CN101220094A - Preparation method for immunoglobulinlg of adenovirus anti-Fi,anti-Pb and anti-Hx - Google Patents
Preparation method for immunoglobulinlg of adenovirus anti-Fi,anti-Pb and anti-Hx Download PDFInfo
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Abstract
The invention discloses a preparation method for the immunoglobulin of an adenoviruses anti-Fi, anti-Pb and anti-Hx, which firstly extracts a fibrin, a pentamer protein and a hexon from the adenoviruses as antigens, then purifies the antigens with discontinuous density centrifugation with the virus antigens prepared through the tissue culture, and then uses the prepared purified antigens for establishing and screening the method for the immunoglobulin of high titer adenoviruses anti-Fi, anti-Pb and anti-Hx and the virus neutralization experiment in vitro, etc. while at last the immunoglobulin from the immunoglobulin blood source of high titer adenoviruses anti-Fi, anti-Pb and anti-Hx by means of low temperature ethanol technique, ion exchange chromatography or affinity chromatography is extracted.
Description
One, technical field
The invention belongs to virusology and field of immunology, relate to a kind of process for preparing medicine, particularly a kind of preparation method of the adenovirus antibody that is used to prevent and treats.More particularly, the present invention relates to the preparation method of the screening method of source, adenovirus specific immunity blood plasma or body fluid by immune animal or people's the antigenic substance that can produce the adenovirus specific immunoglobulin and separation purification of adenovirus specific immunoglobulin etc.
Two, background technology
Adenovirus (Adenovirus) is an a group distribution dna virus very widely.Can cause the disease of human airway, gi tract, urinary system and eye.Minority has carcinogenesis to animal.Adenovirus is divided into infection birds and mammiferous two genus.Human adenovirus is divided into A～G7 group according to physics, chemistry, biological property, and each group comprises some serotypes, totally 42 types.Part type does not have pathogenicly in the adenovirus hominis, and the most normal is 1～7 type.Child's acute upper respiratory tract infection about 5% is caused that by adenovirus the adult infects rare in respiratory tract, and propagating with excrement is main approach flatly, also can propagate by respiratory tract or contaminated articles.Virus involves other internal organs once in a while in pharynx, especially intestinal epithelial cell internal breeding of conjunctiva, and inapparent infection is common.Disease is generally self limiting, can obtain the long-term type specificity immunizing power that continues after the infection, and the neutralizing antibody damaging action is important.But A, B papova are not found carcinogenesis at some new born animal induced tumor to the people.Adenovirus generally infects by respiratory tract.Adenovirus upper respiratory tract infection and pneumonia often take place in collective children mechanism simultaneously.The explanation of crowd's serological research, the initial several months often retains the adenovirus specific antibody that transmits from parent after giving birth to, and ever since to 2 years old antibody deficiency, just increases gradually later in 2 years old.The clinical observation that this and adenovirus pneumonia 80% occur in 7～24 months infants meets fully.It should be noted that local each age group Susceptible population quantity is many more, the number that the adenovirus respiratory tract infection takes place is just many, and that the chance of adenovirus pneumonia takes place the infant is also big more.Adenovirus pneumonia is more common in winter and spring in northern China, and summer, autumn are only accidental, then is more common in autumn in high popular year in Guangzhou.This parapneumonia accounts for 20%～30% of virus pneumonia in Beijing.
The adenovirus of more than 100 type has constituted the Adenoviridae (Adenoviridae) that contains mammalian adenoviruses (Mastadenovirus) and two genus of aviadenovirus (Aviadenovirus), all have common in each Tobamovirus between all members and belong to antigen (antigenic determinant is positioned at hexon), and no antibody cross reaction between different Tobamovirus members.Adenovirus particles diameter 60～90nm does not have cyst membrane, 20 three-dimensional symmetries of body, and capsid is made up of 252 gram particulates, and wherein 240 shell particulates are six adjacent bodies (Hexon), have group-specific α antigen.Being positioned at 20 vertical 12 gram particulates of body is penton (Penton).Each penton by substrate and stretch out the surface an end the fibrous of heading arranged.Substrate has toxin sample activity, can cause cytopathy, and cell is come off from growth, has total on the same group beta antigen.Fiber is relevant with viral aggegation big white mouse or the erythrocytic activity of rhesus monkey, is type specificity γ antigen place.Nucleic acid is with strand form DNA, molecular weight 20～30 * 106 dalton.
