CN108840929A - Anti-human papilloma virus (anti-HPV) small molecular antibody and combinations thereof and preparation method and application - Google Patents
Anti-human papilloma virus (anti-HPV) small molecular antibody and combinations thereof and preparation method and application Download PDFInfo
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- CN108840929A CN108840929A CN201810637337.1A CN201810637337A CN108840929A CN 108840929 A CN108840929 A CN 108840929A CN 201810637337 A CN201810637337 A CN 201810637337A CN 108840929 A CN108840929 A CN 108840929A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
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- C07K2317/35—Valency
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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Abstract
The present invention relates to a kind of anti-human papilloma virus (anti-HPV) small molecular antibody and combinations thereof and preparation method and application.Anti-human papilloma virus (anti-HPV) small molecular antibody Fab of the invention can pass through cell membrane and enter infected cell, attack HPV-E6/E7 oncogene protein, makes its paralysis inactivation and loses cell transformation and carcinogenesis.The present invention according to local HPV popularity, can produce anti-human papilloma virus (anti-HPV) E6, E7, E6/E7 small molecular antibody Fab of all model HPV for covering local prevalence of various combination, with a definite target in view, accurate prevention and control；The present invention also provides liquid crystal microcapsules, gel, suppository, cream, washing lotion, tablet, effervescent tablet, capsule, soft capsule, spray and injection made of a kind of anti-human papilloma virus (anti-HPV) small molecular antibody and combinations thereof simultaneously, the application in drug, sterile products, health care product and medical instrument for preventing and treating human papilloma virus (HPV) infection and/or for preventing and treating cervical carcinoma.
Description
Technical field
The present invention relates to biologics technical fields, and in particular to a kind of anti-human papilloma virus (anti-HPV) small molecular antibody and its group
Close object and preparation method and application.
Background technique
Cervical carcinoma is to seriously endanger the disease of WomanHealth and life, and the whole world has a people to die of cervical carcinoma death for every 2 minutes.
Modern medicine is proved cervix cancer from a kind of human papilloma virus (HPV), and 80% women 50 years old before can be by human milk head
Tumor virus infection, the whole world are infected women up to 3,000,000,000.It recent studies have shown that, 30 years old to 50 years old Chinese women, high-risk HPV is total
Infection rate is 15% to 20.8%, i.e., every 5-6 female middle-aged just has a people to infect the strongest high-risk HPV of carcinogenicity, and deposits
In morbidity increasingly rejuvenation trend.According to statistics, China's cervical cancer pathogenesis rate is 6 times of developed country, accounts for about global patient's sum
1/3.
So far, the world of medicine develops the active drug for treating human papilloma virus (HPV) infection not yet, resists
Raw element and other bactericides, disinfectant absolutely not act on human papilloma virus (HPV).The HPV vaccine listed at present only has
Prevent the effect for once crossing infection.Since human papilloma virus (HPV) is a kind of special " nonenveloped virus ", an intrusion human body
Cell will slough coating coat (capsid protein L 1/L2)；The infected cell surface capsid protein targeted without vaccine
L1 target antigen, causes vaccine failure；Therefore, HPV vaccine has invaded cell to HPV, had been checked out the patient of the HPV infection positive both
There is no prevention effect, more without therapeutic effect.
China shows a research report of 3381 female genital tract human papilloma virus Analysis of test results, women
Middle human papilloma virus (HPV) positive rate is up to 18.75%, calculates accordingly, and China's human papilloma virus (HPV) positive patient is high
Up to 0.9 hundred million people.
The positive patient that these human papilloma virus (HPV) have been entered with cell, other than vaccinating in vain, tradition
Antibody and many Chinese herbal medicines cannot be introduced into infected cell and work due to belonging to macromolecular；Small-molecule chemical medicine and part are small
Molecule Chinese medicine is not then due to having specificity that will necessarily generate strong toxic side effect to normal cell.Therefore, it both pasts medical help at present,
Also without any method of eradication.Most make us terrible, wherein not shifted to an earlier date such as by high-risk human mammilla papillomavirus (HPV) the infected
Intervened, there is 20% may develop into cancer after 5 years.
Therefore, human papilloma virus (HPV) positive patient " sun is turned out cloudy " can be made and can prevent and control by researching and developing one kind
The new drug for treating cervical carcinoma is to be related to the major issue of global hundreds of millions women's healths and life, and pendulum at International Medical science interface
A preceding important topic.
Summary of the invention
The object of the present invention is to provide a kind of anti-human papilloma virus (anti-HPV) small molecular antibody and combinations thereof and preparation methods
And application, solve the problems, such as that there has been no the drugs that can effectively treat cervical carcinoma in the prior art.
The technical proposal for solving the technical problem of the invention is：A kind of anti-human papilloma virus (anti-HPV) small molecular antibody, institute
Anti-human papilloma virus (anti-HPV) small molecular antibody is stated to be prepared by the following method：
A, antigen is prepared：Freund's adjuvant is added in human papillomavirus polypeptides, it is even to be placed in high speed homogenizer high speed
Change, obtains immune human papillomavirus polypeptides antigen；
B, immune egg is prepared：Every two weeks to the human papillomavirus polypeptides antigen of laying hen injection primary immunization, note
It penetrates 2-5 times, takes laying hen to produce immune egg behind at least the 12nd day after last time injection, and carry out coded markings, resisted
Egg is immunized in human papilloma virus-IgY；
C, anti-human papilloma virus (anti-HPV)-IgY crude extract is prepared；
D, anti-human papilloma virus (anti-HPV)-IgY sterling solution is prepared；
E, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is prepared：Anti-human papilloma virus (anti-HPV)-IgY sterling solution is adjusted into pH
To 3.0-5.0, catalytic proteins enzyme is added；Dissolution is sufficiently stirred, generates enzymatic reaction, then low-temperature and high-speed rotating centrifugal, abandons
It goes to precipitate to obtain supernatant, ultrafiltration is carried out to supernatant, obtains concentrate；Concentrate obtained after ultrafiltration is subjected to gel chromatography again,
Chromatography gleanings dialysis is concentrated, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is obtained.
In anti-human papilloma virus (anti-HPV) small molecular antibody of the invention, the anti-human papilloma virus (anti-HPV) that step E is prepared is small
Molecular antibody Fab also obtains the anti-human papilloma of long-actingization using the modification of polyethylene glycol, dextran or polyaminoacid long-actingization
Viral small molecular antibody Fab.
