CN1362418A - Anticholera vibrio, its toxic chicken yolk antibody and the prepn and application - Google Patents
Anticholera vibrio, its toxic chicken yolk antibody and the prepn and application Download PDFInfo
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Abstract
The present invention relates to a fowl egg-yolk antibody (anti-VoIGY and anti-CTIgY) for resisting cholera vibrio and its toxin, its preparation method and application in production of medicine or health-care product for curing related diseases resulted from cholera vibrio infection and preparation for detecting the above-mentioned diseases. Said ivnention utilizes PAGE electrophoresis to detect purity of purified egg-yolk antibody, in the electrophoretic zone only one electrophoretic band is produced, and utilzies UV spectrophotometer to make determination and calculate its protein concentration (mg/ml) which is (1.45XOD 280nm-0.74XOD260nm)X dilution factor. The above-mention method incldues the following steps: preparing cholera vibrio and its toxin antigen, immunizing health hen with said antigen, collecting egg and extracting anti VcIgY and anti-CTIgY from said egg.
Description
(1), technical field
The present invention relates to a kind of yolk antibody material---anti-vibrio cholerae (V.cholerae with antibody activity, Vc) and toxin (cholera toxin, CT) chicken yolk antibody is (anti--VcIgY, anti--CTIgY) product, and relate to its preparation technology and in pharmaceutically application.
(2), technical background
Cholera is by the caused severe intestinal transmissible disease of vibrio cholerae.Morbidity is anxious, propagation is fast, is the major reason of suffering from diarrhoea in the most of area in Asia, Africa.Worldwide being very popular can be taken place, and belongs to the international quarantine transmissible disease.In China's Law on the Prevention and Control of Infectious Diseases, classify the Class A as.The contagium of cholera is patient and carrier, and carrier and light disease patient are many far beyond typical case (in, heavy type) patient, so cholera has more disguise, and control and the difficulty of eliminating are also bigger.This also may be that worldwide being very popular of cholera failed one of major reason of controlling so far.Whether the crowd falls ill after the infection and depends on the quantity of human immunity state (resistibility) and infection pathogen the general susceptible of cholera.This sick inapparent infection accounts for 75%, promptly asymptomatic person, and apparent infection (symptom is arranged) accounts for 25%.Therefore the treatment measure of cholera is the field that people pay close attention to always, at present main treatment measure is fluid replacement therapy and antibiotic treatment, and mainly be the measures such as oral killed vaccine, oral attenuation gene engineering vaccine and natural attenuated live vaccine of adopting in the research of prevention, but the effect of present used vaccine is all undesirable, among comparatively effectively the intestine immunity preparation is being studied at present.
Cholera pathogenic bacteria (comprising vibrio cholerae of classic biotype, El Tor biotype vibrio cholerae epidemic strain, O139 group cholera vibrio) has unique molecular biological characteristics and virulence characteristic.The cholera pathogenesis is after the cholera pathogenic bacteria enters the human small intestine, to adhere to intestinal mucosa, breeds in a large number and produce to cause to rush down extremely strong enterotoxin (CT).CT is made up of two subunits of A, B, and its structural group becomes an A subunit (CTA, Mr:27.2 * 10
3), be positioned at the centre of toxin, connect 5 B subunit (CTB, Mr:11.6 * 10 in the form of a ring on every side
3), B subunit can with the GM1 receptors bind on the small intestinal cell, help A subunit and enter cell performance toxic action; A subunit can activate the adenosine cyclase in the born of the same parents, makes cAMP concentration rising in the born of the same parents, and emiocytosis increases, and a large amount of intestinal juice gather, and form serious watery diarrhea syndromes, so the CT lps molecule is the main virulence factor of vibrio cholerae.
Chicken yolk antibody (IgY) is the IgG (immunoglobulin G) from chicken serum, and the structure of IgY is similar to IgG, has the space conformation of typical immunoglobulin (Ig).Studies show that IgY has the stability of height, to having good tolerance in heat, acid, the alkali environment, IgY also has the advantage that cost is low, be easy to form large-scale production simultaneously.Particulate antigens such as domestic existing virus, bacterium are induced the research report of Ovum Gallus domesticus Flavus expression specificity antibody at present.