(1), hexon, the sedimentation constant of adenovirus six adjacent bodies is made up of 3 identical subunits about 13s, the molecular weight of each polypeptide is 108kD.Hexon is rich in dicarboxylic amino acid, its N-terminal acetylize.The homology of hexon aminoacid sequence is very high between various virus strain.Hexon can stimulate body to produce neutralizing antibody and group and type specificity antibody.
(2), penton albumen, penton is similar to six adjacent bodies on form, is ball-like structure, diameter average out to 8nm, settling ratio approximately is 9.5s, molecular weight is between 246～256kD.Penton is a homotrimer equally, and the molecular weight of each subunit is 85kD, and comparatively responsive to proteolytic enzyme, the homology of penton Argine Monohydrochloride sequence is higher between different strains.Penton albumen participates in the absorption of the mutual identification of virus and cell and virus and penetrates process.
(3), scleroproein, the scleroproein structure of adenovirus is asymmetric, be arranged in order and the non-covalent bonded afterbody of penton basal seat area, handle and 3 structural domains of top spherical junctions by proteinic aminoterminal to carboxyl terminal, the diameter of handle and spherical junctions is respectively 2nm and 4nm, scleroproein also belongs to homotrimer, molecular weight is between 156～207kD, and its settling ratio is generally about 6s.
In the responsing reaction that the humoral immunity of organism system produces adenovirus infection, 3 kinds of major antigen compositions that are primarily aimed at virus produce antibody, these 3 kinds of antibody are inconsistent mutually when occurring in serum, are followed successively by Biciromab (anti-Fi), anti-penton antibody (anti-Pb) and anti-six adjacent body antibody (anti-Hx).
At present anti-virus infection medicine commonly used is generally chemical synthetic drug, and herbal medicine, and biotechnological formulation such as Interferon, rabbit still do not have a kind of at virus and the sure medicine of curative effect in these medicines.Antiviral immunoglobulin is by behind the virus infection or utilize the protein, polysaccharide, nucleic acid, live vector, infectant etc. of virus to induce, in vivo the main specific antiviral substance of Chan Shenging.Because being widely current of adenovirus, will some people be to obtain anti-virus ability by inapparent infection and produce the anti-adenovirus specific immunoglobulin that height is tired.The screening height anti-adenovirus immunoglobulin (Ig) of tiring from the blood source can obtain extensive blood supply source, can effectively satisfy clinical needs.And the immunity system of utilizing the induced animal such as protein, polysaccharide, nucleic acid, live vector, infectant of virus or people produces the protection antibody at adenovirus; by screening; collection has tire animal or human's body fluid or the blood of adenovirus anti-Fi, anti-Pb and anti-Hx of height, purifies and deactivation may exist virus to make specific anti-adenovirus immunoglobulin (Ig) by separating.
Three, summary of the invention
The object of the invention is, proposes the preparation method of preparation method, screening method and the specific immunoglobulin of the people's that a kind of height of anti-adenovirus specific immunoglobulin tires body fluid or blood.