In anti-human papilloma virus (anti-HPV) small molecular antibody of the invention, the anti-human papilloma virus (anti-HPV) that step E is prepared is small
Molecular antibody Fab carries out long-actingization using polyethylene glycol and modifies, and as follows using the process conditions of polyethylene glycol long-actingization modification：
Borate buffer pH value is adjusted to 6.5-10.0, is added and adjusts in anti-human papilloma virus (anti-HPV) small molecular antibody Fab
Borate buffer after pH makes the concentration 0.1-0.5mg/mL of anti-human papilloma virus (anti-HPV) small molecular antibody Fab, and poly- second two is added
Alcohol is reacted, and the ratio of the molar ratio of anti-human papilloma virus (anti-HPV) small molecular antibody Fab and polyethylene glycol is 1/10~1/30,
Reaction temperature is 15 DEG C~30 DEG C, and the reaction time controls in 1h~3h.
In anti-human papilloma virus (anti-HPV) small molecular antibody of the invention, anti-human papilloma virus (anti-HPV) small molecule that step E is obtained
Monoclonal antibody is also by being dried to obtain powdered anti-human papilloma virus (anti-HPV) small molecular antibody Fab；
Powdered anti-human papilloma virus (anti-HPV) small molecular antibody Fab is also by natural CPPs or artificial synthesized CPPs
Coupling obtains the anti-human papilloma virus (anti-HPV) small molecular antibody Fab of the strong coupling of permeation cell film ability, wherein the method packet being coupled
It includes：
Take 1-3 parts powdered of anti-human papilloma virus (anti-HPV) small molecular antibody Fab dry powder, the CPPs- of following mass fraction
EGFP1-3 parts, 5-10 parts of BSA-NS protein microsphere, are dissolved in the PBS of respective pH7.5,0.01mol/L respectively, and obtaining concentration is
The anti-human papilloma virus (anti-HPV) small molecular antibody Fab solution of 0.5-1.5g/L, the CPPs-EGFP solution that concentration is 0.5-1.5g/L,
Concentration is the protein microsphere solution of 2.5-5g/L, then respectively with volume ratio 1:10~1:30 ratio is slowly added to 10-25mol/L
Ethanol solution, be stirred at room temperature reaction 10-30 minute, by the reaction mixture of anti-human papilloma virus (anti-HPV) small molecular antibody Fab with
The reaction mixture of CPPs-EGFP is separately added into ultra-filtration centrifuge tube, respectively with the acetate buffer of pH4.5,0.01mol/L and
The PBS of pH7.5,0.01mol/L are cleaning solution centrifuge washing under the conditions of 4 DEG C；BSA-NS protein microsphere reaction mixture is added
Enter ultra-filtration centrifuge tube, and centrifuge washing is distinguished under the conditions of 4 DEG C to remove remaining SPDP with the PBS of pH7.5,0.01mol/L
Obtain the SPDP derivative of anti-human papilloma virus (anti-HPV) small molecular antibody Fab-PDP, CPPs-EGFP-PDP, BSA-NS-PDP, 4 DEG C
It saves backup；Appropriate DTT, which is added, in obtained anti-human papilloma virus (anti-HPV) small molecular antibody Fab-PDP Acetate Solution makes to react
The final concentration of 50mmol/L of DTT in system, is gently stirred 30 minutes at room temperature, is added in ultra-filtration centrifuge tube, with pH7.5,0.01mol/L
PBS be cleaning solution centrifuge washing under the conditions of 4 DEG C, remove residue DTT, obtain anti-human papilloma virus (anti-HPV) small molecular antibody Fab-
PDP-SH solution, and mixed immediately with CPPs-EG-FP-PDP, BSA-NS-PDP solution, 4 DEG C of stirring 15-20h are centrifuged with PBS
Washing, precipitating, finally obtains CPPs-EG-FP-BSA-NS- anti-human papilloma virus (anti-HPV) small molecular antibody Fab protein conjugate.
In anti-human papilloma virus (anti-HPV) small molecular antibody of the invention, extracted in step C using Aqua pure extract method, chloroform
Method, cold ethanol precipitation method or ammonium sulfate precipitation method prepare anti-human papilloma virus (anti-HPV)-IgY crude extract.
In anti-human papilloma virus (anti-HPV) small molecular antibody of the invention, anti-human cream is prepared using Aqua pure extract method in step C
Head tumor virus-IgY crude extract, specifically includes：Egg is immunized in prepared anti-human papilloma virus (anti-HPV)-IgY to be cleaned with circulating water, wine
Essence cleans disinfection, then smashes anti-human papilloma virus (anti-HPV)-IgY with eggbeater and egg is immunized, and removes egg white using yolk sieve sieving, leaves egg
Huang stirs evenly；Distilled water is added by 3-8 times of egg yolk liquid volume again, be diluted and is uniformly mixed, with 1.0N HCI solution
Adjust pH to 5.5-6.5；The dilution for mixing up pH value is further stirred, is then cooled to 2-6 DEG C, stands 12
- 24 hours hours；By dilution in high speed centrifugation；It takes separating obtained supernatant to set and carries out ultrafiltration concentration 10-20 in ultrafilter
Times；Then the sodium alginate solution that concentration is 1.0-3.0% is added, is slowly added sodium alginate solution to final concentration of 0.1-0.2%,
Stirring is muddy to occurring；Add 1.0-3.0%CaCl2Liquid, until final concentration of 0.1-0.2%, stirs evenly, 3-4 DEG C of standing
8-12 hours；High speed centrifugation simultaneously takes supernatant, obtains anti-human papilloma virus (anti-HPV)-IgY crude extract；
Step D is specifically included：Obtained anti-human papilloma virus (anti-HPV)-IgY crude extract is dissolved in the M of pH7.0,0.01mol/L
In PB phosphate buffer, then ion exchange column and gel chromatography column chromatography are successively crossed respectively, obtain anti-human papilloma virus (anti-HPV)-
IgY sterling solution；
Human papillomavirus polypeptides in step A are HPV-E6, HPV-E7 or HPV-E6-E7；Human papillomavirus polypeptides
Ratio with the mass ratio of Freund's adjuvant is (1-10):(10-1)；
The ratio that mass ratio is added in catalytic protein enzyme in step E is 0.01%-0.1%；Then, low-temperature and high-speed is centrifuged.
The present invention also provides a kind of preparation methods of anti-human papilloma virus (anti-HPV) small molecular antibody, including：
A, antigen is prepared：Freund's adjuvant is added in human papillomavirus polypeptides, it is even to be placed in high speed homogenizer high speed
Change, obtains immune human papillomavirus polypeptides antigen；
B, immune egg is prepared：Every two weeks to the human papillomavirus polypeptides antigen of laying hen injection primary immunization, note
It penetrates 2-5 times, takes laying hen to produce immune egg behind at least the 12nd day after last time injection, and carry out coded markings, resisted
Egg is immunized in human papilloma virus-IgY；
C, anti-human papilloma virus (anti-HPV)-IgY crude extract is prepared；
D, anti-human papilloma virus (anti-HPV)-IgY sterling solution is prepared；
E, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is prepared：Anti-human papilloma virus (anti-HPV)-IgY sterling solution is adjusted into pH
To 3.0-5.0, catalytic proteins enzyme is added；Dissolution is sufficiently stirred, generates enzymatic reaction, then low-temperature and high-speed rotating centrifugal, abandons
It goes to precipitate to obtain supernatant, ultrafiltration is carried out to supernatant, obtains concentrate；Concentrate obtained after ultrafiltration is subjected to gel chromatography again,
Chromatography gleanings dialysis is concentrated, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is obtained.
