CN1944462A - Preparation of anti PV IgY antibody and its use in PV diagnostic reagent and preventing medicine - Google Patents
Preparation of anti PV IgY antibody and its use in PV diagnostic reagent and preventing medicine Download PDFInfo
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Abstract
The present invention discloses the preparation process of anti-PV.IgY antibody and its use in preparing immune diagnosing reagent and preventing medicine for PV relevant diseases. The anti-PV.IgY antibody is prepared through obtaining PV virus protein or its eukaryon expressing vector in molecular biological method, immunizing female fowls with the PV virus protein or its eukaryon expressing vector as the PV virus protein antigen or gene, and separating and purifying the anti-PV.IgY antibody in conventional method. The anti-PV.IgY antibody is used in preparing diagnosis reagent or preventing medicine for PV infection and relevant diseases. Experiment proves the capacity of the specific anti-PV.IgY antibody in combining with corresponding PV antigen specifically. The present invention has the features of high safety, less side effect, obvious curative effect, etc.
Description
Technical field
The invention belongs to a kind of special yolk antibody (yolkimmunoglobulin in the biological technical field, IgY) preparation and application, the preparation method of particularly anti-PV IgY antibody and this anti-PV IgY antibody thereof are used for preparing the purposes of PV infection and relative disease immune diagnostic reagent and prophylactic agent.
Background technology
Papilloma virus (papillomaviruses, PV) be a class nature extensively exist have a liking for epithelium virus groups (epitheliotropic viruses), the no coating small DNA virus that belongs to papovaviridae (Papovaviridae) Papillomavirus, comprise multiple animal teat tumor virus and human papillomavirus (humanpapillomavirus, HPV), PV can infect more than 20 kind of poultry, reptile and comprise human mammal, cause skin, the good neoplasm venereal disease of mucous membrane becomes, have a strong impact on and comprise ox, rabbit, the people causes serious threat in interior different plant species to husbandry and people's life health.
Because the importance of medical research, HPV extremely payes attention to.HPV is the skin and the mucous epithelium of infected person only, causes that epithelial proliferation sexually revises, and is relevant with the pathology of human skin mucous membrane.It is 200 many types of that the HPV that has found at present has, also have many new types just detected with separate, wherein 100 many types of genomes are separated and finish genome sequencing (Hans-Ulrich Bernard.The clinicalimportance of the nomenclature, evolution and taxonomy of humanpapillomaviruses.J Clinical Virology 2005; 32S:S1-S6).
Difference according to the infection epithelium can be divided into HPV skin-type and mucosal pattern.Skin-type such as HPV1 infect the vola and cause sole of the foot wart, and HPV2,4,7 infects the hand skin epithelium, causes epithelium height hyperplasia molluscum; Mucosal pattern HPV infects anus sexual organ mucous epithelium.Tumorigenicity according to HPV can be divided into it low risk and high-risk-type.Low risk is the most common with HPV6 and 11 types, can cause sexual organ papilloma or pointed condyloma; High-risk HPV (HPV16, HPV18, HPV31, HPV45, HPV39, HPV58 and HPV59 etc.) and urodaeum, cervical cancer are relevant in the morbidity of the interior multiple epithelium tumor of human body, can cause the generation of intracutaneous knurl on the uterine cervix and cancerate and the generation of other epithelium tumor, confirm HPV16, the 18th, the carcinogen of cervical cancer.Sickness rate in China's cervical cancer is very high, and women's sum of dying from cervical cancer every year accounts for several nearly half of whole world cervical cancer death person.In recent ten years, the sexually transmitted disease (STD) rapid spread that comprises HPV, 75% property active period crowd all can infect HPV (Koutsky L.Epidemiology of genital human papillomavirusinfection.Am J Med 1997 in life at it; 102:3-8.).And the generation of the malignant tumours such as cervical cancer due to infecting with high-risk HPV will sharply increase after 10～30 years.Therefore, prevention HPV infects extremely urgent, is of great importance for guaranteeing that Chinese national economy grows continuously and fast.
But, the expression of PV virogene is subjected to the regulation and control of host cell to epithelium top layer migration course, and life cycle (virus life the cycle) (Stubenrauch of self is finished in the differentiation of dependent cells, F.and Laimins, L.A. (1999) .Human papillomavirus life cycle:Activeand latent phases.Semin.Cancer Biol.9 (6), 379-386; JohnDoorbar.The papillomavirus life cycle.Journal of Clinical Virology32S (2005) S7-S15).Therefore, have and highly have a liking for host and histocyte specificity, different PV have its special host animal group, HPV can only infect and attack the mucocutaneous of people, in the human body epithelium, duplicate, breed and can not be in external breeding, be the immunobiology research of HPV, the research of the human tumor vaccine that prevention is relevant with treatment HPV has brought huge obstacle.Although, in recent years, utilize the preventative vaccine fast development of HPV virus-like particle, and the formally clinical use of input, but this class vaccine price costliness can not be promoted and use in developing country's big area, therefore, exploring new approach carries out the HPV diagnosis of infection and prevents significant.
HPV enters skin mucosa stratum basale stem cell and sticks the primary event that its surface becomes virus infection by the microlesion of skin or mucous membrane, the beginning that has triggered viral life cycle that combines of HPV L1 albumen and specific receptors (as integrating element, heparin sulfate etc.) has determined the tissue specificity of having a liking for of HPV to a certain extent.As seen, before the microlesion of HPV by skin or mucous membrane entered skin mucosa stratum basale stem cell, specific inhibition HPV virus is sticked host cell will fundamentally block the generation that HPV infects.
In sum, development energy specific inhibition HPV sticks the preparation of host cell, for reducing the HPV infection rate significantly, and then the incidence that reduces the various human body HPV associated malignancies comprise cervical cancer all has crucial meaning, and this strategy obviously also is the prevention that is used for other PV infection except that HPV simultaneously.
Existing studies confirm that, the combination of biomacromolecule is mainly carried out specific combination by ligand-receptor and Ag-Ab dual mode, and wherein the combination between the Ag-Ab belongs to immune combination, has high specificity and avidity, therefore, by preparing the specific antibody of corresponding antigens.Just be based on this principle, many immunology diagnosis technology and reagent and biological targeting methods of treatment and medicine arise at the historic moment, and are widely used in the middle of the diagnosis and treatment of nearly all humans and animals relative disease.
