CN101186648A - Pseudomonas aeruginosa resisting Fab' fragment - Google Patents
Pseudomonas aeruginosa resisting Fab' fragment Download PDFInfo
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- CN101186648A CN101186648A CNA2007100931304A CN200710093130A CN101186648A CN 101186648 A CN101186648 A CN 101186648A CN A2007100931304 A CNA2007100931304 A CN A2007100931304A CN 200710093130 A CN200710093130 A CN 200710093130A CN 101186648 A CN101186648 A CN 101186648A
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Abstract
The invention provides a specific antibody of bacillus pyocyaneus, in particular to a bacillus pyocyaneus resistant Fab' fragment, which is extracted from the egg of laying hens immunized via bacillus pyocyaneus antigen, with high yield, better effect, stable physicochemical property, high specificity, simple preparation, better repeatability, and industrialization production support. The inventive bacillus pyocyaneus resistant Fab' fragment can significantly resist the growth of bacillus pyocyaneus, by external measurement, which can be used to prepare the drug treating bacillus pyocyaneus resistant infection.
Description
Technical field
The present invention relates to a kind of immunoglobulin (Ig), be specifically related to a kind of Fab ' fragment of immunoglobulin (Ig).
Background technology
Pseudomonas aeruginosa is the most a kind of conditioned pathogens that see in the Rhodopseudomonas more, is distributed widely in occurring in nature, can propagate by approach such as environmental pollution, cross infection, autogenous infection and nosocomial infections.Pseudomonas aeruginosa resistibility to external world is strong than other bacteriums, and at humidity place energy long-term survival, strong to dry resistibility, nutritional requirement is low, because of features such as its multi-drug resistants, has become the main pathogenic bacterium of nosocomial infection at present.The relation of charrin's disease and burn, immunologic hypofunction, septicemia, lower respiratory infection etc. is very close, and case fatality rate is high.Though bring in constant renewal at the microbiotic that charrin's disease adopts, because Pseudomonas aeruginosa has the wide spectrum resistance, result of treatment is often not good, makes searching microbiotic alternative medicine become and press for.
Unique advantages such as Fab ' fragment has that cost is low, output is high, easy preparation, homogeneity and good stability, high specificity, curative effect is reliable simultaneously, price suitable.Successfully extract anti-small protein, antiviral Fab ' at present, and find that it has good biological effect, be used for oral control enteropathogen and obtained good result.Along with increasing the weight of gradually of bacterial resistance phenomenon, seek the focus that a kind of novel antibacterial treatment approach that can antibioticly can evade bacterial resistance has again become research.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of Fab ' fragment of anti Bacillus pyocyaneu Flugge.
The technical scheme that the present invention solves the problems of the technologies described above is:
Pseudomonas aeruginosa resisting Fab ' fragment is characterized in that: (1) described pseudomonas aeruginosa resisting Fab ' fragment energy specificity is in conjunction with Pseudomonas aeruginosa; (2) described pseudomonas aeruginosa resisting Fab ' fragment is to obtain by the following method: collect the egg that is produced with Pseudomonas aeruginosa after as the antigen immune laying hen, from its yolk, separate water-soluble part then, and precipitate with sodium sulfate, taking precipitate, use the pepsin hydrolysis throw out at last, collecting molecular weight is the hydrolysate of 44KDa.