The present invention also provides above-mentioned anti-human papilloma virus (anti-HPV) small molecular antibody as preparation for preventing and treating human papilloma
Answering in viral (HPV) infection, and/or the drug for preventing and treating cervical carcinoma, sterile products, health care product or medical instrument
With.
It include above-mentioned anti-human papilloma virus (anti-HPV) small molecular antibody and at least the present invention also provides a kind of composition
A kind of other pharmaceutically acceptable components.
In the present compositions, the anti-human papilloma virus (anti-HPV) small molecular antibody addition auxiliary material or base-material or chemistry
Medicine, Chinese medicine, be made liquid crystal microcapsules, gel, suppository, cream, washing lotion, tablet, effervescent tablet, capsule, soft capsule, in spray
At least one, or be made anti-human papilloma virus (anti-HPV) (HPV) E6, E7, E6/E7 small molecular antibody Fab water for injection injection and
At least one of powder-injection.
The present invention also provides combinations of the above object as preparation for prevent and treat human papilloma virus (HPV) infection, and/or
The application in drug, sterile products, health care product or medical instrument for preventing and treating cervical carcinoma.
Implement anti-human papilloma virus (anti-HPV) small molecular antibody of the invention and combinations thereof and preparation method and application, has
Following beneficial effect：Anti-human papilloma virus (anti-HPV) small molecular antibody Fab of the invention can pass through cell membrane and enter infected cell, attack
HPV-E6/E7 oncogene protein is hit, makes its paralysis inactivation and loses cell transformation and carcinogenesis；And it can be according to locality
HPV popularity, produce all model HPV for covering local prevalence of various combination anti-human papilloma virus (anti-HPV) E6, E7,
E6/E7 small molecular antibody Fab, some put mistake accurate prevention and control.
Specific embodiment
Below with reference to embodiment, to anti-human papilloma virus (anti-HPV) small molecular antibody of the invention and combinations thereof and preparation side
Method and application are described further：
The present invention relates to a kind of anti-human papilloma virus (anti-HPV) small molecular antibodies, and the anti-human papilloma virus (anti-HPV) small molecular antibody is by such as
Lower section method is prepared.
The preparation method of anti-human papilloma virus (anti-HPV) small molecular antibody is specific as follows：
Specifically, A, prepare antigen：Freund's adjuvant is added in human papillomavirus polypeptides, is placed in high speed homogenizer
High speed homogenizes, and obtains immune human papillomavirus polypeptides antigen；Wherein, human papillomavirus polypeptides HPV-E6, HPV-
E7 or HPV-E6-E7；The ratio of the mass ratio of human papillomavirus polypeptides and Freund's adjuvant is (1-10):(10-1).
B, immune egg is prepared：Every two weeks to the human papillomavirus polypeptides antigen of laying hen injection primary immunization, altogether
Injection 2-5 times takes laying hen to produce immune egg, and carries out coded markings behind at least the 12nd day after injection for the last time, obtains
Egg is immunized in anti-human papilloma virus (anti-HPV)-IgY；Preferably, it injects 3 times, takes laying hen produced behind the 15th day after third time injection
Immune egg.
C, anti-human papilloma virus (anti-HPV)-IgY crude extract is prepared, it can be heavy using Aqua pure extract method, chloroform extraction method, cold ethyl alcohol
The methods of shallow lake method or ammonium sulfate precipitation method etc. prepare anti-human papilloma virus (anti-HPV)-IgY crude extract；It is following by taking Aqua pure extract method as an example into
Row is described in detail, but is realized extracted in this hair using the simple transformation of other methods progress to those skilled in the art
Within bright protection scope.Anti-human papilloma virus (anti-HPV)-IgY crude extract is prepared using Aqua pure extract method, is specifically included：Prepared by
Anti-human papilloma virus (anti-HPV)-IgY egg be immunized cleaned with circulating water, wipes of alcohol wash disinfection, then smash anti-human papilloma with eggbeater
Egg is immunized in virus-IgY, removes egg white using yolk sieve sieving, leaves yolk, stir evenly；Again extraordinarily by the 3-8 of egg yolk liquid volume
Enter distilled water, be diluted and be uniformly mixed, with 1.0N HCI solution tune pH to 5.5-6.5；To mix up the dilution of pH value into
One step stirs, and is then cooled to 2-6 DEG C, stands -24 hours 12 hours；By dilution in high speed centrifugation；It takes
Separating obtained supernatant, which is set, to carry out being concentrated by ultrafiltration 10-20 times in ultrafilter；Then the alginic acid that concentration is 1.0-3.0% is added
Sodium liquid is slowly added sodium alginate solution to final concentration of 0.1-0.2%, and stirring is muddy to occurring；Add 1.0-3.0%
CaCl2Liquid, until final concentration of 0.1-0.2%, stirs evenly, 3-4 DEG C standing 8-12 hours；High speed centrifugation simultaneously takes supernatant, obtains
To anti-human papilloma virus (anti-HPV)-IgY crude extract.
D, anti-human papilloma virus (anti-HPV)-IgY sterling solution is prepared, is specifically included：Obtained anti-human papilloma virus (anti-HPV)-IgY is thick
Extract is dissolved in the MPB phosphate buffer of pH7.0,0.01mol/L, then successively crosses ion exchange column and gel chromatography respectively
Column chromatography, obtains anti-human papilloma virus (anti-HPV)-IgY sterling solution.Anti-human papilloma virus (anti-HPV)-IgY sterling solution may also pass through cold
Dry or middle low temperature spray drying or fluidized bed drying is lyophilized and other drying modes for not influencing antibody activity are made anti-human
Papillomavirus-IgY sterling dry powder.
E, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is prepared：Anti-human papilloma virus (anti-HPV)-IgY sterling solution is adjusted into pH
Ratio to 3.0-5.0, then in mass ratio is that catalytic proteins enzyme is added in 0.01%-0.1%；Dissolution is sufficiently stirred, generates enzyme
Promote reaction, then low-temperature and high-speed rotating centrifugal, discards and precipitate to obtain supernatant, carries out ultrafiltration to supernatant, obtains concentrate；Again will
Obtained concentrate carries out gel chromatography after ultrafiltration, and by chromatography gleanings dialysis concentration, it is anti-to obtain anti-human papilloma virus (anti-HPV) small molecule
Body Fab.Wherein, catalytic protein enzyme can be papain or pepsin etc..Obtained anti-human papilloma virus (anti-HPV) is small
Molecular antibody Fab can also be further across freeze-drying or middle low temperature spray drying or fluidized bed drying and other not shadows
Anti-human papilloma virus (anti-HPV) small molecular antibody Fab dry powder is made in the drying mode for ringing antibody activity.It should be noted that biotechnology
There are many methods for cutting transformation macromolecular antibody, and the present invention is illustrated with one of method and implementation steps, but is not limited to this
Method and implementation steps, those skilled in the art can be made various changes or modifications with above explained method and implementation steps, this
A little equivalent forms are within the scope of the present invention.
F, anti-human papilloma virus (anti-HPV) small molecular antibody Fab also utilizes polyethylene glycol, dextran or polyaminoacid long-actingization
Modification obtains the anti-human papilloma virus (anti-HPV) small molecular antibody Fab of long-actingization, realizes that long-term effect, reduction poison pair are made to extend half-life period
With, increase stability, reduce immunogenicity and antigenicity.Illustrate by taking PEG method of modifying and implementation steps as an example below, but unlimited
In this method and implementation steps, those skilled in the art can be made various changes or be repaired with above explained method and implementation steps
Change, such equivalent forms are within the scope of the present invention.Those skilled in the art are also easy transformation according to this method and adopt
Take the other long-actingization methods of dextran or polyaminoacid etc. to carry out long-actingization to modify, protection scope of the present invention it
It is interior.The process conditions modified using polyethylene glycol long-actingization are as follows：Borate buffer pH value is adjusted to 6.5-10.0, in anti-human cream
The borate buffer after adjusting pH is added in head tumor virus small molecular antibody Fab, makes anti-human papilloma virus (anti-HPV) small molecular antibody Fab
Concentration be 0.1-0.5mg/mL, polyethylene glycol is added and is reacted, and anti-human papilloma virus (anti-HPV) small molecular antibody Fab and poly- second
The ratio of the molar ratio of glycol is 1/10~1/30, and reaction temperature is 15 DEG C~30 DEG C, and the reaction time controls in 1h~3h.Wherein
Polyethylene glycol is the PEG, preferably PEG5000, or PEG4000 or PEG2000 etc. of macromolecule.
G, the present invention is coupled small point of anti-human papilloma virus (anti-HPV) using the artificial synthesized CPPs of a variety of natural origin CPPs or a variety of
Sub- monoclonal antibody.Such as the small peptide and poly of ANTP (Antennapedia), VP22 (viral protein 22) and various synthesis
Arginine, transportan (sequence gWTLN-SAgYLLgKINLKALAALAKKIL) etc..Below with cell-penetrating peptide-small molecule
Illustrate for monoclonal antibody-microballoon conjugate preparation method and implementation steps, but is not limited to this method and implementation steps.Its
The method of middle coupling includes：Take the powdered anti-human papilloma virus (anti-HPV) small molecular antibody Fab dry powder 1-3 of following mass fraction
Part, CPPs-EGFP1-3 parts, 5-10 parts of BSA-NS protein microsphere, are dissolved in the PBS of respective pH7.5,0.01mol/L respectively, obtain
To concentration be 0.5-1.5g/L anti-human papilloma virus (anti-HPV) small molecular antibody Fab solution, concentration be 0.5-1.5g/L CPPs-
EGFP solution, the protein microsphere solution that concentration is 2.5-5g/L, then respectively with volume ratio 1:10~1:30 ratio is slowly added to
Reaction 10-30 minutes is stirred at room temperature, by the anti-of anti-human papilloma virus (anti-HPV) small molecular antibody Fab in the ethanol solution of 10-25mol/L
The reaction mixture of mixture and CPPs-EGFP is answered to be separately added into ultra-filtration centrifuge tube, respectively with the acetic acid of pH4.5,0.01mol/L
The PBS of salt buffer and pH7.5,0.01mol/L are cleaning solution centrifuge washing under the conditions of 4 DEG C；BSA-NS protein microsphere is anti-
Answer mixture be added ultra-filtration centrifuge tube, and with the PBS of pH7.5,0.01mol/L under the conditions of 4 DEG C centrifuge washing, to remove residue
SPDP, the SPDP for respectively obtaining anti-human papilloma virus (anti-HPV) small molecular antibody Fab-PDP, CPPs-EGFP-PDP, BSA-NS-PDP spread out
Biology, 4 DEG C save backup；It is added in obtained anti-human papilloma virus (anti-HPV) small molecular antibody Fab-PDP Acetate Solution appropriate
DTT makes the final concentration of 50mmol/L of DTT in reaction system, gently stirs at room temperature 30 minutes, be added ultra-filtration centrifuge tube in, with pH7.5,
The PBS of 0.01mol/L is cleaning solution centrifuge washing under the conditions of 4 DEG C, removes residue DTT, obtains small point of anti-human papilloma virus (anti-HPV)
Sub- monoclonal antibody-PDP-SH solution, and mixed immediately with CPPs-EG-FP-PDP, BSA-NS-PDP solution, 4 DEG C of stirring 15-20h,
With PBS centrifuge washing, precipitating finally obtains CPPs-EG-FP-BSA-NS- anti-human papilloma virus (anti-HPV) small molecular antibody Fab albumen
Conjugate.
It is described in detail below by specific embodiment.