Birds are the same with Mammals to have the ability that maternal immunity power is passed to filial generation.As far back as twentieth century sixties, after just finding that hen is subjected to immunostimulation, produce immune response, in ovarian follicle, generate in yolk and the uterine tube in the sophisticated process of yolk, IgG can optionally be transferred in the yolk in the blood, and in these a large amount of enrichments, and be immunoglobulin (Ig) unique in the yolk, IgY (Leslie thus gains the name, G.A.and Clem, L.W.Phylogeny of immunoglobulim structure andfunction.3.Immunoglobulins of the chiken.J.EXP.Med.1969; 130:1337-1352; Zhang WW.The use of gene-specific IgY antibody fordrug target discovery[J] .Research Focus.2003; 8 (8): 364-371).Hen is easy to raise, and expense is low; Immunization method is simple, and the chicken egg laying amount is big, and it is convenient to collect, and purifying is simple, and the antibody production height meets modern protection of animal rule.Can from the ovum gallinaceum in same individual source, prepare the good polyclonal antibody of homogeneity in a large number.The specific IgY that chicken of immunity can obtain high yield reached for 9 week～28 weeks, the content of IgY is up to 10g/L～20g/L in the yolk, from egg, can separate very easily, the purifying corresponding antibodies, each yolk can extract the pure product antibody of IgY of 10mg, every hen can obtain the above homogeneity IgY of 30g in 1 year, considerably beyond the antibody amount of a rabbit anteserum preparation, and the antibody activity height.Therefore, think that hen is the immunoglobulin (Ig) of a kind of high yield, high-quality, economy " source mill ".Simultaneously, hen is easy to raise, expense is low, and immunity is simple, output is big, collection makes things convenient for, must not draw blood, purifying is simple, antibody production is high, meets the protection of animal rule.Particularly closely during the last ten years, along with the foundation of extracting method, the research of relevant this respect is emerge in multitude like the mushrooms after rain just, and separation and purification antibody is a kind of efficient, economy, fine preparation method of polyclonal antibody from yolk.
On immunology, IgY and serum IgG homology, it is a kind of 7S immunoglobulin, slightly different with Mammals IgG, this protein molecular weight is about 180kDa, contains two subunits, the i.e. light chain of the heavy chain of 67～70kDa and 22～30kDa, have acidproof, heat-resisting, stable performance, be convenient to separate, characteristics (Janson AK, Smith CI, the Hammarstrom L.Biologicalproperties of yolk immunoglobulins.Adv Exp Med Biol.1995 of purifying; 371A:685-90.).
Recently find, IgY has biological characteristics (the Krief A that is different from mammal IgG, Letessonb JJ, Billena D, et al.Comparison between ' IgY technology ' from chickens and ' IgG technology ' from mice for production oftailor-made antibodies.Tetrahedron Letters.2002; 43:1843-1846):
As IgY the complement component of mammal there is not fixed action, also not with the Fc receptors bind, do not react with serum composition, effectively avoid the caused false positive/false negative result of mammal endogenous antibody, and, chicken antibody is acidproof, heat-resisting, stable performance, chicken prepares antibody as immune host and has caused extensive concern, and in the ELISA detection kit, widely used, at present, IgY has been widely used in comprising immunoprecipitation, immunoelectrophoresis, ELISA, immuno-electron microscope, a series of immune diagnostic techniques such as immunoblotting, and the immunology diagnosis that has the gesture of the IgG of alternative mammalian source, IgY to be used for disease has greatly become the development trend that immune diagnostic method is learned.(Warr?GW，Magor?KE，HigginsDA.IgY：clues?to?the?origins?of?modern?antibodies[J].ImmunologyToday.1995；16(8)：392-39)。
In addition, the IgY good stability can use repeatedly as the aglucon of affinity column, and significantly improve the separation efficiency of affinity chromatography.Become affinity chromatography aglucon with good research and application prospect, and be used for the target material rapidly, high efficiency separation, especially has very high application potential (Shelver WL for some purpose products that adopt additive method to be difficult to separation and purification, Larsen GL, Huwe JK.Use ofan immunoaffinity column for tetrachlorodibenzo-p-dioxin serumsample cleanup.J Chromatogr B Biomed Sci Appl.1998 Feb 13; 705 (2): 261-8; Kim HO, Durance TD, Li-Chan EC.Reusability ofavidin-biotinylated immunoglobulin Y columns in immunoaffinitychromatography.Anal Biochem.1999Mar?15；268(2)：383-97)。With the specific IgY is that aglucon is coupled on the Sepharose 4B medium of cyanogen bromide-activated, BSA in the affine separation foetal calf serum, products obtained therefrom purity is apparently higher than the BSA product of ROCHE company, and elution requirement is simple, only use 0.1mol/L Gly-HCl (pH2.8) as eluent, a step is promptly reached the separating effect of expection.The entire operation process is simple fast, only needs a few hours can finish the regeneration of sample, wash-out separation and post.Simultaneously, because Yolk immunoglobulin character is comparatively stable, handle affine filler well and can preserve more than half a year under 4 ℃ of conditions, and separating power is almost constant.After using for several times, chromatography column still can keep separating power preferably.
Moreover, aspect medicinal application, oral IgY can obtain the passive immunization protection, young animal or human infectious intestinal disease have been used at present prevent or treat, as infant's intestinal tract disease control food, the prevention of dental caries, the prevention of newborn sucking pig lethality coli-infection, and the fish disease treatment etc., when particularly the use of microbiotic or other medicines has problems, IgY becomes first-selection (Lee SB, Mine Y, Stevenson RM.Effects of hen egg yolk immunoglobulin in passiveprotection of rainbow trout against Yersinia ruckeri.J Agric FoodChem.2000 Jan; 48 (1): 110-5; Davalos-Pantoja L, Ortega-Vinuesa JL, Bastos-Gonzalez D, etal.Oral administration of specific yolkantibodies (IgY) may prevent Pseudomonas aeruginosa infections inpatients with cystic fibrosis:A phase I feasibility study.PediatrPulmonol.2003 Jun; 35 (6): 433-40; De Almeida CM, Quintana-FloresVM, Medina-Acosta E, et al.Egg yolk anti-BfpA antibodies as a toolfor recognizing and identifying enteropathogenic Escherichia coli.Scand J Immunol.2003Jun; 57 (6): 573-82; Shin JH, Yang M, NamSW, et al.Use of egg yolk-derived immunoglobulin as an alternativeto antibiotic treatment for control of Helicobacter pylori infection.Clin Diagn Lab Immunol.2002 Sep; 9 (5): 1061-6.).
In a word, after the eighties in 20th century, utilize specific antibody (IgY) in the yolk to carry out that passive immunization prevents and the some diseases for the treatment of the people has been subjected to the attention of countries in the world, now be widely used in fields such as immunology diagnosis, medicine, healthcare products, breed.China has begun this research on the one hand after the nineties in 20th century, and has obtained certain achievement.