Not seeing in the prior art has by adopting vibrio cholerae and toxin thereof as antigen, and the immune health hen is collected the ovum that it produces, and extracts the report of anti--VcIgY and anti--CTIgY method again from ovum.
(3), Fa Ming content
The present invention is antigen by with Vc or extraction Vc cytotoxin PROTEIN C T with them, the immune health bird inlay, the extraction activeconstituents (anti--VcIgY, anti--CTIgY, anti--CTIgY).Provide new approaches and novel method for treatment Vc infects, and be that the immunity biological agent that further prepares anti-Vc lays the foundation.Therefore:
One of technical problem to be solved by this invention is: at the prior art above shortcomings, provide a kind of anti-vibrio cholerae with antibody activity and toxin chicken yolk antibody thereof (anti--VcIgY, anti--CTIgY).
Two of technical problem to be solved by this invention is: provide above-mentioned anti-vibrio cholerae and toxin chicken yolk antibody thereof (anti--VcIgY, anti--CTIgY) preparation method.
Three of technical problem to be solved by this invention is: provide above-mentioned anti-vibrio cholerae and toxin chicken yolk antibody thereof (anti--VcIgY, anti--as CTIgY) to be used for using because of the prevention of infecting the severe intestinal transmissible disease that vibrio cholerae causes or the pharmaceutical preparation or the healthcare products of treatment in preparation, and the application that is used for the detection reagent of the clinical diagnosis of vibrio cholerae or detection in preparation.
The present invention one of solve the problems of the technologies described above the anti-vibrio cholerae that provided and toxin chicken yolk antibody thereof (anti--VcIgY, anti--CTIgY), it is characterized in that: described resisting-VcIgY, anti--CTIgY press YSDS-PAGE electrophoresis (7.5% separation gel) and detect, and collection of illustrative plates presents single district band; Be blue through electrophoresis Coomassie brilliant blue R250 dyeing; High molecular band place clear single specific enzymes occurred and dyes band during immunity marking Western-blotting detected; Carrying out dual wavelength mensuration with ultraviolet spectrophotometer to calculate protein concentration (mg/ml) is: (1.45 * OD
280nm-0.74 * OD
260nm) * extension rate.
What the present invention solved the problems of the technologies described above two is by adopting such technical scheme to realize, promptly a kind of anti-vibrio cholerae of preparation and toxin chicken yolk antibody thereof (anti--VcIgY, anti--CTIgY) method, it is characterized in that adopting following step to finish:
1. prepare the Vc O antigen
Produce cholera pathogenic bacteria (vibrio cholerae of classic biotype, El Tor biotype vibrio cholerae epidemic strain, O139 group cholera vibrio) bacterial strain, coat on the TCBS agar, 37C cultivates 24h, must pure strain after, enlarged culturing 24h in the TCBS broth culture.Collect the Vc bacterium liquid of thalline, ultrasonic disruption added complete freund adjuvant by weight 1: 1, and emulsification gets O antigen.
2. prepare CT toxin antigen
(1) extraction method
1. the cultivation of .Vc cell strain:
Produce cholera pathogenic bacteria bacterial strain, coat on the TCBS agar, cultivate 24h for 37 ℃, behind the pure strain, enlarged culturing 24h in the TCBS broth culture.Collect culture supernatant, separate obtaining toxin by ultrafiltration with affinity chromatography, added complete freund adjuvant by weight 1: 1, emulsification gets CT toxin antigen.
2.. the preparation of toxic protein: A. ultrafiltration process: according to the molecular weight (78000) of the CT that has known, the method preparation of employing membrane filtration.B, chromatographic separation method: with reference to the method for Kelstein etc., adopt affinity column separation and Extraction CT toxin, last lyophilize is preserved.
Or (2) synthesis method: make up the gene expression plasmid that contains CT cytotoxin A subunit and B subunit, in the intestinal bacteria system, express, expression product is through separation and purification, preparation CT toxin A subunit (CTA) and B subunit (CTB) toxin, independent CTB or mixed as immunizing antigen by weight 1: 1, add isopyknic freund adjuvant, the emulsification mixing gets CT toxin antigen.