Pseudomonas aeruginosa resisting Fab ' fragment of the present invention is from obtaining as extracting the egg of the laying hen of antigen immune with Pseudomonas aeruginosa, and concrete grammar is:
(1) with the laying hen of Pseudomonas aeruginosa, gets its egg, isolate egg yolk liquid as antigen immune;
(2) add the water dissolution egg yolk liquid of 9 times of volumes of egg yolk liquid, regulate pH5.0,4 ℃ and placed 6 hours;
(3) 4 ℃ of following 10000 rev/mins of centrifugal 25min get supernatant liquor; (4) add 18% sodium sulfate, dissolving, room temperature is placed 1h;
(5) the following 10000 rev/mins of centrifugal 15min of normal temperature get precipitation;
(6) with and PBS dissolution precipitation egg yolk liquid isopyknic 0.01M, pH7.4 used, add 14% sodium sulfate with step (2), dissolving, room temperature is placed 1h;
(7) the following 10000 rev/mins of centrifugal 15min of normal temperature get precipitation;
(8) set by step 20 of (7) gained precipitation quality extraordinarily go into stomach en-, and use water dissolution, and regulator solution pH3.5 adds an amount of toluene, 30 ℃ of water-bath 1.5h;
(9) regulate pH5.0,57 ℃ of heating 30min behind adding 15% ammonium sulfate, be cooled to below 45 ℃ then rapidly;
(10) 4 ℃ of following 10000 rev/mins of centrifugal 15min get supernatant liquor;
(11) regulate supernatant liquor pH7.2, add 20% ammonium sulfate, 4 ℃ of following 8000 rev/mins of centrifugal 15min get precipitation;
(12) use the deionized water dissolution precipitation, adding alum to the concentration of alum in solution is 0.8～1g/100ml, regulates pH7.8, and the following 8000 rev/mins of centrifugal 15min of 4C get supernatant liquor;
(13) add 38% ammonium sulfate, 4 ℃ of following 8000 rev/mins of centrifugal 15min get the degerming of precipitation diafiltration, get final product;
Material percentage concentration in the aforesaid method is mass/volume per-cent (W/V).
Advantages such as pseudomonas aeruginosa resisting Fab ' fragment of the present invention has the output height, tires, physico-chemical property is stable, high specificity, and pass through the growth of the relevant remarkable antagonism Pseudomonas aeruginosa of experiment in vitro proof pseudomonas aeruginosa resisting Fab ' of the present invention, can be used for preparing the medicine for the treatment of charrin's disease.
Description of drawings
Fig. 1 is the pseudomonas aeruginosa resisting Fab ' fragment anti Bacillus pyocyaneu Flugge of the present invention detection curve of tiring.
Fig. 2 is pseudomonas aeruginosa resisting Fab ' fragment immunoblotting of the present invention figure as a result.
Fig. 3 is the growth curve of Pseudomonas aeruginosa under pseudomonas aeruginosa resisting Fab ' fragment effect of the present invention, curve wherein ◆ be the growth curve of the Pseudomonas aeruginosa of normal growth, curve ■ is the growth curve of Pseudomonas aeruginosa under 5mg/ml pseudomonas aeruginosa resisting Fab ' fragment effect of the present invention, curve ▲ be the growth curve of Pseudomonas aeruginosa under 10mg/ml pseudomonas aeruginosa resisting Fab ' fragment effect of the present invention, curve ■ is the growth curve of Pseudomonas aeruginosa under 20mg/ml pseudomonas aeruginosa resisting Fab ' fragment effect of the present invention.
The embodiment epidemic focus is aided with that complete freund adjuvant is subcutaneous in the dipteron wing, immunity is carried out in the injection of both thighs muscle many places, and per two all booster immunizations are 1 time after the first immunisation, amount to immunity 4 times.Begin to collect egg after the first immunisation 2 weeks and preserve standby.