Therefore, use the IgY antibody of the anti-PV of preparation, will in the prevention that the diagnosis of PV, antigenic purifying and PV infect, play a significant role.
Chinese patent (application number is 03139687.9) discloses a kind of " resistance disease-specific composite yolk antibody and the application in treatment for the sexually transmitted diseases thereof ", in its claim 1 " (A) preferably cause the The main pathogenic fungi of characteristic of disease, prepare two or more single pathogenic pathogenic agent; Claim 2 shows that described pathogenic agent is Treponoma palladium, gonorrhea diplococcus, human papillomavirus and hsv." " wherein, steps A is according to the epidemiology survey situation, and screening causes the pathogenic agent of venereal disease, prepares single pathogenic pathogenic agent respectively as antigen, breeds in a large number, collects culture, purifies the dilution packing in its specification sheets; The various single venereal disease pathogenic agent that cause are added freund adjuvant, pulverize and make milk-like slurry, make the pathogenic former isoantigen of single venereal disease ".Be used to separate papilloma virus owing to obtaining relatively large papilloma knurl body in the specificity host animal body that infects from PV, for example separablely in the cow teats knurl go out bovine papilloma virus (BPV) antigen, be used for the immunity of animal, but, human papillomavirus (humanpapillomavirus, HPV) comprise more than 200 type, the HPV expression of gene is subjected to the regulation and control of host cell to epithelium top layer migration course, and the life cycle (virus life cycle) of self is finished in the differentiation of dependent cells, HPV has strict species specificity and has a liking for the epithelium characteristic, can not infect other species except that the people.(Stubenrauch，F，and?Laimins，L.A.Humanpapillomavirus?life?cycle：Active?and?latent?phases.Semin.CancerBiol.1999；9(6)，379-386；John?Doorbar.The?papillomavirus?lifecycle.Journal?of?Clinical?Virology?32S(2005)S7-S15)。Up to the present, from heterograft (Bonnez et al., 1993 of natural damage location (Crawford and Crawford, 1963), the immunodeficient mouse that infects; Brown et al., 1998; Kreideret al., 1987), raft sample culture systems (Meyers et al, 1992,1997; Ozbun, 2002) all can not obtain enough HPV viral loads and carry out research (Clinical and AppliedImmunology Reviews 5 (2005) 65-76 that HPV infects.The road to new antiviraltherapies.Keith R.Jerome), therefore, the pathogenic agent that the claim of this patent disclosure and technical scheme can not obtain enough papilloma viruss prepares resistance disease-specific composite yolk antibody as the antigen immune hen, realizes its purpose.Thereby this patent does not have exploitativeness.
Chinese patent (application number: 03153633.6) disclose preparation method and the combination preparation thereof of a kind of resistance disease-specific IgY, in its claim " 1 preferably causes the The main pathogenic fungi of characteristic of disease, and preparation causes the single or complex antigen of the various pathogenic agent of venereal disease respectively; Prepare immune egg ... "; In the claim 2 " screening causes the pathogenic agent of venereal disease; according to the cultural characters and the optimal culture conditions of these pathogenic agent; select the suitable culture base respectively; cultivate breeding; collect culture, purification, dilution packing, respectively various pathogenic agent some amounts is added freund adjuvant, puts in the high-speed homogenization machine with 10000r/min～30000r/min homogenize; make into water-in-oil emulsion, promptly makes various venereal disease pathogen antigen ".In claims 3 " pathogenic agent that causes venereal disease comprises pale rice spirochete (TP), gonorrhea diplococcus (GC), human papillomavirus (HPV) and II herpes simplex virus type (HSV-2) ", in the claim 6 " antigen prepd: choose TP, GC, HPV, HSV-2 type strain; respectively according to their cultural characters and optimal culture conditions; select the suitable culture base; cultivate in a large number and breed; collect culture, purify, dilute packing then with ordinary method; " because above-mentioned same reason, HPV can not obtain in vitro culture.Therefore, this patent does not have exploitativeness equally.
Chinese patent (application number: 03104002.0) disclose the preparation and the application of the relevant yolk antibody of a kind of infectious agent, but in its claims, do not comprised the human papillomavirus that causes pointed condyloma and papilloma and cervical cancer.In addition, describe among the embodiment 6 in this patent specification " by 36 routine hepatitis B patients (chronic viral hepatitis B 26 examples wherein; reactivity hepatitis B 12 examples, hepatitis B virus carriers's 8 examples) are given drug alone (Interferon, rabbit 3,000,000 units), hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine targeted therapy, hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine and hepatitis B reorganization surface antibody specificity yolk antiidiotypic antibody ab2 β combination therapy respectively and carry out observation of curative effect and comparison.Experimental result shows: hepatitis B virus surface antigen HBsAg negative conversion rate combination therapy group (71.2%) is apparently higher than targeted therapy group (67.5%) and drug alone group (12.4%); Hepatitis B virus HBeAg negative conversion rate combination therapy group (89.4%) obviously is better than drug alone group (42.5%); Two groups (59.2%) obviously is better than drug alone group (6.5%) behind the hepatitis B viruses (HBV) DNA negative conversion rate; Back two groups of patients serum ALT, DBIL and TNF-alpha levels are starkly lower than the drug alone group.Surface hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine targeted therapy has advantages such as high specificity, toxic side effect are little; Hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine and hepatitis B reorganization surface antibody specificity yolk antiidiotypic antibody ab2 β combination therapy have the effect that activates somatocyte disease humoral immunization, thereby reach the purpose to hepatitis B virus resisting protection liver organization cell.Though " this patent does not spell out hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine and hepatitis B reorganization surface antibody specificity yolk antiidiotypic antibody ab2 β is by oral or injection administration; by its purpose, obviously by injection administration (and should be intravenous injection) as the biological targeting treatment.But, up to the present, China and in the world other country all do not ratify IgY and related preparations thereof are directly used in human experimentation as injection formulations.Therefore, the exploitativeness of this patent is doubtful too.
Summary of the invention
One object of the present invention is, a kind of preparation method of anti-PV IgY antibody is provided.
The anti-PV IgY antibody that second purpose of the present invention provides the method preparation is used to prepare PV infection and relative disease immune diagnostic reagent and prophylactic agent purposes.
The 3rd purpose of the present invention provides the purposes of the antigenic IgY antibody of above-mentioned anti-PV in the PV antigen purification.