Technical thought of the present invention is, adenovirus is the common pathogenic agent of infantile viral pneumonia, virus infects by respiratory tract, obvious seasonal is arranged, based on winter, spring, about 80% adenovirus pneumonia occurs in 6 months to 2 years old children's, and older youngster's state of an illness is lighter, or only show as the symptom of upper respiratory tract infection, but the case fatality rate of severe infant is up to 33.6%.Behind virus infection, the scleroproein of adenovirus, penton albumen and hexon are as the activation of antigenic stimulation mature B cell, propagation, under T cell auxiliary, become cell justacrine Ig, after after a while, after body is subjected to adenovirus infection once more, is subjected to scleroproein, penton albumen and the hexon stimulation of adenovirus, the very fast generation antibody of body, and antibody titer is very high, and the time length, lowering speed was also very slow more than 1 month.Normal population all once one or many infected ADV, and ubiquity ADV inapparent infection exists so have tire adenovirus anti-Fi, anti-Pb and anti-Hx antibody of height in some people colony.The viral fragment of the scleroproein that contains adenovirus that employing is extracted from adenovirus, penton albumen and hexon is as antigen, set up the ELISA method and in health is donated blood the source, screen the immunoglobulin (Ig) of the high anti-adenovirus of tiring, through haematogenous transmissible diseases such as hepatitis virus, virus of AIDS, syphilis after the assay was approved, extract immunoglobulin (Ig) with the cold ethanol method, be prepared into and contain the tire immunoglobulin preparation of adenovirus anti-Fi, anti-Pb and anti-Hx of height.
The technical solution adopted in the present invention is, the tire preparation method for immunoglobulinlg of adenovirus anti-Fi, anti-Pb and anti-Hx of height is characterized in that, may further comprise the steps:
1) preparation contains the antigen of scleroproein, penton albumen and the hexon of adenovirus
Adenovirus is inoculated in Hep-2 cell or hela cell or HEKC or KB cell or the human embryonic lung diploid fibroblast or the Vero cell of the individual layer of growing up, in incubator, cultivate, every day observation of cell pathology situation, when cytopathogenic effect (CPE) occurs +++(being that cytopathy accounts for whole monolayer cell 75%), collecting cell and supernatant, inactivation of virus, at-70 ℃ of multigelations, again through ultrasonicly smash, stored frozen;
2) purified virus antigen
Use cesium chloride, sucrose and saccharosan form a continuous or discrete density gradient as medium in centrifuge tube, and the effect by gravity or centrifuge field makes viral fragment layering, separation, or chromatography adsorption column method purified virus antigen; With the ultrasonic adenovirus nutrient solution high speed centrifugation of smashing 20 minutes, discard sediment and stay supernatant, pack into the discontinuous gradient density centrifugation pipe of 5%, 15%, 30%, 45%, 60% sucrose preparation, ultracentrifugation 2 hours, determine this centrifuge tube position, virus place, sucking-off stored frozen according to the molecular weight of virus;
3) set up the method for screening high tire adenovirus anti-Fi, anti-Pb and anti-Hx immunoglobulin (Ig)
Viral fragment with the scleroproein that contains adenovirus that extracts from adenovirus, penton albumen and hexon mixes as antigen, preferred 1: 1: 1 mixed, wrap by the enzyme plate with 10 μ g/ml～20 μ g/ml, the antigen that utilization is combined in surface of solid phase carriers still keeps its immunologic competence, when measuring, the scleroproein of the adenovirus of the scleroproein of adenovirus, penton albumen and hexon antibody and surface of solid phase carriers, penton albumen and hexon react in plasma serum and the body fluid.With the method for washing the immune complex that forms on the solid phase carrier and other materials in the liquid are separated again.Add the antibody of enzyme labelling again, also be combined on the solid phase carrier by reaction.The amount of examined object matter is certain ratio in enzyme amount on the solid phase and the sample at this moment.After adding the substrate of enzyme reaction, substrate is become coloured product by enzyme catalysis, and the amount of product is directly related with the amount of examined object matter in the sample, so can carry out qualitative or quantitative analysis according to the depth of colour generation.Because the catalytic efficiency of enzyme is very high, has amplified immunoreactive result indirectly, makes measuring method reach very high susceptibility.
4) with the raw material of aforesaid method screening with ammonium sulfate precipitation method or polyethylene glycol precipitation or cold ethanol method or/and the ion exchange chromatography method or/and affinity chromatography extracts the immunoglobulin (Ig) of high tire adenovirus anti-Fi, anti-Pb and anti-Hx;
A) ammonium sulfate precipitation method is that to utilize it be neutral salt, solubleness temperature influence in water is little, can not cause proteic sex change inactivation, be that the ammonium sulfate saturation ratio that adopts is 20%～60% in the present invention, PH6.5～8.5, can repeat the ammonium sulfate precipitation of one or many, to reach the purpose that improves purity of protein.