(2) get the egg yolk liquid of step (1) gained egg, add the water dissolution of 9 times of volumes of egg yolk liquid, regulate pH5.0,4 ℃ and placed 6 hours;
(3) 4 ℃ of following 10000 rev/mins of centrifugal 25min get supernatant liquor;
(4) add 18% sodium sulfate, dissolving, room temperature is placed 1h;
(5) the following 10000 rev/mins of centrifugal 15min of normal temperature get precipitation;
(6) with the PBS dissolution precipitation of the isopyknic 0.01M of the used egg yolk liquid of step (2), pH7.4, add 14% sodium sulfate, dissolving, room temperature is placed 1h;
(7) the following 10000 rev/mins of centrifugal 15min of normal temperature get precipitation;
(8) set by step 20 of (7) gained precipitation quality extraordinarily go into stomach en-, and use water dissolution, and regulator solution pH3.5 adds an amount of toluene, 30 ℃ of water-bath 1.5h;
(9) regulate pH5.0,57 ℃ of heating 30min behind adding 15% ammonium sulfate, be cooled to below 45 ℃ then rapidly;
(10) 4 ℃ of following 10000 rev/mins of centrifugal 15min get supernatant liquor;
(11) regulate supernatant liquor pH7.2, add 20% ammonium sulfate, 4 ℃ of following 8000 rev/mins of centrifugal 15min get precipitation;
(12) use the deionized water dissolution precipitation, adding alum to the concentration of alum in solution is 0.8～1g/100ml, regulates pH7.8, and 4 ℃ of following 8000 rev/mins of centrifugal 15min get supernatant liquor;
(13) add 38% ammonium sulfate, 4 ℃ of following 8000 rev/mins of centrifugal 15min get the degerming of precipitation diafiltration and get final product.
The egg yolk liquid extract that aforesaid method is obtained determines the original yolk liquid of protein content average out to 2.85mg/ml in the extract by the UV-light dual wavelength; SDS-PAGE electrophoresis showed yolk liquid extract is rendered as ten minutes protein band clearly substantially, shows its purity up to more than 95% by the scanning of gel imaging instrument, and comparison standard protein electrophoretic band is measured its molecular weight and is about 44kDa.
The evaluation of example 2 pseudomonas aeruginosa resisting Fab ' fragments of the present invention
1, to detect that the highest positive of pseudomonas aeruginosa resisting Fab ' tires be 1: 25600 to indirect elisa method.
With the pseudomonas aeruginosa resisting Fab ' doubling dilution is 1/400,1/1600,1/6400,1/25600,1/51200,1/102400, its absorbance under 450nm of indirect ELISA reaction detection, concrete outcome as shown in Figure 1, logarithmic value with extension rate is a transverse axis, with the absorbancy is that the longitudinal axis is made a smooth curve, and the absorbancy change is an oblique line substantially with extension rate as can be seen).Indirect elisa method detects the positive judgement of tiring of antiendotoxic Fab ': (actual measurement OD value-blank OD value)/(negative control OD value-blank OD value)＞2.1.Negative control is not immune chicken Fab ' (1: 100).It is 1: 25600 that the highest positive that draws Fab ' according to positive criterion is tired.
The preparation of example 1 pseudomonas aeruginosa resisting Fab ' fragment of the present invention
Pseudomonas aeruginosa resisting Fab ' fragment of the present invention is from obtaining as extracting the egg of the laying hen of antigen immune with Pseudomonas aeruginosa, and concrete grammar is:
(1) Pseudomonas aeruginosa (ATCC27853, southwestern hospital laboratory is preserved the standard bacterial classification) is standby as immunogen after ultrasonication behind furnishing 1.5 * 108/ml bacterium liquid after the pure culture.Began immunity after 25 ages in week, the leghorn routine was raised a week.With immunogen be aided with that complete freund adjuvant is subcutaneous in the dipteron wing, the injection of both thighs muscle many places carries out immunity, per two all booster immunizations are 1 time after the first immunisation, amount to immunity 4 times.Begin to collect egg after the first immunisation 2 weeks and preserve standby.