The 4th purpose of the present invention provides the purposes of the antigenic IgY antibody of above-mentioned anti-PV in prevention and treatment PV infection.
In order to realize above-mentioned task, the present invention takes following technical scheme:
A kind of preparation method of anti-PV IgY antibody is characterized in that, specifically comprises the following steps:
(a) selecting PV is the caused papilloma knurl of parillomarvirus infections body tissue, carry out fragmentation by physics, chemistry, biological method, separate the PV viral protein, and will be above-mentioned one or more corresponding PV viral protein mix composition antigen suspension with arbitrary proportion;
(b) above-mentioned PV viral protein adopts molecular biology method can obtain PVL1, L2, E6, E7, E4, E5, E1 or E2 albumen by eukaryotic expression system, prokaryotic expression system, yeast expression system, the virus expression systems of routine, and above-mentioned one or more corresponding PV viral protein mixed with arbitrary proportion, form the antigen suspension;
(c) above-mentioned PV viral protein adopts the molecular biology method preparation can express one or more PVL1, L2, E6, E7, E4, E5, the proteic carrier for expression of eukaryon of E1, E2, and above-mentioned one or more carrier for expression of eukaryon mixed with arbitrary proportion, form the antigen suspension; As the genetic immunization vaccine, the mode by genetic immunization produces corresponding PV antigen;
(B), immunization method:
(a) initial immunity uses the antigen suspension of Freund's complete adjuvant emulsive A (a) or the preparation of A (b) step every female poultry, especially the distributed intramuscularly of 4 points on the hen chest is annotated, after 2 weeks, antigen suspension with Freund's incomplete adjuvant emulsive A (a) or the preparation of A (b) step carries out the immunity second time, later on every 30 days left and right sides booster immunizations once, immunity amount is the same; After one week, collect female poultry egg (egg) at initial immunity, preserve standby down for 4 ℃;
(b) adopt the method for intramuscular injection, particle gun directly the carrier for expression of eukaryon of expressing one or more PVL1, L2, E6, E7, E4, E5, L1, L2 albumen and gene thereof of A (c) step preparation to be injected in the female poultry body (hen), immunity female poultry (hen), after 2 weeks, carry out the immunity second time; Later on every 30 days left and right sides booster immunizations once, the immunity amount is the same, after one week, collects female poultry egg (egg) at initial immunity, preserves standby down for 4 ℃;
(C), the purifying of IgY:
The separation of IgY and purifying employing water dilution method or polyoxyethylene glycol method or asuro method or xanthan gum method or ultrafiltration process are carried out;
(I) method of water dilution method separation and purification IgY is:
The female poultry egg of collecting (egg) is got its yolk, thin up, water: yolk=9: 1, hatch 6h for 4 ℃, 4 ℃ of following centrifugal 25min get supernatant liquor, add 19% sodium sulfate precipitation, through ethanol sedimentation, gel-filtration/ion-exchange can obtain anti-PVIgY antibody;
(II) polyoxyethylene glycol method separation and purification IgY:
The female poultry egg of collecting (egg) is got its yolk, thin up, water: yolk=4: 1, the PEG of adding 3.5%, incubated at room 20min, 4 ℃ of following centrifugal 25min, get supernatant liquor, add the 12%PEG precipitation, add 12% PEG after the PBS dissolving and precipitate once more, add precooling 50% ethanol sedimentation, centrifugal 25min under-5 ℃, taking precipitate with PBS dissolving back filter membrane analysis, can obtain anti-PV IgY antibody;
(III) asuro method separation and purification IgY:
The female poultry egg of collecting (egg) is got its yolk, thin up, water: yolk=9: 1, hatch 6h for 4 ℃, centrifugal 25min under 4 ℃, get supernatant liquor, add the 6ml asuro, 15ml 1M calcium chloride is hatched 30min and is got supernatant, will add 100ml PBS and get supernatant after centrifugal, supernatant liquor is mixed adding PBS to 200M, add 19% sodium sulfate precipitation, centrifugal back adds PBS dissolves once more, adds 14% sodium sulfate taking precipitate once more, PBS dissolving back filter membrane analysis can obtain anti-PV IgY antibody;
(IV) xanthan gum method separation and purification IgY:
The female poultry egg of collecting (egg) is got its yolk, thin up, and water: yolk=1: 1, mix with xanthan gum solution and to hatch; The proportioning of described xanthan gum solution is: distilled water/xanthan gum=80ml/120mg, centrifugal 15min under 20 ℃ gets supernatant liquor and adds 19% sodium sulfate precipitation, and PBS dissolving back adds 14% sodium sulfate and centrifuging and taking throw out, with PBS dissolving back filter membrane analysis, can obtain anti-PV IgY antibody.
The anti-PV IgY antibody of method for preparing is used to prepare PV to be infected and relative disease immune diagnostic reagent and prophylactic agent purposes.
The immunodiagnosis that PV infection and relative disease immune diagnostic reagent thereof are used for ELISA, immunocytology and immuning tissue's cytology, antibody chip method detects.
Anti-PV IgY antibody with the auxiliary material of 0.1～90% concentration and 10～99.9% according to the conventional formulation method, be mixed with the different pharmaceutical preparation, pharmaceutical preparation comprises ointment, sprays, washing liquid, gel preparation, dry powder formulations, effervescent tablet or capsule, directly or indirectly be used in each link or the PV predisposing infection area or the infection site of PV propagation/route of infection, infection or the propagation of prevention PV.
Described anti-PV IgY antibody mixes with 10～99.9% auxiliary material with 0.1～90% concentration, method according to routine is mixed with ointment, sprays, gel preparation or dry powder formulations, directly be applied on the various external application sexually transmitted disease (STD) prevention utensils, directly blocking-up PV propagates through sexual behaviour in the sexuality process, is used to prevent the infection of HPV.
Described anti-PV IgY antibody is used for the antigenic affinity purification of corresponding PV as affinity ligand.
The present invention is by separating PV antigen or obtain above-mentioned PV albumen by genetic engineering technique by prokaryotic expression system or eukaryotic expression system (comprising baculovirus expression system) or yeast expression system from animal (ox etc.) PV focus of infection, and the mixture that above-mentioned one or more corresponding antigens is formed with the arbitrary proportion mixing; But the antigenic carrier for expression of eukaryon of above-mentioned one or more PV of construction expression also, the mode by genetic immunization produces corresponding PV antigen.Immunity poultry, especially hen obtain the anti-PV IgY of specificity antibody, will play a significant role in the prevention that the diagnosis of PV, antigenic purifying and PV infect.