Human papilloma virus (HPV) has 100 Multiple Types, and the present invention selects 18 kinds of common models to cut using biotechnology
The method cut prepares anti-human papilloma virus (anti-HPV) small molecular antibody Fab, including HPV16 type, HPV18 type, HPV26 type, HPV31
Type, HPV33 type, HPV35 type, HPV39 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type,
HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV82 type；The human papilloma other than this 18 kinds of models can also in addition be selected
Viral (HPV) prepares anti-human papilloma virus (anti-HPV) small molecular antibody Fab using the method for biotechnology cutting.Below wherein to use
Biotechnology patterning method is illustrated for preparing anti-HPV16 type E6 small molecular antibody Fab, is prepared using biotechnology patterning method
Anti- HPV16 type E7, E6/E7 small molecular antibody Fab and using biotechnology patterning method prepare the other models of anti-HPV E6, E7,
It is same to can refer to the following method for preparing anti-HPV16 type E6 small molecular antibody Fab for the operating procedure of E6/E7 small molecular antibody Fab
It carries out, only the antigen of immunization laying hen will change HPV16 type E7 polypeptide antigen or E6/E7 complex polypeptide antigen and difference into
Corresponding E6, E7 polypeptide antigen of HPV model or E6/E7 complex polypeptide antigen.It, can also be by 2-18 kind or more when actual implementation
Corresponding E6, E7 polypeptide antigen of different HPV models or E6/E7 complex polypeptide antigen optimize combination, and that complex polypeptide is made is anti-
It is former；Then, using this combined complex polypeptide antigen immunization laying hen, anti-multivalence human papilloma virus E6, E7 or E6/ is made
E7-IgY.Can also by a variety of anti-human papilloma virus (anti-HPV) E6, E7 or E6/E7-IgY obtained or anti-multivalence human papilloma virus E6,
E7 or E6/E7-IgY optimizes combination according to local HPV popularity, and the institute for covering local prevalence of various combination is made
Have model HPV a variety of anti-human papilloma virus (anti-HPV) E6, E7 or E6/E7 composite IgYs or anti-multivalence human papilloma virus E6, E7 or
E6/E7 composite IgY.Again by made anti-human papilloma virus (anti-HPV) E6, E7 or E6/E7-IgY or anti-multivalence human papilloma virus
E6, E7 or E6/E7-IgY or a variety of anti-multivalence human papilloma virus E6s, E7 or E6/E7 composite IgY are cut by biotechnology
The method preparation all model HPV that can cover local prevalence a variety of anti-human papilloma virus (anti-HPV) E6, E7 or E6/E7 small molecules it is anti-
Body Fab or anti-multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody Fab, detailed process just do not repeat here.
One, anti-HPV-E6-IgY is prepared
1.HPV16 type-E6 antigen polypeptide designs
According to HPV16E6 amino acid sequence (GenBank：AKN79013.1)： HPV16 is constructed by the homologous modeling of Modeller using MOE software
Type-E6 antigen polypeptide.Other anti-multivalence human papilloma virus E6s, E7 or E6/E7 amino acid sequence, for example, the ammonia of HPV16E7
Base acid sequence is The amino acid sequence of HPV18E6 is The amino acid sequence of HPV18E6 is Etc., it is here only schematically, just no longer to all anti-multivalence human papillomas
Viral E6, E7 or E6/E7 amino acid sequence enumerates.
2.HPV16 prepared by type-E6 polypeptide antigen
(the 1-10 in mass ratio in HPV16 type-E6 antigen polypeptide:Freund adjuvant, merging high speed is added in ratio 10-1)
In homogenizer, is homogenized with 8,000rpm high speed, obtain immune HPV16 type-E6 polypeptide antigen.
3. preparing anti-HPV16 type-E6-IgY is immunized egg
Laying hen is immunized using HPV16 type-E6 polypeptide antigen prepared by the above method；Strengthened note again every two weeks
It penetrates once, planned immunization epidemic disease is three times；After third time is 15 days immune, laying hen is searched respectively and produces immune egg, and carries out coded markings.I.e.
Anti- HPV16 type-E6-IgY is made, egg is immunized.
4. preparing anti-HPV16 type-E6-IgY
The present invention illustrates by taking Aqua pure extract method as an example, and when practical application is not limited to this method, and chloroform extraction method can be used
The methods of with cold ethanol precipitation method and ammonium sulfate precipitation method.
(1) it prepares anti-HPV16 type-E6-IgY and egg crude extract is immunized
Egg is immunized in prepared anti-HPV16 type-E6-IgY first to be cleaned with circulating water, wipes of alcohol wash disinfection, then with beating
Egg machine smashes immune egg, removes egg white using yolk sieve sieving, leaves yolk, stir evenly；Again extraordinarily by the 3-8 of egg yolk liquid volume
Enter distilled water, be diluted and be uniformly mixed, with 1.0N HCI solution tune pH to 5.5-6.5.
The dilution for adjusting pH value is further stirred, is then cooled to 2-6 DEG C, it is small to stand 12
When -24 hours；Dilution is centrifuged 20 minutes in 10,000rpm；Separating obtained supernatant is taken to set progress ultrafiltration in ultrafilter dense
10-20 times of contracting, obtain concentrate；Then 1.0-3.0% sodium alginate solution is slowly added into concentrate, until final concentration of 0.1-
0.2%, stirring is muddy to occurring；Add 1.0-3.0%CaCl2Liquid, until final concentration of 0.1-0.2%, stirs evenly, 3-4
DEG C stand 8-12 hours；8,000rpm centrifugations 20 minutes, take supernatant；0.45 μm of film 0.22 μm of film filtration sterilization of series winding；Ultipor
VFTMDV50 removes virus removal except virus filter, obtains anti-HPV16 type-E6-IgY crude extract.
(2) anti-HPV16 type-E6-IgY sterling is prepared
Anti- HPV16 type-E6-IgY crude extract will be made and be dissolved in pH7.0, in 0.01M PB (phosphate buffer) liquid, then
Ion exchange column and gel chromatography column chromatography are successively crossed respectively, obtain anti-HPV16 type-E6-IgY sterling；By freeze-drying
Or middle low temperature spray drying or fluidized bed drying and other drying modes for not influencing antibody activity obtain anti-HPV16
Type-E6-IgY sterling dry powder.
Two, biotechnology cutting transformation prepares anti-multivalence human papilloma virus small molecular antibody Fab
There are many method, the present invention is said biotechnology cutting transformation macromolecular antibody with one of method and implementation steps
It is bright, but it is not limited to this method and implementation steps.As noted, those skilled in the art can be to method described below
It is made various changes or modifications with implementation steps, such equivalent forms equally fall within claims of the present invention limited range.
1. being that representative biotechnology decomposes transformation with enzymatic hydrolysis
Adjust obtained anti-HPV16 type-E6-IgY solution or other anti-multivalence human papilloma virus E6, E7 or E6/E7-
The pH value of IgY solution is to 3.0-5.0, then ratio in mass ratio is that papain or pepsin is added in 0.01%-0.1%
Equal catalytic proteins enzyme；Dissolution is sufficiently stirred, generates enzymatic reaction, obtains crude anti-HPV16 type-E6 small molecular antibody Fab
Solution or other anti-multivalence human papilloma virus E6s, E7 or E6/E7 small molecular antibody Fab solution.