B) polyethylene glycol precipitation is a characteristic of utilizing the protein precipitant that polyoxyethylene glycol (PEG) is nontoxic, character is gentle, and the molecular weight that PEG selects for use is 3000～8000Da, and the solubility scope of PEG is 3%～10%.
C) the cold ethanol partition method is to utilize ethanol regulator solution specific inductivity, under alcohol concn 15%-25% condition, regulating pH is 5.1～7.2, controlled temperature is at-3～-7 ℃, isolating Cohn ' s component I I+III (FII+III) or Cohn ' s component I+II+III (FI+II+III) are raw material, through the ultrafiltration and concentration dealcoholysis.
D) ion exchange chromatography is to be selected from the impurity of the direct absorption of the ion exchange resin (as DEAE-sepharose, DEAE-sepharose FF, S-sepharose, S-sepharose FF and DEAE-sephadx A-50) that contains DEAE-or S-group except that the specificity antivirus immunoglobulin (Ig), thereby directly wear the antiviral immunoglobulin that liquid obtains purifying from stream, wherein condition is that pH is 4.0～7.8.
E) affinity chromatography is to have the reversible bonded material of many specificitys mutually and the method set up according to biomacromolecule, the selected affinity chromatography aglucon of the present invention is Protein A and two kinds of proteic aglucons of Protein G, mainly contain rProtein A Sephrose Fast Flow, Protein A Sephrose CL-4B, Protein G Sephrose CL-4B, the salt concn of damping fluid is 0.004～0.04; PH is 4.8～9.2.
5) the anti-adenovirus immunoglobulin (Ig) after the purification is through 60 ± 1 ℃, 10 hours inactivation of viruses and/or in pH value 4.1 ± 0.3, placement 21 days and/or nano-film filtration and/or solvent (S/D) method and/or dry heating method are (80 ℃ under 24 ± 1 ℃ of conditions, 72 hours) remove, inactivation of viruses, through Sterile Filtration, be distributed into or be lyophilized into the immunoglobulin (Ig) of adenovirus anti-Fi, anti-Pb and anti-Hx again.
Four, embodiment
The present invention is described in further detail below in conjunction with embodiment that the contriver provides.
Embodiment 1: preparation contains the antigen of scleroproein, penton albumen and the hexon of adenovirus
Get the fertilized eggs of hatching 10～20d, the air chamber and the position of foetus are drawn, and near the embryo, look for the less position of a blood vessel, behind the tincture of iodine, the ethanol disinfection, after boring an aperture on the air chamber, in the middle of egg membrane, sting a crack again, and with the air sucking-off in the air chamber, the fine hair allantois is sunk separate, splash into ADV (1 * 10 with air chamber
7TCID50/ml) behind the 0.2ml, seal and paraffin envelope pore, after the inoculation chicken embryo is positioned over 37 ℃ of hatchings 7 days to melt with the sterilization adhesive plaster.After hatching finishes,, along the air chamber edge eggshell is thrown off, mentioned chorioallantoic membrane, puncture, draw allantoic fluid and collect chorioallantoic membrane,, be the tissue juice that is rich in ADV its fragmentation with aseptic capillary pipet with aseptic nipper with the tincture of iodine, ethanol disinfection eggshell.
Embodiment 2: preparation contains the antigen of scleroproein, penton albumen and the hexon of adenovirus
Cell cultures routinely, treat that the Hep-2 cell has been grown in flakes after, splash into ADV (1 * 10
7TCID50/ml) be inoculated in the Hep-2 cell that grows up to individual layer, hatch for 33 ℃, every day observation of cell pathology situation, treat that cytopathy (CPE) occurs ++ the time, supernatant liquor is discarded, and the liquid of keeping that does not contain calf serum that adds half amount continues to cultivate, when cytopathy (CPE) occurs +++above after, collecting cell and supernatant are put the refrigerator stored frozen.