IgY antibody provided by the invention has safe, nontoxic, easy to use reliable for effect, with low cost characteristics than other antiviral and preparation, is particularly suitable for developing country and uses in the larger context.
Description of drawings
Fig. 1 is that anti-HPV16L1 IgY antibody suppresses the mouse red blood cell that HPV16L1 VLP causes: wherein, (A) cause the mouse red blood cell aggegation for HPV16L1 VLP; (B) for suppressing HPV16L1 VLP, anti-HPV16L1 IgY antibody causes the mouse red blood cell aggegation; (C) negative contrast.
Fig. 2 is the HPV16L1 albumen of expressing in the anti-HPV16L1 IgY antibody test Chinese hamster ovary celI: wherein (A) is the expression of HPV16L1 albumen in Chinese hamster ovary celI; (B) negative contrast.
The present invention is described in further detail below in conjunction with embodiment that accompanying drawing and contriver provide.
Embodiment:
The application's embodiment has provided the example that female poultry is a hen, but all female poultries all can prepare antigen by method of the present invention, and as duck, goose or the like, as space is limited, the application does not provide one by one.
The anti-PV IgY of specificity of the present invention antibody can prepare by following method:
(A), the antigenic preparation of PV:
(a) select PV to infect caused papilloma knurl body tissue, carry out fragmentation, separate the PV virus antigen by physics, chemistry, biological method;
(b) adopt molecular biology method by eukaryotic expression system, prokaryotic expression system, yeast expression system, virus expression systems obtain PV L1 or (with) L2 or (with) E6 or (with) E7 or (with) E4 or (with) E5 or (with) E1 or (with) E2 antigen;
(c) above-mentioned antigen adopt the molecular biology method preparation can express one or more PVL1 or (with) L2 or (with) E6 or (with) E7 or (with) E4 or (with) E5 or (with) E1 or (with) the proteic carrier for expression of eukaryon of E2.
(B), immunization method:
(a) initial immunity is annotated with the distributed intramuscularly of 4 points of antigen suspension in every pigeon breast portion of Freund's complete adjuvant emulsive A (a) or the preparation of A (b) method.After 2 weeks, the antigen supernatant liquid for preparing with Freund's incomplete adjuvant emulsive A (a) or A (b) method carries out the immunity second time.Later on every 30 days left and right sides booster immunizations once, immunity amount is the same.After one week, collect egg at initial immunity, preserve standby down for 4 ℃.
(b) adopt methods such as intramuscular injection, particle gun directly with the preparation of A (c) method express one or more PV L1 or (with) L2 or (with) E6 or (with) E7 or (with) E4 or (with) E5 or (with) E1 or (with) the proteic carrier for expression of eukaryon of E2 injects immune hen in the hen body, after 2 weeks, carry out the immunity second time.Later on every 30 days left and right sides booster immunizations once, immunity amount is the same.After one week, collect egg at initial immunity, preserve standby down for 4 ℃.
(C), the purifying of IgY:
The separation of IgY and purifying can adopt water dilution method (WD) or polyoxyethylene glycol method (PEG) or asuro method (DS) or xanthan gum method (Xan) (Bai Xiaoli, Liang Houming. the research of four kinds of Immunoglobulin of Yolk purification process. Guangzhou foodstuffs industry science and technology .2003; 19 (4): 72-74.) or ultrafiltration process.
(a) water dilution method (WD) separation and purification IgY:
The egg of collecting is got yolk, and (water: yolk=9: 1), hatch 6h for 4 ℃, centrifugal (10000g, 25min, 4 ℃) get supernatant liquor (WD-SN) to thin up, add 19% sodium sulfate precipitation (WD-SS), and through ethanol sedimentation, gel-filtration/ion-exchange obtains IgY.
(b) polyoxyethylene glycol method (PEG) separation and purification IgY:
The egg of collecting is got yolk, thin up (water: yolk=4: 1), add PEG (3.5%), incubated at room 20min, centrifugal (10000g, 25min, 4 ℃) get supernatant liquor (PEG-SN), add the 12%PEG precipitation, add PEG (12%) after the PBS dissolving and precipitate once more, add precooling 50% ethanol sedimentation, centrifugal (10000g, 25min ,-5 ℃) get precipitation (PEG-Alc), PBS dissolving back filter membrane analysis obtains IgY.
(c) asuro method (DS) separation and purification IgY:
The egg of collecting is got yolk, thin up (water: yolk=9: 1), hatch 6h for 4 ℃, centrifugal (10000g, 25min, 4 ℃) get supernatant liquor, add the 6ml asuro, 15ml 1M calcium chloride is hatched 30min and is got supernatant, will add 100mlPBS and get supernatant (DN-SN) after centrifugal, supernatant liquor is mixed adding PBS to 200m, add 19% sodium sulfate precipitation (DS-SS), centrifugal back adds PBS dissolves once more, adds 14% sodium sulfate and gets precipitation (DS-As2) once more, PBS dissolving back filter membrane analysis obtains IgY.
(d) xanthan gum method (Xan) separation and purification IgY:
The egg of collecting is got yolk, thin up (water: yolk=1: 1), (distilled water/xanthan gum=80ml/120mg) is mixed and is hatched centrifugal (10000g, 15min with xanthan gum solution, 20 ℃) get supernatant liquor (Xan-SN), add 19% sodium sulfate precipitation (Xan-SS), PBS dissolving back adds 14% sodium sulfate and centrifuging and taking precipitation (Xan-As2), PBS dissolving back filter membrane analysis obtains IgY.
The present invention is by above-mentioned antigen prepd and immune programme for children, and through separate and purifying after can obtain the antigenic IgY antibody of the anti-PV of specificity.
Anti-PV IgY antibody corresponding PV antigen purification with separate in purposes:
The present invention is by after above-mentioned antigen prepd and the immune programme for children, and the aglucon that the IgY antibody of separation and purifying can be used as affinity column is used for the antigenic separation and purification of corresponding PV.Can significantly improve the separation efficiency of affinity chromatography, and can use repeatedly.
The purposes of anti-PV IgY antibody in the preparation immunologic function test reagent:
The present invention is by after above-mentioned antigen prepd and the immune programme for children, separating also, the IgY antibody of purifying can be widely used in comprising ELISA, immunoprecipitation, immunoelectrophoresis, ELISA, immunoblotting, antibody chip immunocytology and a series of immune diagnostic techniques such as immuning tissue's cytology, antibody chip are in order to detect corresponding PV antigen.