2. gel chromatography
Will above crude anti-HPV16 type-E6 small molecular antibody Fab solution or other anti-multivalence human papilloma virus E6s,
E7 or E6/E7 small molecular antibody Fab solution is through any conventional method high speed centrifugation and ultrafiltration, and then gel chromatography, chromatography is received
Collect object dialysis concentration, is handled using bacterial virus filtering equipment, filter out bacterial virus, that is, the anti-HPV16 type-E6 of purifying is made
Small molecular antibody Fab solution or other anti-multivalence human papilloma virus E6s, E7 or E6/E7 small molecular antibody Fab solution.It will purifying
Anti- HPV16 type-E6 small molecular antibody Fab solution or other anti-multivalence human papilloma virus E6s, E7 or E6/E7 small molecular antibody
Fab solution, which passes through freeze-drying or middle low temperature spray drying or fluidized bed drying and other, does not influence the drying of antibody activity
Mode be made purifying anti-HPV16 type-E6 small molecular antibody Fab dry powder or other anti-multivalence human papilloma virus E6s, E7 or
E6/E7 small molecular antibody Fab dry powder.U.S. PallUltrafine Filtration can be used in bacterial virus filtering equipment
Company manufacture or the manufacture of other companies bacterial virus filtering device, first of bacterium filtering device is with 0.22 μm
It is to remove branch with 0.1 μm of film that film sterilizing filter, which removes bacteriums, second mycoplasma filtering devices such as detection of Salmonella (Salmonella),
Substance filter removes mycoplasma, and third road virus filtering device is to be removed virus filter with Ultipor VFTMDV50 and removed to prevent or cure a disease
Poison.
Three, long-actingization modifies anti-multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody Fab
The present invention is using anti-multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody prepared by protein modification
Fab realizes long-term effect, reduction toxic side effect, increase stability, reduction immunogenicity and antigenicity to extend half-life period.
The macromoleculars such as polyethylene glycol, dextran, polyaminoacid dressing agent can be used and carry out modification transformation, the present embodiment with
Illustrate for protein PEG method of modifying and implementation steps, but is not limited to this method and implementation steps.
Use PEG modify anti-multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody Fab process conditions for：
1. adjusting the pH value of borate buffer to 6.5-10.0；
2. anti-multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody Fab are added by 0.1-0.5mg/mL concentration
Borate buffer；
3. by the molar ratio of anti-multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody Fab and polyethylene glycol 5000
It is 1：10~1：30 are added polyethylene glycol 5000.
4. 15-30 DEG C of reaction temperature, the reaction time controls in 1-3h.
Four, coupled cell cell-penetrating peptide enhancing small molecular antibody Fab penetrating cell film enters the ability of infected cell
Anti- multivalence human papilloma virus E6, E7 can be coupled using the artificial synthesized CPPs of a variety of natural origin CPPs or a variety of
Or E6/E7 small molecular antibody Fab.Such as ANTP (Antennapedia), VP22 (viral protein 22) and various synthesis
Small peptide and poly arginine, transportan (sequence gWTLN-SAgYLLgKINLKALAALAKKIL) etc..Below to wear
Illustrate for film peptide-small molecular antibody Fab- microballoon conjugate preparation method and implementation steps, but be not limited to this method and
Implementation steps.
Take anti-multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody Fab pure powder 1.0-3.0g, CPPs-
EGFP1.0-3.0g, BSA-NS protein microsphere 5.0-10.0g are dissolved in 2L PBS (pH7.5,0.01mol/L), respectively with volume
Than 1：10-1:30 ratio is slowly added to 10-25mol/L ethanol solution, reaction 10-30 minutes is stirred at room temperature, by anti-multivalence people
Papillomavirus E6, E7 or E6/E7 small molecular antibody Fab, CPPs-EGFP reaction mixture is separately added into Amicon Ultra-
15 ultra-filtration centrifuge tubes, with acetate buffer (pH4.5,0.01mol/L), PBS (pH7.5,0.01mol/L) be cleaning solution from
The heart washs 3 times (4 DEG C, 3500g/min, 15min/ times), BSA-NS reaction mixture, with pH7.5PBS centrifuge washing 4 times
(10000rpm/min, 1min/ times) respectively obtains anti-multivalence human papilloma virus E6, E7 or E6/E7 to remove remaining SPDP
The SPDP derivative of small molecular antibody Fab-PDP, CPPs-EGFP-PDP, BSA-NS-PDP, 4 DEG C save backup；It is anti-what is obtained
Appropriate DTT, which is added, in multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody Fab-PDP Acetate Solution (makes reactant
The final concentration of 50mmol/L of DTT in system), room temperature is gently stirred 30 minutes, Amicon Ultra-15 ultra-filtration centrifuge tube is added, with PBS
(pH7.5,0.01mol/L) is cleaning solution centrifuge washing 3 times (4 DEG C, 3500g/min, 15min/ times), removes residue DTT, obtains
Anti- multivalence human papilloma virus E6, E7 or E6/E7 small molecular antibody Fab-PDP-SH solution, immediately with CPPs-EG-FP-PDP,
BSA-NS-PDP (suspended with PBS) solution mixing, 4 DEG C of stirring 15-20h, with PBS centrifuge washing 4 times, precipitate, finally obtain
The anti-multivalence human papilloma virus E6 of CPPs-EG-FP-BSA-NS-, E7 or E6/E7 small molecular antibody Fab protein conjugate.
Since the patient that HPV vaccine invaded cell to HPV, is checked out the HPV infection positive is invalid, IgY and IgG and
The conventional antibodies such as common monoclonal antibody belong to macromolecular, cannot be introduced into infected cell and work；Therefore, the whole world does not have still so far
There is the specific drug that can really solve HPV " sun is turned out cloudy " this global problem, this is also each positive patient by HPV infection
Have to the cruel reality faced.
HPV display form variation and innovative point of the invention in human body is as shown in the table
Long-actingization anti-human papilloma virus (anti-HPV) E6, E7, E6/E7 small molecular antibody Fab as made by the present invention or anti-is more
Valence human papilloma virus E6, E7, E6/E7 small molecular antibody Fab can pass through cell membrane and enter infected cell, attack HPV-E6/
E7 oncogene protein makes its paralysis inactivation and loses cell transformation and carcinogenesis；To inhibit lesion process and block disease
Malicious high copy expression and barrier virus are in spread between cells.Therefore, can by prepared anti-human papilloma virus (anti-HPV) E6, E7,
Various stable systems are made in E6/E7 small molecular antibody Fab or anti-multivalence human papilloma virus E6, E7, E6/E7 small molecular antibody Fab
Agent.It can be according to local human papilloma virus (HPV) popularity, by local popular all or representational several models
Anti-human papilloma virus (anti-HPV) E6, E7, E6/E7 small molecular antibody Fab or anti-multivalence human papilloma virus E6, E7, E6/E7 small molecule are anti-
Various stabilization formulations are made in body Fab combined application (such as divalent, 4 valences, 8 valences, 9 valences, 15 valences, 18 valences, 21 valences, 27 valences).The system
Agent includes but is not limited to these preparations：
Preferably, said preparation further includes one in excipient, filler, solvent, cosolvent, surfactant and capsule auxiliary material
Kind is a variety of.
Preferably, said preparation is vaginal jellies, suppository, washing lotion etc..