Embodiment 3: preparation contains the antigen of scleroproein, penton albumen and the hexon of adenovirus
Cell cultures routinely, treat that the Vero cell has been grown in flakes after, splash into ADV (1 * 10
7TCID50/ml) be inoculated in the Vero cell that grows up to individual layer, hatch for 33 ℃, every day observation of cell pathology situation, treat that cytopathy (CPE) occurs ++ the time, supernatant liquor is discarded, and the liquid of keeping that does not contain calf serum that adds half amount continues to cultivate, when cytopathy (CPE) occurs +++above after, collecting cell and supernatant are put the refrigerator stored frozen.
Embodiment 4: the scleroproein of adenovirus, penton albumen and the antigenic of hexon separate
With the nutrient solution of above collection behind-70 ℃ of refrigerator multigelations 3 times, again under the ultrasound condition of 20MHz, ultrasonication 20 minutes; Tissue juice after ultrasonic is centrifugal 20 minutes in 10000 rev/mins, discard sediment and stay supernatant, pack into the discontinuous gradient density centrifugation pipe of 5%, 15%, 30%, 5%, 60% sucrose preparation, ultracentrifugation 2h in 150000 rev/mins whizzer, determine the position of scleroproein, penton albumen and the hexon place centrifuge tube of adenovirus respectively, sucking-off and stored frozen are standby respectively.
Embodiment 5: the screening of the immunoglobulin (Ig) blood plasma of adenovirus anti-Fi, anti-Pb and anti-Hx
Scleroproein, penton albumen and hexon with the adenovirus of separation and purification, press the protein content balanced mix, be cushioned liquid (PH9.6 0.05M carbonate buffer solution) with bag scleroproein, penton albumen and the hexon mixed solution of adenovirus is diluted to 10 μ g/ml, with polystyrene shrinkage pool plate as solid phase carrier, every hole adds mixed liquid of protein 0.1ml, and 4 ℃ are spent the night.Wash next day 3 times.Add 1: 20 the dilution blood plasma 0.1ml to be checked in above-mentioned wrapped by reacting hole in, put 37 ℃ and hatched 1 hour, the washing.Do blank, feminine gender and positive hole simultaneously.The washing back adds the enzyme mark anti-antibody 0.1ml of fresh dilution (extent of dilution after titration) in reacting hole, hatched 30 minutes, and washed with DDW at last for 37 ℃.The tmb substrate solution 0.1ml that in each reacting hole, adds interim preparation again, 37 ℃ 30 minutes, in each reacting hole, add 2M sulfuric acid 0.05ml termination reaction at last.On the ELISA detector, in the 450nm place, survey each hole OD value with zeroing back, blank hole, if it is greater than 2.1 times of the negative control OD value of stipulating, promptly positive.
Embodiment 6: the immunoglobulin (Ig) that extracts high tire adenovirus anti-Fi, anti-Pb and anti-Hx
The height that the filters out adenovirus anti-Fi that tires, the immunoglobulin (Ig) blood plasma of anti-Pb and anti-Hx under agitation adds ammonium sulfate, making the whole saturation ratio of reaction solution ammonium sulfate is 50% ± 10%, after temperature is to leave standstill 3 hours under 4 ℃, again 3000 rev/mins centrifugal minute, abandon supernatant, dissolution precipitation, adding ammonium sulfate again, to make the whole saturation ratio of reaction solution ammonium sulfate be 30% ± 10%, after temperature is to leave standstill 3 hours under 4 ℃, again 3000 rev/mins centrifugal minute, abandon supernatant, dissolution precipitation, dialysis lysate, the height that the is purifying adenovirus anti-Fi that tires, the immunoglobulin (Ig) of anti-Pb and anti-Hx.