The purposes of anti-PV IgY antibody in preparation prevention PV infection medicine:
The present invention is by after above-mentioned antigen prepd and the immune programme for children, the IgY antibody of separation and purifying can join with 0.1～90% concentration and contain 10～99.9% auxiliary material, and is mixed with the directly or indirectly prophylactic agent formulation of the prevention HPV infection of use such as ointment, sprays, washing liquid, gel preparation, dry powder formulations, bubble rattan effervescent tablet (capsule); IgY antibody or the above-mentioned different preparations that comprise IgY antibody can also be applied on the contraceptive devices such as condom, Diaphragm contraceptive, in sexual life is usefulness, can reach the purpose that blocking-up PV propagates between Different Individual to greatest extent.Route of infection according to PV, directly or indirectly IgY or the above-mentioned different preparations that comprise IgY are smeared or are sprayed at relevant predisposing infection area and PV infection site, mode by passive immunization, improve the titre of local specific antibody, specificity combines with the PV antigen immune, blocking-up PV sticks host cell, reduces PV between the Different Individual or the propagation between the same individual different sites, reaches the purpose that prevention PV infects.
Following examples that the contriver provides only are used for understanding, describing the present invention, and the present invention is not limited to these embodiment.
Related in an embodiment plasmid, bacterial classification, phage random peptide library, host bacterium, cell, laboratory animal and main agents are expressed as follows respectively:
PT-L1 plasmid (reorganization pGEM-T plasmid contains the HPV16L1 gene) (Zheng Bin, Wang Jianwei, Jiang Huiying, etc., utilize insect-baculovirus expression system to express HPV 16 L1 albumen. " China's experiment and clinical virology magazine " 2001; 15 (4): 314-316.) (comprise the HPV16L1 gene with the pCDNA-HPV16L1 plasmid.Sun Xiangle, department carry out and give birth to, Cao's a ceremonial jade-ladle, used in libation grandson etc., and " structure of HPV 16-L1 eukaryon expression plasmid and expression and laboratory animal are to the immune response of naked DNA.The virus journal ", 1999; 15 (3): 270-274.).The shuttle vectors pFastBac-of baculovirus expression system
TMHTb, DH10Bac competent cell, Sf-9 cell are available from Invitrogen company.Grace`s substratum, cellfectin transfection reagent are available from GIBCO company.25 week～30 laying hen in all ages, body weight 1.5Kg～2.0Kg is provided by strong Po Cun poulty house, Chang'an, Xi'an county; New zealand white rabbit, C57BL/6 mouse, Balb/C mouse are provided by Xi'an Communications University medical animal experiment center; Protein G MagneticBeads is available from New England BioLabs company; The HPV-16L1 monoclonal antibody is available from Neomarker company; HRP-IgG is available from DAKO company; PCR beads purchases the company in phamasia.
The configuration of all ingredients that relates in the present embodiment and method are referring to " molecular cloning ", " Bac-to-Bac of GIBCO company baculovirus expression system operational manual ", " ProBondTM purification system specification sheets ".
Embodiment 1: the preparation of anti-PV IgY antibody
1) the antigenic preparation of PV:
PV antigen can obtain by following three kinds of different approach:
(a) select PV to infect caused papilloma knurl body tissue, carry out fragmentation, separate the PV virus antigen by physics, chemistry, biological method;
(b) adopt molecular biology method by eukaryotic expression system, prokaryotic expression system, yeast expression system, virus expression systems obtain PV L1 or (with) L2 or (with) E6 or (with) E7 or (with) E4 or (with) E5 or (with) E1 or (with) E2 antigen;
(c) adopt the molecular biology method preparation can express one or more PV L1 or (with) L2 or (with) E6 or (with) E7 or (with) E4 or (with) E5 or (with) E1 or (with) the proteic carrier for expression of eukaryon of E2.
Present embodiment only adopts (b) method to express HPV16L1 albumen by insect baculovirus expression system, with the antigenic obtain manner of its explanation PV, need to prove, the invention is not restricted to this embodiment.
Utilize reorganization Bac/B-HPV16L1 baculovirus strain to infect the Sf9 cell and cultivate 72h, ProBond for 27 ℃
TMThe non-sex change condition of Column purifying HPV16L1 albumen (method is seen ProBondTM purification system specification sheets); The quantitative HPV16L1 albumen of biuret method, Western blot detects purifying protein.
2) immune programme for children:
Initial immunity was that HPV16L1 albumen (400 μ g) adds equal-volume Freund's complete adjuvant (Freundscomplete adjuvant originally, FCA) fully be mixed into emulsion, 4 the distributed intramuscularly 1ml of some emulsions under the conventional tincture of iodine, alcohol disinfecting pigeon breast portion and the wing carry out immunity.The antigen of booster immunization is that HPV16L1 albumen (200 μ g) adds Freund's incomplete adjuvant (Freunds incomplete adjuvant, FIA) abundant mixing 2 all booster immunizations at interval subsequently.After one week, collect egg at initial immunity, preserve standby down for 4 ℃.
3) separation of IgY and purifying (present embodiment is that example is illustrated with the polyoxyethylene glycol method)
With ratio (v/v) the dilution yolk liquid of phosphoric acid buffer, add the polyethylene glycol 6000 (PEG-6000) of 3.5% (w/v) in the mixed solution after dilution, centrifugal (10 ℃, 5000g, 20min) with 4: 1; The absorbent cotton filtering supernatant, the PEG-6000 to wherein adding 8.5% (w/v) leaves standstill 10min, centrifugal (4 ℃, 10000g, 25min); In precipitation, add the long-pending phosphoric acid buffer of 2.5 times of initial yolk liquids, in lysate, add the PEG-6000 of 12% (w/v), centrifugal (4 ℃, 10000g, 25min); Resolution of precipitate is in the long-pending phosphoric acid buffer of 0.25 times of initial egg yolk liquid, place 10min on ice, add the ethanol (being chilled to-20 ℃ in advance) of equal-volume 50% (v/v) again, centrifugal (0 ℃, 10000g, 30min), use the phosphoric acid buffer dissolution precipitation, in 4 ℃ refrigerator, preserve standby.