Preferably, said preparation is tablet, spray, pulvis, oral solutions or capsule.
Preferably, said preparation is anti-multivalence human papilloma virus (HPV) E6, E7, E6/E7 small molecular antibody Fab injection
At least one of liquid drugs injection and powder-injection.
Anti-human papilloma virus (anti-HPV) (HPV) E6, E7, E6/E7 small molecular antibody Fab or anti-multivalence human papilloma virus
E6, E7, E6/E7 small molecular antibody Fab add auxiliary material or base-material or chemical drugs, Chinese medicine, and liquid crystal microcapsules, liposome liquid crystal are made
At least one of micro-capsule, gel, suppository, cream, washing lotion, tablet, effervescent tablet, capsule, soft capsule, spray, or
It is made at least one in anti-human papilloma virus (anti-HPV) (HPV) E6, E7, E6/E7 small molecular antibody Fab water for injection injection and powder-injection
Kind.As a kind of elimination human papilloma virus (HPV) infection and the biological medicament of prevention and treatment cervical carcinoma, including use below
On the way：Can be used as preparation for by human papilloma virus (HPV) the infected's " sun is turned out cloudy ", prevent cervical cancer patient perform the operation after
Or the application of the drug or corresponding product of cervical carcinoma is recurred, prevented and treated after chemicotherapy.
Below with reference to test example and embodiment, to anti-human papilloma virus (anti-HPV) small molecular antibody Fab and combinations thereof of the invention
Object, preparation method and application are described further：
It is described in detail below by specific test example.
Test example 1：
Anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab resists corresponding human papilloma virus (HPV) E6 albumen
Body combination bioactivity.
HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV31 type, HPV33 type, HPV45 type, HPV52 are selected respectively
The E6 albumen of the types such as type, HPV53 type, HPV58 type is detected made as detection antigen with " ELISA " method (enzyme-linked immunization)
The antibody titer of the anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab obtained, as a result as shown in the table：
Note：Anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab antibody solution concentration in test sample is 1mg/
mL。
It can be seen that from the above testing result, prepared anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab is to corresponding
Human papilloma virus (HPV) E6 proteantigen have very high antibody combination potency.
Test example 2：
Anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab resists corresponding human papilloma virus (HPV) E7 albumen
Body combination bioactivity.
HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV31 type, HPV33 type, HPV45 type, HPV52 are selected respectively
The E7 albumen of the types such as type, HPV53 type, HPV58 type is detected made as detection antigen with " ELISA " method (enzyme-linked immunization)
The antibody titer of the anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab obtained, as a result as shown in the table：
Note：Anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab antibody solution concentration in test sample is 1mg/
mL。
It can be seen that from the above testing result, prepared anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab is to corresponding
Human papilloma virus (HPV) L7 proteantigen have very high antibody combination potency.
Embodiment 1：Anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab gel makes women's " sun of HPV test positive
Turn out cloudy " test
(1) subjects：Age 21~45 years old, without great organ disease person, medicine-less allergy history and without allergic constitution
Person), the women of sexual intercourse history is diagnosed as through HPV antibody test and liquid-based cytology as positive patient.Totally 260
Example.It is randomly divided into treatment group and control group each 130.
(2) test method
Treatment group：It is treated using anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab gynecological gel.It first uses daily
The cleaning of anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab washing lotion, then vagina is perfused small point of anti-human papilloma virus (anti-HPV) E6/E7
Sub- monoclonal antibody gynecological gel uses one, each course for the treatment of is used continuously totally 9, three boxes once a day every time.Continuous three treatments
Journey.Receive track of the whole process follow-up observation as required, patient enters group further consultation after 35 days, and observation HPV infection is turned out cloudy situation.
Control group：It is treated using without the common gynecological gel of anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab.
Daily vagina perfusion is free of the common gynecological gel of anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab, once a day, every time
Using one, each course for the treatment of is used continuously totally 9, three boxes.Continuous three courses for the treatment of.Receive track of the whole process follow-up observation as required, suffers from
Person enters group further consultation after 35 days, and observation HPV infection is turned out cloudy situation.
(3) follow-up study
After patient enters group as required, receives oral informed and fill in every clinical observation table, the 35th heaven-made corresponding inspection in detail
It looks into, and result is inserted in detail in observation table.It will be in detail in filling table, out if occurring adverse drug reaction over the course for the treatment of
Existing severe reaction, reports corresponding department immediately and handles in time.
(4) curative effect determinate standard is treated
HPV testing result point：(1) HPV concentration is basically unchanged, (2) HPV concentration be remarkably decreased to test preceding 30% hereinafter,
(3) HPV detection is completely negative.It is in vain：HPV is detected as (1) HPV concentration and is basically unchanged, and (2) result remains positive.
(5) result
Further consultation after test 35 days, treatment group's check, HPVHPV concentration are remarkably decreased to testing first 30% or less 79, turn out cloudy
51, invalid 0, effective percentage is 60%, negative conversion rate 39%.Control group check HPV turns out cloudy 6, negative conversion rate 4.6%；Nothing
Effect 124.
Embodiment 2：Prepare anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab liposome liquid crystal microcapsules.
Specific implementation step is as follows：
(1) lecithin and cholesterol each 50% is pressed 1：5 ratios are dissolved in ether；
(2) obtained anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab dry powder is pressed 1：100 ratios are added
4mmol/L phosphate buffer (PBS) is made into the monoclonal antibody solution of concentration 1%；
(3) PBS solution of lecithin and cholesterol diethyl ether solution and monoclonal antibody is pressed 3：1 ratio is uniformly mixed；
(4) it is ultrasonically treated 2min (every processing 0.5min, interval .0.5min)；
(5) for decompression rotary evaporation to being in gel, vortex oscillation makes gel phase inversion in a water bath, is further continued for evaporation and eliminates second
Ether；
(6) ultracentrifugation (35000r/min, 30min) is separated off the monoclonal antibody not being packed in；
(7) precipitating is washed with water secondary, and merging supercentrifuge centrifugation must precipitate；
(8) resulting precipitating 10mmol/L PBS is diluted, obtains anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody
Fab liposome liquid crystal microcapsules.