3. O antigen and CT toxin antigen are prepared antibody by following step:
(1). to the immunity of the hen that lays eggs:
Select the hen that lays eggs (health) isolated rearing for use.Carry out multi-point injection under the cock skin, as 5 points, thalline is 10
9CFU/ml, the toxin protein amount is equivalent to 10
10The CFU/ml bacterium produces.Per two all booster immunizations once amount to immunity 8 times later on; Began to collect egg after 7 days, 4 ℃ store for future use.
(2). the extraction of yolk antibody
1.. adopt water-soluble method to extract yolk antibody.From immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-VcIgY, anti--CTIgY.Or 2. adopt membrane filter method to extract yolk antibody.Separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, lyophilize is resisted-VcIgY, anti--CTIgY.
Above-mentioned resisting provided by the present invention-VcIgY and anti--CTIgY product are laid eggs hen by vibrio cholerae and toxin immunity thereof and are got, and have specificity, the control vibrio cholerae is infected the severe intestinal transmissible disease that causes have good effect.It is carrier that the present invention adopts ovum gallinaceum that hen produces, and safety non-toxic is easy to industrialization.
(4), the description of the drawings
Accompanying drawing has provided preparation method's of the present invention process flow sheet.
(5), embodiment
Below in conjunction with accompanying drawing embodiments of the invention are further described this:
Embodiment 1: the product with vibrio cholerae cytotoxin yolk antibody of antibody activity, can obtain by following steps: preparation vibrio cholerae thalline and/or cytotoxin antigen, with the antigen immune healthy hens, collect it then and lay eggs, from it is laid eggs, extract anti--VcIgY and anti--CTIgY again.
The preparation of embodiment 2:Vc O antigen is produced cholera pathogenic bacteria bacterial strain, enlarged culturing 24h in the TCBS broth culture.Collect the Vc bacterium liquid of thalline, ultrasonic disruption added complete freund adjuvant by weight 1: 1, and emulsification gets O antigen.
Embodiment 3: the antigenic preparation of cytotoxin CT, separate and obtain toxogenic CT bacterial strain, coat on the TCBS agar, cultivate 24h for 37 ℃, behind the pure strain, enlarged culturing 24h in the TCBS broth culture, collect culture supernatant, obtain toxin by ultrafiltration and chromatographic separation, added complete freund adjuvant by weight 1: 1, emulsification gets CT toxin antigen.
Embodiment 4: make up the gene expression plasmid that contains CT cytotoxin A subunit and B subunit, in the intestinal bacteria system, express, expression product is through separation and purification, preparation CT toxin A subunit (CTA) and B subunit (CTB) toxin, independent CTB or mixed as immunizing antigen by weight 1: 1, add isopyknic freund adjuvant, the emulsification mixing gets CT toxin antigen.
Embodiment 5: in the following way separation and Extraction anti--VcIgY and anti--CTIgY: from immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-VcIgY and resisting-CTIgY.
Embodiment 6: separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, lyophilize is resisted-VcIgY and anti--CTIgY.
Embodiment 7: the product of getting among the embodiment 1,5,6 carries out the IgY purity test: use the purity of the yolk antibody of PAGE electrophoresis detection purifying, only occur an electrophoresis band in the electrophoresis, be the yolk antibody of purification.
Embodiment 8: the product of getting among the embodiment 1,5,6 carries out the inspection of IgY concentration: that will extract does 5-10 dilution doubly with PBS, measures to calculate protein concentration with ultraviolet spectrophotometer then.
Protein concentration (mg/ml)=(1.45 * OD
280nm-0.74 * OD
260nm) * extension rate
Embodiment 9: the product of getting among the embodiment 1,5,6 carries out the IgY titration: adopt Ovshinsky double diffusion method to carry out the IgY titration, it is 1 that IgY tires: 640-1: 25600.