Embodiment 2: anti-PV IgY antibody activity detects
1) mensuration of IgY antibody titer
Detect with enzyme-linked immunosorbent assay, method is as follows: after being washed plate with the HPV16Ll protein solution 150 μ L/ holes bag that contains 10 μ g/ml by droplet plate and sealing, add the purifying IgY of different extension rates, hatch 1h for 37 ℃, TBST washes plate, 3 * 5min; Add the anti-chicken IgG of the rabbit 100 μ L/ holes of horseradish peroxidase (HRP) mark of dilution in 1: 5000, hatch 1.5h for 37 ℃, TBST washes plate, 3 * 5min; Add the substrate colour developing, 1mol H
2SO
4Termination reaction.Enzyme mark automatic analyser 450nm wavelength is measured its absorbance value (OD 450nm), is contrast with the egg yolk liquid of immune egg not.The result shows: 1 week behind the initial immunity, can detect anti-HPV16L1IgY antibody in the egg yolk, and behind the booster immunization 2 times, tiring of IgY raises rapidly in the egg yolk, and the highest tiring reaches 1: 10240, and reaches a plateau in this level, continues more than 4 weeks.
2) mouse red blood cell agglutination inhibition test
Get blood behind the C57BL/6 eyeball of mouse, the heparin sodium anti-freezing, PBS washed cell 3 * 5min, 500 * g is centrifugal, and 3 * 5min is again by 1: 100 volume ratio suspension cell.The HPV16L1 VLP albumen 50 μ l/ holes that add doubling dilution during, B capable at 96 hole circle base plate A is capable add PBS50 μ l/ hole during C is capable; The anti-HPV16L1 IgY antibody 50 μ l/ holes that add dilution in 1: 200 in B is capable add PBS 50 μ l/ holes during A, C are capable, educate 30min altogether for 37 ℃; Add 100 μ l mouse red blood cell suspensions respectively, mixing, educate 3h altogether, the result for 4 ℃: anti-HPVL1 IgY antibody can suppress the mouse red blood cell agglutination reaction (Fig. 1) that HPV16L1 albumen causes.
Embodiment 3: anti-PV IgY antibody is used for the antigenic purifying of corresponding PV and separates as affinity ligand
Sepharose 4B to cyanogen bromide-activated handles, and it is crosslinked to resist proteic IgY antibody of HPV16L1 and Sepharose 4B to carry out with the concentration of 7.2mg albumen/ml glue, dress post (XK16) 10ml.The proteic Sf-9 insect cell of HPV16Ll is expressed in non-degeneration methods cracking, get supernatant after centrifugal, with 25mmol/L phosphate buffered saline buffer (pH7.0) is sample-loading buffer and level pad, 0.1mol/LGly-HCl (pH2.8) be elutriant, affine separation HPV16L1 albumen on the TA explorer 100 protein chromatographic instrument.One step was promptly reached the separating effect of expection.The entire operation process is simple fast, only needs a few hours can finish the regeneration of sample, wash-out separation and post.Simultaneously, because Yolk immunoglobulin character is comparatively stable, handle affine filler well and can preserve more than half a year under 4 ℃ of conditions, and separating power is almost constant.After using for several times, post still can keep separating power preferably.
Embodiment 4: the application of anti-PV IgY antibody in immunology reagent
Can be used as PV relative disease immune diagnostic reagent and be used for ELISA, immunoprecipitation, immunoelectrophoresis, ELISA, immunoblotting, antibody chip immunocytology and immuning tissue's cytology, antibody chip etc.
Utilize reorganization pcDNA-HPV16L1 plasmid transfection Chinese hamster ovary celI (to comprise the HPV16L1 gene, Sun Xiangle, department carries out and gives birth to, Cao's a ceremonial jade-ladle, used in libation grandson, etc. the structure of HPV 16-L1 eukaryon expression plasmid and expression and laboratory animal are to the immune response of naked DNA. viral journal .1999; 15 (3): 270-274), ordinary method prepares cell climbing sheet, through 3%H
2O
2Handle 10min, 5% normal goats serum room temperature sealing 10min, it is anti-as one to add anti-HPV16L1 IgY antibody, and 4 ℃ are spent the night, and TBST washes plate, 3 * 5min; Add the anti-chicken igg antibody of rabbit and hatch 1h for 37 ℃, TBST washes plate, 3 * 5min; Biotin labeled second antibody is hatched 1h for 37 ℃, adds horseradish peroxidase complex and hatches 1h for 37 ℃, and TBST washes plate, 3 * 5min; The DAB colour developing, Hematorylin is redyed, dehydration of alcohol, transparent, the gummy mounting of dimethylbenzene.Substituting one with TBS resists as negative control.Microscopically is observed.Anti-HPVL1IgY antibody can with the HPV16L1 albumen generation specificity association reaction that is expressed in the Chinese hamster ovary celI, pale brown look positive particle is positioned at nucleus and cytoplasm, negative control sheet non-coloring (Fig. 2).
In addition, the people's cervical mucosa tissue that also can select HPV16 to infect, conventional formalin fixed, paraffin embedding, section dewaxes to water, through 3%H
2O
2Handle 10min, 5% normal goats serum room temperature sealing 10min, it is anti-as one to add anti-HPV16L1 IgY antibody, and 4 ℃ are spent the night, and TBST washes plate, 3 * 5min; Add the anti-chicken igg antibody of rabbit and hatch 1h for 37 ℃, TBST washes plate, 3 * 5min; Biotin labeled second antibody is hatched 1h for 37 ℃, adds horseradish peroxidase complex and hatches 1h for 37 ℃, and TBST washes plate, 3 * 5min; The DAB colour developing, Hematorylin is redyed, dehydration of alcohol, transparent, the gummy mounting of dimethylbenzene.Substituting one with TBS resists as negative control.Microscopically is observed.Found that to occur pale brown look positive particle in the cervical mucosa epithelial nucleus, negative control sheet non-coloring, illustrate anti-HPV16L1 IgY antibody can be expressed in HPV16L1 fusion rotein generation specificity association reaction in the cervical mucosa epithelial nucleus.
Embodiment 5: the application of anti-PV IgY antibody in different preparations
Anti-PV IgY antibody can 0.1～90% concentration join and contain 10～99.9% auxiliary material, and the prophylactic agent formulation that the prevention PV that is mixed with direct or indirect use infects, as ointment, sprays, washing liquid, gel preparation, dry powder formulations, bubble rattan tablet (capsule) etc.
Embodiment 6: the application of anti-PV IgY antibody in different external contraceptive utensils
According to embodiment 5 described methods, concentration that can 0.1～90% with anti-PV IgY antibody joins and contains 10%～99.9% auxiliary material, and be mixed with as ointment, sprays, gel preparation, dry powder formulations etc., it is applied on the external contraceptive utensil, can in the process that sexual life is carried out, directly blocks the propagation of PV.
