of salmonella flagellin Flagellin derivant albumin except the DPPH free radical, can may further comprise the steps:
1) salmonella flagellin Flagellin derivant protein sample to be measured is made into the sample solution of 10,25,50,75,100,125 μ g/mL; With the methyl alcohol of analyzing pure level just DPPH be dissolved into the DPPH free-atom aqueous solution of 0.1mM;
2) six centrifuge tube difference labels, the DPPH free-atom aqueous solution that adds equal-volume 470 μ l, then the salmonella flagellin Flagellin derivant protein solution that adds successively respectively above-mentioned variable concentrations, measure its absorbance in the 517nm place behind the reaction 5min under the room temperature, its value is respectively blank A 0, and sample A 1
3) calculate removing hydroxy radical activity: be calculated as follows the DPPH free radical scavenging activity:
DPPH removes ratio=(1-A 1/ A 0) * 100%.
8. detection method according to claim 7, it is characterized in that: described salmonella flagellin Flagellin derivant albumen is to the DPPH free radical scavenging activity〉1.5% have the standard of removing the hydroxy radical activity for salmonella flagellin Flagellin derivant albumen.
9. each described method of claim 1-8 is to the tracking of salmonella flagellin Flagellin derivant albumen at protein biological activity, the activity of salmonella flagellin Flagellin derivant albumen is confirmed in the preparation of salmonella flagellin Flagellin derivant protein production, storage, the transportation, and the application of the activity that contains the biological products of salmonella flagellin Flagell in derivant albumen in detecting.
10. the salmonella flagellin Flagellin derivant albumen that comprises CBLB502 albumen and CZLC331 albumen has anti-oxidant application with removing in the free radical function medicament in preparation, it is characterized in that: described have a drug candidate that medicine anti-oxidant and that remove the free radical function comprises the panimmunity systemic disease that the treatment oxidative stress is induced, medicine in the disease of immune system that the treatment oxidative stress is induced, the panimmunity systemic disease that the treatment oxidative stress is induced etc. causes the medicine of the relevant disease that the body free radical increases, and also comprises their medical product.
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