Embodiment 10: gather 50 on the immune egg of anti--VcIgY, separate and obtain 800ml yolk, thin up is to 5000ml, the centrifugal clear liquid 4000ml that obtains, the employing Hollow Fiber Ultrafiltration extracts, it is stand-by to be concentrated into 400ml at last, wherein contain essence carry (promptly refer to purity the IgY more than 99.5% anti--VcIgY) 12 grams.Maltose 10 grams are added the 5ml water dissolution, sterilized 30 minutes for 100 ℃, auxiliary material and the 50ml ultrafiltrated of handling mixed, lyophilize, aseptic pulverizing is packaged into bag, and every pouch includes 0.5 gram, is used for the treatment of the severe intestinal transmissible disease that causes because of the CT infection.
Embodiment 11: gather 50 on the immune egg of anti--CTIgY, separate and obtain 800ml yolk, thin up is to 5000ml, the centrifugal clear liquid 4000ml that obtains, the employing Hollow Fiber Ultrafiltration extracts, it is stand-by to be concentrated into 400ml at last, wherein contain essence carry (promptly refer to purity the IgY more than 99.5% anti--CTIgY) 10 grams.Medical starch 10 grams are added the 5ml water dissolution, sterilized 30 minutes for 100 ℃, auxiliary material and the 50ml ultrafiltrated of handling mixed, lyophilize, aseptic pulverizing is packaged into bag, and every pouch includes 0.5 gram, is used for the treatment of the severe intestinal transmissible disease that causes because of the CT infection.
Embodiment 12: gather 100 on the immune egg of anti--VcIgY, obtain 1600ml yolk, add 2 times of PBS diluents, and add 3.5%PEG6000 and stir and left standstill 2 hours, the centrifugal clear liquid 3500ml that obtains, add again PEG6000 to final concentration be 12%, left standstill centrifugal collecting precipitation 2 hours.With 100ml dissolved in distilled water IgY precipitation, by the biofilter degerming, lyophilize is slightly carried anti--VcIgY with each conventional capsule dress 200mg and is carried out can, and products obtained therefrom is used to prevent CT to infect, human body instructions of taking every day 2-3 time, each 3 capsules.
Embodiment 13: gather 100 on the immune egg of anti--CTIgY, obtain 1600ml yolk, add 2 times of PBS diluents, and add 3.5%PEG6000 and stir and left standstill 2 hours, the centrifugal clear liquid 3500ml that obtains, add again PEG6000 to final concentration be 12%, left standstill centrifugal collecting precipitation 2 hours.With 100ml dissolved in distilled water IgY precipitation, by the biofilter degerming, lyophilize is slightly carried anti--CTIgY with each conventional capsule dress 250mg and is carried out can, and products obtained therefrom is used to prevent CT to infect, human body instructions of taking every day 2-3 time, each 4 capsules.
Embodiment 14: experiment in vitro:
1.. molecular weight detection: SDS-PAGE electricity arteries and veins, separation gel length 8% concentrates gum concentration 5%, current stabilization 11mA electricity arteries and veins 5h, Marker adopts the low relative molecular mass standard protein, takes off gel and is used for conventional silver staining and electrotransfer electricity arteries and veins.
2.. purity check: immunoblotting, reference literature carries out.
3.. the active detection: ELISA indirect test, working method is with reference to conventional ELISA indirect method, used antigen, anti--VcIgY (or anti--CTIgY), enzyme labelled antibody all determines working concentration by the chessboard volumetry, the toxin antigen concentration is 100ug/ml, bag is by the every hole of plate application of sample 100ml, tested anti--VcIgY concentration is 5ug/ml (anti--CTIgY is 8ug/ml), every hole application of sample 100ml, it is 1: 1000 that peppery plate peroxidase is marked anti-IgY-IgG (Sigma company) working dilution, substrate is OPD, 2ml/ vitriol oil termination reaction, microplate reader 492nm measures the OD value down.
Embodiment 15:CT cytotoxin is to IEC cell strain toxicity and the provide protection of IgY antibody:
1.. morphological observation: normal IEC cell is adherent polymorphism growth, and refractive power is good.Behind the CT detoxifying function, cell is spherical to be become, and occurs many particles in the endochylema, and when concentration＞50ug/ml, the IEC cell is many with cracking, the obvious shrinkage of residual cells volume.Electronic Speculum presents similar change when saturating.