Embodiment 7: the immunoglobulin (Ig) that extracts high tire adenovirus anti-Fi, anti-Pb and anti-Hx
The height that the filters out adenovirus anti-Fi that tires, the immunoglobulin (Ig) blood plasma of anti-Pb and anti-Hx is in PH7.5～6.0, temperature-2 ℃～-6 ℃, end reaction liquid alcohol concn is 15%～25%, isolating Cohn ' s component I I+III (F II+III) or Cohn ' s component I+II+III (F I+II+III) are raw material, with 20 times of water for injection dissolution precipitations, and under alcohol concn 15%-25% condition, regulating pH is 5.1～7.2, controlled temperature is at-3～7 ℃, the height that centrifugal gained supernatant the is purification adenovirus anti-Fi that tires, the immunoglobulin (Ig) of anti-Pb and anti-Hx.
Embodiment 8: the immunoglobulin (Ig) that extracts high tire adenovirus anti-Fi, anti-Pb and anti-Hx
In order further to improve the purity of the immunoglobulin (Ig) of adenovirus anti-Fi, anti-Pb and anti-Hx, can in the processing step of ammonium sulfate precipitation method, polyethylene glycol precipitation, the cold ethanol precipitator method etc., add ion exchange chromatography, collective's step is: it is 6.8 ± 0.4 that solution is adjusted PH, specific conductivity is 1.5 ± 0.5ms/cm, temperature is 22 ± 3 ℃, by DEAESephrose FF chromatography column, the protein peak that stream is worn is the tire immunoglobulin (Ig) of adenovirus anti-Fi, anti-Pb and anti-Hx of highly purified height.
Embodiment 9: the immunoglobulin (Ig) that extracts high tire adenovirus anti-Fi, anti-Pb and anti-Hx
The adenovirus anti-Fi that filters out, the blood plasma of the immunoglobulin (Ig) of anti-Pb and anti-Hx is introduced equilibrated DEAE-sepharose CL-6B or DEAE-sepharose FF post, condition is pH5.0～6.0, ionic strength is 0.01～0.03, the high adenovirus anti-Fi that tires, the immunoglobulin (Ig) stream of anti-Pb and anti-Hx passes post, stream passes the immunoglobulin (Ig) of post again by Lysine-Sepharose 4B, to penetrate liquid and washings merges, and after adjust pH is 6.0～5.4, adding SP-Sephadex C50 or CM-sepharose FF uses the glycine of pH11 to parse adenovirus anti-Fi, the immunoglobulin (Ig) of anti-Pb and anti-Hx, the isolating immunoglobulin purity of this separation method is nearly 100%, and polymer content is low.
Claims (16)
1. the preparation method for immunoglobulinlg of adenovirus anti-Fi, anti-Pb and anti-Hx is characterized in that, may further comprise the steps:
1) preparation contains the antigen of scleroproein, penton albumen and the hexon of adenovirus
Adenovirus is inoculated in Hep-2 cell or hela cell or HEKC or KB cell or the human embryonic lung diploid fibroblast or the Vero cell of the individual layer of growing up, in incubator, cultivate, every day observation of cell pathology situation, when cytopathogenic effect (CPE) occurs +++(being that cytopathy accounts for whole monolayer cell 75%), collecting cell and supernatant, inactivation of virus, at-70 ℃ of multigelations, again through ultrasonicly smash, stored frozen;
2) purified virus antigen
Form a continuous or discrete density gradient with cesium chloride or sucrose or saccharosan as medium in centrifuge tube, the effect by gravity or centrifuge field makes viral fragment layering, separation, or chromatography adsorption column method purified virus antigen; With the ultrasonic adenovirus nutrient solution high speed centrifugation of smashing 20 minutes, discard sediment and stay supernatant, pack into the discontinuous gradient density centrifugation pipe of 5%, 15%, 30%, 45%, 60% sucrose preparation, ultracentrifugation 2 hours, determine this centrifuge tube position, virus place, sucking-off stored frozen according to the molecular weight of virus;
3) set up the immune globulin whitening method that screens high tire adenovirus anti-Fi, anti-Pb and anti-Hx