(2) get the egg yolk liquid of step (1) gained egg, add the water dissolution of 9 times of volumes of egg yolk liquid, regulate pH5.0,4 ℃ and placed 6 hours;
(3) 4 ℃ of following 10000 rev/mins of centrifugal 25min get supernatant liquor;
(4) add 18% sodium sulfate, dissolving, room temperature is placed 1h;
(5) the following 10000 rev/mins of centrifugal 15min of normal temperature get precipitation;
(6) with the PBS dissolution precipitation of the isopyknic 0.01M of the used egg yolk liquid of step (2), pH7.4, add 14% sodium sulfate, dissolving, room temperature is placed 1h;
(7) the following 10000 rev/mins of centrifugal 15min of normal temperature get precipitation;
(8) set by step 20 of (7) gained precipitation quality extraordinarily go into stomach en-, and use water dissolution, and regulator solution pH3.5 adds an amount of toluene, 30 ℃ of water-bath 1.5h;
(9) regulate pH5.0,57 ℃ of heating 30min behind adding 15% ammonium sulfate, be cooled to below 45 ℃ then rapidly;
(10) 4 ℃ of following 10000 rev/mins of centrifugal 15min get supernatant liquor;
(11) regulate supernatant liquor pH7.2, add 20% ammonium sulfate, 4 ℃ of following 8000 rev/mins of centrifugal 15min get precipitation;
(12) use the deionized water dissolution precipitation, adding alum to the concentration of alum in solution is 0.8～1g/100ml, regulates pH7.8, and 4 ℃ of following 8000 rev/mins of centrifugal 15min get supernatant liquor;
(13) add 38% ammonium sulfate, 4 ℃ of following 8000 rev/mins of centrifugal 15min get the degerming of precipitation diafiltration and get final product.
The egg yolk liquid extract that aforesaid method is obtained determines the original yolk liquid of protein content average out to 2.85mg/m1 in the extract by the UV-light dual wavelength; SDS-PAGE electrophoresis showed yolk liquid extract is rendered as ten minutes protein band clearly substantially, shows its purity up to more than 95% by the scanning of gel imaging instrument, and comparison standard protein electrophoretic band is measured its molecular weight and is about 44kDa.
The evaluation of example 2 pseudomonas aeruginosa resisting Fab ' fragments of the present invention
1, to detect that the highest positive of pseudomonas aeruginosa resisting Fab ' tires be 1: 25600 to indirect elisa method.
With the pseudomonas aeruginosa resisting Fab ' doubling dilution is 1/400,1/1600,1/6400,1/25600,1/51200,1/102400, its absorbance under 450nm of indirect ELISA reaction detection, concrete outcome as shown in Figure 1, logarithmic value with extension rate is a transverse axis, with the absorbancy is that the longitudinal axis is made a smooth curve, and the absorbancy change is an oblique line substantially with extension rate as can be seen).Indirect elisa method detects the positive judgement of tiring of antiendotoxic Fab ': (actual measurement OD value-blank OD value)/(negative control OD value-blank OD value)＞2.1.Negative control is not immune chicken Fab ' (1: 100).It is 1: 25600 that the highest positive that draws Fab ' according to positive criterion is tired.2, western blot test detects
(1) be ready to transfer device and pvdf membrane, stack successively from the positive pole to the negative pole in the following order: 1 thickening filter paper+pvdf membrane+gel+1 thickening filter paper is put in negative pole on the interlayer thing, Yi Bian graphite down.Connect power supply, change 20min with the 120mA electricity.
(2) deenergization and pull up plug on the groove is dismantled transfer device from top to bottom, gel is transferred in the Xylene Brilliant Cyanine G dye liquor dyes, so that check whether protein shifts complete.
(3) cut the pvdf membrane lower left corner, with label orientation.Pvdf membrane is put into the plastics bag (confining liquid is arranged) that can add heat-seal, get rid of the bubble of the inside as far as possible, airtight then sack lies on the shaking table that shakes gently in 37 ℃ of incubation 1h.
4. the taking-up pvdf membrane is used deionized water wash 5min again.With pvdf membrane and 37 ℃ of incubation 1h of the anti-chicken IgY of rabbit ELIAS secondary antibody (diluting 1: 1000), add substrate solution colour developing 20min, air-dry preservation.
As shown in Figure 2, pseudomonas aeruginosa resisting Fab ' fragment of the present invention can combine the back with the anti-chicken IgG of rabbit ELIAS secondary antibody and react with substrate, shows that pseudomonas aeruginosa resisting Fab ' and chicken IgG have same antigen, have good specificity to the antigen Pseudomonas aeruginosa simultaneously.