2.. biochemistry detection: by after measuring cell MTT, LDH (serum lactic dehydrogenase), cAMP and also finding the CT detoxifying function, MTT is the decline of carrying out property, and LDH and cAMP concentration continue to increase.
3.. the activity of the release of cytokine (inflammatory mediator): IL-6 (interleukin-6) TNF-2 (tumour necrosis factor-2) detects, and found that the rising that all has in various degree.
Embodiment 16: anti--the external antagonism toxin of CTIgY biological activity test:
It is anti--CTIgY that ((30～90ug/ml) mix under aseptic condition at 1: 1 for 10ug～200ug/ml) and CT toxin.Behind 37 ℃ of 2h, get the 0.1ml supernatant and add in the IEC cell cultures, discovery IgY concentration can be blocked the toxic action of CT toxin to the IEC cell fully at 100～200ug/ml; Thereafter serial dilution, its protection activity also lowers thereupon, and the microscope morphological observation also obtains identical result: normal IEC cell is adherent polymorphism growth, no particle in the cell, and refractive power is good.Behind the CT detoxifying function, cell is spherical to be become, and occurs many particles in the endochylema, and when concentration＞50ug/ml, the IEC cell is many with cracking, the obvious shrinkage of residual cells volume.Behind the antagonism toxin, the form of cell changes and alleviates.
Embodiment 17: anti--VcIgY and anti--CTIgY observe in the body biologic activity: set up the model that rodent Vc infects with reference to pertinent literature, test be divided into the treatment group (low dosage (10mg/Kg), middle dosage (25mg/Kg), high dosage (50mg/Kg) be anti--VcIgY), control group; Treat after 10,20,30 days, observe the diarrhoea situation of animal, intestinal mucosa pathological examination, indexs such as Vc Bacteria Detection and CT content of toxins mensuration.Found that: middle and high dosage group is after treatment 5 days, and symptom of diarrhea alleviates, and the mucous membrane content of toxins obviously descends, and the intestinal mucosa pathological examination index of animal has clear improvement after 10 days, and content of toxins is further to reduce.
Embodiment 18: anti--VcIgY and anti--CTIgY observe with microbiotic combined utilization biologic activity: set up the model that rodent Vc infects with reference to pertinent literature, test be divided into simple anti--VcIgY treatment group, simple anti--CTIgY treatment group, merely microbiotic (Fu side's Sulfamethoxazole) treatment group, unite group.Treat after 5,10,15 days, observe animal diarrhoea situation, intestinal mucosa pathological examination, indexs such as Vc Bacteria Detection and CT content of toxins mensuration.Found that: unite group than two simple groups after treatment 5 days, the diarrhoea situation of animal, the improvement of intestinal mucosa pathology, mucous membrane content of toxins and Vc quantity all have obvious decline; The intestinal mucosa content of toxins of simple resisting-CTIgY treatment group is less than antibiotic therapy group and anti--VcIgY treatment group merely merely.
Claims (4)
1, (anti--VcIgY and resist-CTIgY), it is characterized in that: above-mentioned antibody is pressed YSDS-PAGE electrophoresis (7.5% separation gel) detection to the chicken yolk antibody of a kind of anti-vibrio cholerae and toxin thereof, and collection of illustrative plates presents single district band; Be blue through electrophoresis Coomassie brilliant blue R250 dyeing; High molecular band place clear single specific enzymes occurred and dyes band during immunity marking Western-blotting detected; Carrying out dual wavelength mensuration with ultraviolet spectrophotometer to calculate protein concentration (mg/ml) is: (1.45 * OD
280nm-0.74 * OD
260nm) * extension rate.
2, anti-vibrio cholerae of a kind of preparation and toxin chicken yolk antibody thereof, the method for promptly anti--VcIgY and anti--CTIgY is characterized in that: adopt following processing step:
(1) preparation O antigen
Produce cholera pathogenic bacteria (vibrio cholerae of classic biotype, El Tor biotype vibrio cholerae epidemic strain, O139 group cholera vibrio) bacterial strain, coat on the TCBS agar, cultivate 24h for 37 ℃, behind the pure strain, enlarged culturing 24h in the TCBS broth culture; Collect the VC bacterium liquid of thalline, ultrasonic disruption added complete freund adjuvant by weight 1: 1, and emulsification gets O antigen.