With the scleroproein that contains adenovirus that from adenovirus, extracts, penton albumen and hexon virus fragment mix as antigen, wrap by the enzyme plate with 10 μ g/ml～20 μ g/ml, the antigen that utilization is combined in surface of solid phase carriers still keeps its immunologic competence, blood plasma from the human or animal, the scleroproein of adenovirus in serum and the body fluid, the scleroproein of the adenovirus of penton albumen and hexon antibody and surface of solid phase carriers, penton albumen and hexon react, with the method for washing the immune complex that forms on the solid phase carrier and other materials in the liquid are separated again, the antibody that adds enzyme labelling again, also be combined on the solid phase carrier by reaction, the amount of examined object matter is certain ratio in enzyme amount on the solid phase and the sample at this moment, after adding the substrate of enzyme reaction, substrate is become coloured product by enzyme catalysis, the amount of product is directly related with the amount of examined object matter in the sample, so can carry out qualitative or quantitative analysis according to the depth of colour generation, because the catalytic efficiency of enzyme is very high, amplified immunoreactive result indirectly, made measuring method reach very high susceptibility;
4) with the product of aforesaid method screening with ammonium sulfate precipitation method or polyethylene glycol precipitation or cold ethanol method or/and the ion exchange chromatography method or/and affinity chromatography extracts the immunoglobulin (Ig) of high tire adenovirus anti-Fi, anti-Pb and anti-Hx;
5) immunoglobulin (Ig) of adenovirus anti-Fi, anti-Pb after the purification and anti-Hx is through 60 ± 1 ℃, 10 hours inactivation of viruses and/or in pH value 4.1 ± 0.3, placement 21 days and/or nano-film filtration and/or solvent (S/D) method and/or dry heating method are (80 ℃ under 24 ± 1 ℃ of conditions, 72 hours) remove, inactivation of viruses, through Sterile Filtration, be distributed into or be lyophilized into the immunoglobulin (Ig) of adenovirus anti-Fi, anti-Pb and anti-Hx again.
2. as right 1 described method, the antigenic cell that preparation contains scleroproein, penton albumen and the hexon of adenovirus is Hep-2 cell or Vero cell.
3. as right 1 described method, the used medium of purified virus antigen is a sucrose, and gradient is 15%, 30%, 45%.
4. as right 1 described method, set up in the immune globulin whitening method of high tire adenovirus anti-Fi, anti-Pb and anti-Hx of screening, the scleroproein of the adenovirus of separation and purification, penton albumen and hexon are to become 10 μ g/ml～20 μ g/ml by 1: 1: 1 balanced mix of protein content.
5. as right 1 described method, blood plasma and serum that the raw material sources of the immunoglobulin (Ig) of adenovirus anti-Fi, anti-Pb and anti-Hx are behaved.
6. as right 1 described method, the alcohol concn that separates the immunoglobulin (Ig) of adenovirus anti-Fi, anti-Pb and anti-Hx with the cold ethanol partition method is 15%～25%; PH is 5.1～7.2; Temperature is-3 ℃～-7 ℃.
7. the method described as right 1, improve purity with ion exchange chromatography, use contains the ion exchange resin of DEAE-or S-group, directly adsorb the impurity except that the specificity antivirus immunoglobulin (Ig), thereby directly wear the immunoglobulin (Ig) that liquid obtains purifying from stream, wherein condition is that pH is 5.0～6.8;
Described ion exchange resin is selected from: DEAE-Sepharose, DEAE-Sepharose FF, S-Sepharose, S-Sepharose FF and DEAE-Sephadx A-50.
8. method as claimed in claim 7, it is characterized in that: when extracting the immunoglobulin (Ig) of high tire adenovirus anti-Fi, anti-Pb and anti-Hx with ion exchange chromatography, raw material is through DEAE-Sepharose CL-6B or DEAE-Sepharose FF post, and the immunoglobulin (Ig) stream of adenovirus anti-Fi, anti-Pb and anti-Hx passes post; Equilibrium conditions is pH5.0～6.0, and ionic strength is 0.01～0.03.