Claims (8)
1. the preparation method of an anti-PV IgY antibody is characterized in that, specifically comprises the following steps: (A), the antigenic preparation of PV:
(a) selecting PV is the caused papilloma knurl of parillomarvirus infections body tissue, carry out fragmentation by physics, chemistry, biological method, separate the PV viral protein, and will be above-mentioned one or more corresponding PV viral protein mix composition antigen suspension with arbitrary proportion;
(b) above-mentioned PV viral protein adopts molecular biology method can obtain PV L1, L2, E6, E7, E4, E5, E1 or E2 albumen by eukaryotic expression system, prokaryotic expression system, yeast expression system, the virus expression systems of routine, and above-mentioned one or more corresponding PV viral protein mixed with arbitrary proportion, form the antigen suspension;
(c) above-mentioned PV viral protein adopts the molecular biology method preparation can express one or more PV L1, L2, E6, E7, E4, E5, the proteic carrier for expression of eukaryon of E1, E2, and above-mentioned one or more carrier for expression of eukaryon mixed with arbitrary proportion, form suspension, as the genetic immunization vaccine, the mode by genetic immunization produces corresponding PV antigen;
(B), immunization method:
(a) initial immunity is annotated with the distributed intramuscularly of 4 points of antigen suspension on female poultry chest of Freund's complete adjuvant emulsive A (a) or the preparation of A (b) step, after 2 weeks, antigen suspension with Freund's incomplete adjuvant emulsive A (a) or the preparation of A (b) step carries out the immunity second time, later on every 30 days left and right sides booster immunizations once, immunity amount is the same; After one week, collect female poultry egg at initial immunity, preserve standby down for 4 ℃;
(b) adopt the method for intramuscular injection, particle gun directly the carrier for expression of eukaryon of expressing one or more PV L1, L2, E6, E7, E4, E5, L1, L2 albumen and gene thereof of A (c) step preparation to be injected in the female poultry body, the female poultry of immunity, after 2 weeks, carry out the immunity second time; Later on every 30 days left and right sides booster immunizations once, the immunity amount is the same, after one week, collects female poultry egg at initial immunity, preserves standby down for 4 ℃;
(C), the purifying of IgY:
The separation of IgY and purifying employing water dilution method or polyoxyethylene glycol method or asuro method or xanthan gum method or ultrafiltration process are carried out;
(I) method of water dilution method separation and purification IgY is:
The female poultry egg of collecting is got its yolk, thin up, and water: yolk=9: 1, hatch 6h for 4 ℃, 4 ℃ of following centrifugal 25min get supernatant liquor, add 19% sodium sulfate precipitation, and through ethanol sedimentation, gel-filtration/ion-exchange can obtain anti-PVIgY antibody;
(II) polyoxyethylene glycol method separation and purification IgY:
The female poultry egg of collecting is got its yolk, thin up, water: yolk=4: 1, the PEG of adding 3.5%, incubated at room 20min, 4 ℃ of following centrifugal 25min, get supernatant liquor, add the 12%PEG precipitation, add 12% PEG after the PBS dissolving and precipitate once more, add precooling 50% ethanol sedimentation, centrifugal 25min under-5 ℃, taking precipitate with PBS dissolving back filter membrane analysis, can obtain anti-PVIgY antibody;
(III) asuro method separation and purification IgY:
The female poultry egg of collecting is got its yolk, thin up, water: yolk=9: 1, hatch 6h for 4 ℃, 4 ℃ of following centrifugal 25min get supernatant liquor, add the 6ml asuro, 15ml 1M calcium chloride is hatched 30min and is got supernatant, will add 100ml PBS and get supernatant after centrifugal, supernatant liquor is mixed adding PBS to 200M, add 19% sodium sulfate precipitation, centrifugal back adds PBS dissolves once more, adds 14% sodium sulfate taking precipitate once more, PBS dissolving back filter membrane analysis can obtain anti-PVIgY antibody;
(IV) xanthan gum method separation and purification IgY:
The female poultry egg of collecting is got its yolk, thin up, and water: yolk=1: 1, mix with xanthan gum solution and to hatch; The proportioning of described xanthan gum solution is: distilled water/xanthan gum=80ml/120mg, centrifugal 15min under 20 ℃ gets supernatant liquor and adds 19% sodium sulfate precipitation, and PBS dissolving back adds 14% sodium sulfate and centrifuging and taking throw out, with PBS dissolving back filter membrane analysis, can obtain anti-PVIgY antibody.
2. the method for claim 1 is characterized in that, described female poultry is a hen.
3. the preparation method of a kind of anti-PV IgY antibody according to claim 1, it is characterized in that, described PV is present all infection animals found and people's PV hypotype, comprise BPV-1, CRPV, HPV1, HPV2, HPV 4, HPV 3, HPV 10, HPV 16, HPV 18, HPV31, HPV 45, HPV 6, HPV 11, HPV 42, HPV 43, HPV 44, HPV 33, HPV35, HPV 39, HPV 51, HPV 52, HPV 56, HPV58, HPV 59, HPV68 one of them; The PV viral protein is meant PV structural protein L1, L2 or functional protein E1, E2, E5, E6, E7; The PV virus antigen is meant PV structural protein L1, L2 albumen or its gene and functional protein E1, E2, E5, E6, E7 albumen or its gene.
4. the anti-PV IgY antibody of the described method preparation of claim 1 is used to prepare PV infection and relative disease immune diagnostic reagent and prophylactic agent purposes.
5. purposes as claimed in claim 4 is characterized in that: the immunodiagnosis that described PV infection and relative disease immune diagnostic reagent thereof are used for ELISA, immunocytology and immuning tissue's cytology, antibody chip method detects.
6. purposes as claimed in claim 4, it is characterized in that, described anti-PV IgY antibody with the auxiliary material of 0.1～90% concentration and 10～99.9% according to the conventional formulation method, be mixed with the different pharmaceutical preparation, pharmaceutical preparation comprises ointment, sprays, washing liquid, gel preparation, dry powder formulations, effervescent tablet or capsule, directly or indirectly be used in each link or the PV predisposing infection area or the infection site of PV propagation/route of infection, infection or the propagation of prevention PV.
7. purposes as claimed in claim 4, it is characterized in that, described anti-PV IgY antibody mixes with 10～99.9% auxiliary material with 0.1～90% concentration, method according to routine is mixed with ointment, sprays, gel preparation or dry powder formulations, directly be applied on the various external application sexually transmitted disease (STD) prevention utensils, directly blocking-up PV propagates through sexual behaviour in the sexuality process, is used to prevent the infection of PV.
8. purposes as claimed in claim 4 is characterized in that, described anti-PV IgY antibody is used for the antigenic affinity purification of corresponding PV as affinity ligand.
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