(2) preparation CT toxin antigen:
1.. extraction method: produce malicious cholera pathogenic bacteria bacterial strain and cultivate 24h in the TCBS broth culture, collect culture supernatant, separate obtaining toxin by ultrafiltration with affinity chromatography, added complete freund adjuvant by weight 1: 1, emulsification gets CT toxin antigen.
Or 2.. synthesis method: make up the gene expression plasmid that contains CT cytotoxin A subunit and B subunit, in the intestinal bacteria system, express, expression product is through separation and purification, preparation CT toxin A subunit (CTA) and B subunit (CTB) toxin, independent CTB or mixed as immunizing antigen by weight 1: 1, add isopyknic freund adjuvant, the emulsification mixing gets CT toxin antigen.
(3) again the thalline and the CT toxin antigen of gained is prepared antibody by following step:
1.. to the immunity of the hen that lays eggs:
Select the hen that lays eggs (health) isolated rearing for use, carry out injection under the cock skin, thalline is 10
9CFU/ml, the toxin protein amount is equivalent to 10
10The CFU/ml bacterium produces, and later per two all booster immunizations once amount to immunity 8 times, and 4 ℃ store for future use;
2.. the extraction of yolk antibody
A. adopt water-soluble method to extract yolk antibody; From immune ovum gallinaceum, separate yolk, with distilled water diluting, adjust suitable pH value and leave standstill 8h for 4 ℃, the centrifuging and taking supernatant concentrates, lyophilize, is resisted-VcIgY and anti--CTIgY;
Or B. adopts membrane filter method to extract yolk antibody: separate yolk, with distilled water diluting, 4 ℃ leave standstill 12h after, be that the film of 200KD and 150KD filters respectively with the molecular weight, collect the proteic substance of 150KD-200KD, lyophilize is resisted-VcIgY and anti--CTIgY.
3, anti-vibrio cholerae and toxin chicken yolk antibody thereof, promptly anti--VcIgY and anti--CTIgY are used for the treatment of because of vibrio cholerae in preparation and infect the relative disease medicine that causes or the application in the healthcare products.
4, anti-vibrio cholerae and toxin chicken yolk antibody thereof, promptly anti--VcIgY and anti--CTIgY are used for detecting the application of infecting the preparation of the relative disease that causes because of vibrio cholerae in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343320B (en) * | 2008-07-01 | 2012-06-27 | 华南农业大学 | Anti-vibrio parahaemolyticus chicken yolk antibody, preparation method and application thereof |
| CN103890173A (en) * | 2011-08-19 | 2014-06-25 | 鸵鸟制药株式会社 | Antibody and antibody-containing composition |
-
2001
- 2001-12-21 CN CN 01133725 patent/CN1362418A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343320B (en) * | 2008-07-01 | 2012-06-27 | 华南农业大学 | Anti-vibrio parahaemolyticus chicken yolk antibody, preparation method and application thereof |
| CN103890173A (en) * | 2011-08-19 | 2014-06-25 | 鸵鸟制药株式会社 | Antibody and antibody-containing composition |
| CN103890173B (en) * | 2011-08-19 | 2016-02-03 | 鸵鸟制药株式会社 | Antibody and the composition containing antibody |
| US9828419B2 (en) | 2011-08-19 | 2017-11-28 | Ostrich Pharma Kk | Antibody and antibody-containing composition |
| US10106599B2 (en) | 2011-08-19 | 2018-10-23 | Ostrich Pharma Kk | Antibody and antibody-containing composition |
| US10428138B2 (en) | 2011-08-19 | 2019-10-01 | Ostrich Pharma Kk | Antibody and antibody-containing composition |
| US11041016B2 (en) | 2011-08-19 | 2021-06-22 | Ostrich Pharma Kk | Compositions containing anti-HIV ostrich antibodies |
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