9. as right 7 described methods, described ion exchange chromatography stream passes the antiviral immunoglobulin of post again by Lysine-Sepharose 4B, after will penetrating the merging of liquid and washings, add SP-Sephadex C50 or CM-SepharoseFF absorption, use the glycine of pH11 to parse the specificity antivirus immunoglobulin (Ig); Equilibrium conditions is a pH value 6.0～5.4; Analysis condition is the glycine of pH11.
10. the method for claim 1, the solid carrier of affinity chromatography is selected from: Sephadex, PDX, Sephacryl, Sepharose, Sepharose CL, Sepharose FF, Superdex, Superose, Tri saceyl, Tri sacylplus, Ultrogel A, Ultrogel AcA, highly porous regenerated cellulose pearl; The affinity chromatography aglucon is ProteinA and two kinds of proteic aglucons of Protein G.
11. as described in claim 10, the affinity chromatography resin is preferably rProtein A Sephrose Fast Flow, ProteinA Sephrose CL-4B, Protein G Sephrose CL-4B, and equilibrium conditions is that the salt concn of damping fluid is that 0.004～0.04:pH is 4.8～9.2.
12. the method for claim 1, the immunoglobulin (Ig) of adenovirus anti-Fi after the purification, anti-Pb and anti-Hx is through 60 ± 1 ℃, 10 hours inactivation of viruses and/or in pH value 4.1 ± 0.3, the removal of placement 21 days and/or nano-film filtration and/or solvent (S/D) method and/or dry heating method (80 ℃, 72 hours), inactivation of viruses under 24 ± 1 ℃ of conditions.
13. method as claimed in claim 12, the combination of the several method of removal, inactivation of viruses, be preferably 60 ± 1 ℃, 10 hours, 60 ± 1 ℃, 10 hours and nano-film filtration, pH value 4.1 ± 0.3, under 24 ± 1 ℃ of conditions, placed 21 days and nano-film filtration, solvent (S/D) method and nano-film filtration, solvent (S/D) method and dry heating method (80 ℃, 72 hours).
14. method as claimed in claim 13, the aperture of nanometer film are 20 nanometers～50 nanometers.
15. method as claimed in claim 13, solvent (S/D) method is TNBP/Tween 80 or TNBP/Triton X-100, concentration is preferably TNBP0.3%～2% (w/w), and Tween 80 is 1% (w/w), and Triton X-100 is 1% (w/w).
16. as the described method of claim 1～15, related immunoglobulin (Ig) is IgG, the IgG that it is contained
1Be 55%～65%, IgG
2Be 30%～40%, IgG
3Be 2～5%, IgG
4Be 0.8%～4%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102445548A (en) * | 2011-11-15 | 2012-05-09 | 广西壮族自治区兽医研究所 | Detection kit for indirect ELISA of FAVI antibody based on penton protein |
| CN106755106A (en) * | 2017-01-06 | 2017-05-31 | 华中农业大学 | The preparation method and application of Ankara subunit vaccine |
| CN111499698A (en) * | 2020-03-19 | 2020-08-07 | 中国农业科学院特产研究所 | Canine type I adenovirus subunit vaccine and preparation method thereof |
-
2008
- 2008-01-22 CN CNA200810019531XA patent/CN101220094A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102445548A (en) * | 2011-11-15 | 2012-05-09 | 广西壮族自治区兽医研究所 | Detection kit for indirect ELISA of FAVI antibody based on penton protein |
| CN106755106A (en) * | 2017-01-06 | 2017-05-31 | 华中农业大学 | The preparation method and application of Ankara subunit vaccine |
| CN111499698A (en) * | 2020-03-19 | 2020-08-07 | 中国农业科学院特产研究所 | Canine type I adenovirus subunit vaccine and preparation method thereof |
| CN111499698B (en) * | 2020-03-19 | 2023-05-16 | 中国农业科学院特产研究所 | Canine I type adenovirus subunit vaccine and preparation method thereof |
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