The extracorporeal biology effect experiment of example 3 pseudomonas aeruginosa resisting Fab ' fragments of the present invention
1, experimental technique:
Experiment divides 2 groups, and wherein one group is control group: 3.9 * 105cfu/ml Pseudomonas aeruginosa liquid is hatched in 37 ℃ of thermostat containers, in 6,12,24,36h, and sampling back live bacterial count.Another group is for Fab ' fragment antagonism group of the present invention: with Pseudomonas aeruginosa (3.9 * 105cfu/ml) add Fab ' fragment 5,10 of the present invention, 20mg/ml respectively, in 6,12,24,36h, sampling back live bacterial count.
2, experimental result: as shown in Figure 3, pseudomonas aeruginosa resisting Fab ' can obviously suppress the growth of Pseudomonas aeruginosa, has good extracorporeal biology effect.
Claims (3)
1. pseudomonas aeruginosa resisting Fab ' fragment is characterized in that: (1) described pseudomonas aeruginosa resisting Fab ' fragment can specificity in conjunction with Pseudomonas aeruginosa; (2) described pseudomonas aeruginosa resisting Fab ' fragment is to obtain by the following method: collect the egg that is produced with Pseudomonas aeruginosa after as the antigen immune laying hen, from its yolk, separate water-soluble part then, and precipitate with sodium sulfate, taking precipitate, use the pepsin hydrolysis throw out at last, collecting molecular weight is 44 daltonian hydrolysates.
2. the preparation method of the described pseudomonas aeruginosa resisting Fab ' fragment of claim 1, this method is made up of following steps:
(1) with the laying hen of Pseudomonas aeruginosa, gets its egg, isolate egg yolk liquid as antigen immune;
(2) add the water dissolution egg yolk liquid of 9 times of volumes of egg yolk liquid, regulate pH5.0,4 ℃ and placed 6 hours;
(3) 4 ℃ of following 10000 rev/mins of centrifugal 25min get supernatant liquor;
(4) add 18% sodium sulfate, dissolving, room temperature is placed 1h;
(5) the following 10000 rev/mins of centrifugal 15min of normal temperature get precipitation;
(6) with the PBS dissolution precipitation of the isopyknic 0.01M of the used egg yolk liquid of step (2), pH7.4, add 14% sodium sulfate, dissolving, room temperature is placed 1h;
(7) the following 10000 rev/mins of centrifugal 15min of normal temperature get precipitation;
(8) set by step 20 of (7) gained precipitation quality extraordinarily go into stomach en-, and use water dissolution, and regulator solution pH3.5 adds an amount of toluene, 30 ℃ of water-bath 1.5h;
(9) regulate pH5.0,57 ℃ of heating 30min behind adding 15% ammonium sulfate, be cooled to below 45 ℃ then rapidly;
(10) 4 ℃ of following 10000 rev/mins of centrifugal 15min get supernatant liquor;
(11) regulate supernatant liquor pH7.2, add 20% ammonium sulfate, 4 ℃ of following 8000 rev/mins of centrifugal 15min get precipitation;
(12) use the deionized water dissolution precipitation, adding alum to the concentration of alum in solution is 0.8～1g/100ml, regulates pH7.8, and 4 ℃ of following 8000 rev/mins of centrifugal 15min get supernatant liquor;
(13) add 38% ammonium sulfate, 4 ℃ of following 8000 rev/mins of centrifugal 15min get the degerming of precipitation diafiltration and get final product;
Material percentage concentration in the aforesaid method is mass/volume per-cent.
3. the application of the described pseudomonas aeruginosa resisting Fab ' fragment of claim 1 in preparation treatment charrin's disease medicine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497909B (en) * | 2009-03-05 | 2012-07-04 | 中国人民解放军军事医学科学院生物工程研究所 | Method for preparing anti-A type botulinus toxin immunoglobulin antibody |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101497909B (en) * | 2009-03-05 | 2012-07-04 | 中国人民解放军军事医学科学院生物工程研究所 | Method for preparing anti-A type botulinus toxin immunoglobulin antibody |
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