Embodiment 3：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab gel
Formula：
Technique：
(1) formula ratio lactose, hydroxyethyl cellulose (HEC), peppermint oil, essence is small with ultraviolet light sterilization 24
When, sterile sealing is spare；
(2) it is heated to by formula ratio distilled water to 90 DEG C, then is separately added into S-40 and K-30 dispersing agent and lactose, stir
It 30 minutes or more, is uniformly dissolved；60 DEG C are cooled to, tween (dropwise addition) and peppermint oil and glycerol (drop are respectively added slowly to
Add), low speed whisks 60 minutes to abundant dissolution；60 DEG C are maintained the temperature at, solution A is formed；
(3) solution A is heated to 60 DEG C, maintains the temperature at 60 DEG C, it is while stirring that the addition of pharmaceutical grade allantoin is molten
In liquid A, 60min is stirred at low speed, forms solution B；
(4) appropriate 95% alcohol of formula ratio epidermal growth factor is dissolved, 60 DEG C is heated to while stirring, directly
To being completely dissolved；60 DEG C are maintained the temperature at, epidermal growth factor alcoholic solution C is obtained；
(5) solution C is added dropwise in solution B while stirring, continues high-speed stirred 60min, temperature controls always
It 50-60 DEG C, until being completely dissolved, then cools to room temperature, obtains solution D；
(6) will resist while stirring small point of E6/E7 of a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV58)
Sub- monoclonal antibody is added in solution D, stirs at low speed 60min, until being mixed thoroughly, obtains solution E；
(7) essence (rose essence) is added in solution E while stirring, stirs at low speed 60min, until completely molten
Solution, obtains solution F；
(8) formula ratio hydroxyethyl cellulose (HEC) is added in solution F while stirring, stirs at low speed 60min, directly
To being completely dissolved, the solution of isotropic stable is formed, tests solution viscosity, after being suitble to, obtains solution G；
(9) pH value that solution G is measured with pH meter, with lemon acid for adjusting pH value to 4.5 ± 0.1；
(10) stand a night, until top foam all disappear it is molten after, then solution G is divided in the plastic barrel that cleaning and sterilizing is crossed
In, labelled factory.
Embodiment 4：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab cream
Formula：
Technique：
(1) it is heated to by formula ratio distilled water to 90 DEG C, then is separately added into Span-80 and tween K-30 and lactose, stir
It mixes 30 minutes or more, is uniformly dissolved；60 DEG C are cooled to, is respectively added slowly to potassium sorbate and glycerol (dropwise addition), low speed whisks
It is dissolved to abundant within 60 minutes；60 DEG C are maintained the temperature at, solution A is formed；
(2) appropriate 95% alcohol of formula ratio menthol is dissolved, 60 DEG C is heated to while stirring, until completely
Dissolution；Obtain solution B；
(3) solution A is heated to 60 DEG C, maintains the temperature at 60 DEG C, solution B is added dropwise to solution A while stirring, it is low
Speed whisks 30 minutes to abundant dissolution；60 DEG C are maintained the temperature at, solution C is formed；
(4) atoleine is heated to 60 DEG C, maintains the temperature at 60 DEG C, distinguish while stirring at low speed by vaseline,
Octadecanoid acid, octadecanol are added, and heating while stirring at low speed makes its melting, form solution D；
(5) solution D is heated to 60 DEG C, maintains the temperature at 60 DEG C, solution C is added in solution D while stirring, it is low
Speed stirring 60min, forms solution E；
(6) mixing speed is reduced, certain time is emulsified, stops heating after molding, is cooled to 40 DEG C or so, it is then low on one side
Speed stirring will resist a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV58) E6/E7 small molecular antibody Fab to be added on one side
It in solution E, is fully ground and stirs evenly, o/w emulsifiable paste is made, standing is cooled to room temperature, and obtains solution F；
(7) with the pH value of pH meter measurement solution F, pH value is adjusted to 4.5 ± 0.1；
(8) night is stood, solution F is divided in the plastic tube that cleaning and sterilizing is crossed, labelled loading packing box factory.
Embodiment 5：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab vaginal suppository
Formula：
Technique：
(1) it is heated to by 90% distilled water of formula ratio to 90 DEG C, gelatin swelling is added.60 DEG C are cooled to, solution A is formed；
(2) it keeps solution A temperature at 60 DEG C, solution A is added in formula ratio glycerol while stirring, mix, be cooled to 40
DEG C hereinafter, solution B；
(3) a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV58) E6/E7 small molecular antibody Fab will be resisted to add
Enter solution B, low speed whisks 30 minutes to abundant dissolution；It is cooled to room temperature, obtains solution C；
(4) with the pH value of pH meter measurement solution C, pH value is adjusted to 4.5 ± 0.1；
(5) night is stood, solution C is divided in the plastic tube that cleaning and sterilizing is crossed, labelled loading packing box factory.
Embodiment 6：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab washing lotion
Formula：
Technique：
(1) by formula ratio seaweed essence, pharmaceutical grade glycerol, peppermint oil, essence and 403,503 foaming agent ultraviolet lightings
Penetrate sterilization 24 hours, sterile sealing is spare；
(2) formula ratio distilled water is heated to 90 DEG C, stopped 15 minutes；60 DEG C are cooled to, is successiveed while stirring
Seaweed essence is added and pharmaceutical grade glycerol, low speed whisk 30 minutes, until being completely dissolved, is cooled to room temperature, obtains solution A；
(3) essence (rose essence) is added in solution A while stirring, stirs at low speed 60min, until completely molten
Solution, obtains solution B；
(4) 80 DEG C are heated to by 403, are then added dropwise to in 403, stir at low speed slowly by 503 while stirring
30min obtains solution C；
(5) it keeps solution C temperature at 80 DEG C, is added dropwise to peppermint oil in solution C slowly while stirring, low speed stirs
60min is mixed, solution D is obtained；
(6) solution D is heated to 90 DEG C of high-temperature sterilization 5min, then cooled to room temperature；
(7) solution B is slowly added in solution D while stirring, stirs at low speed 60min；As not formed homogeneous is steady
Fixed milky solution then needs to extend mixing time, obtained solution E；
(8) will resist while stirring small point of E6/E7 of a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV33)
Sub- monoclonal antibody is slowly added in solution E, stirs at low speed 60min；Such as the milky solution of not formed isotropic stable, then need to extend
Mixing time, obtained solution F；
(9) it with the pH value of pH meter measurement solution F, is adjusted using citric acid or pH4.5 disodium hydrogen phosphate-citrate buffer solution
PH value is to 4.5 ± 0.1；
(10) stand a night, until top foam all disappear it is molten after, then solution F liquid is divided in the plastics that cleaning and sterilizing is crossed
In container, labelled factory.
Embodiment 7：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab effervescent tablet
Formula：
Technique：
(1) by NaHCO360 DEG C of dry 2h, add 20%PEG ethanol softwood processed in right amount, and 14 mesh nylon mesh are pelletized 60 DEG C and done
It is dry spare；
(2) citric acid, lactose, MCC mix 80 mesh and crush, and are added in 2%HPMC alcohol liquid and stir evenly；
(3) be added resist a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV33) E6/E7 small molecular antibody Fab,
Aluminium hydroxide mixed 14 meshes, then stiffened fatty acid magnesium mixes tabletting to obtain the final product；
(4) blister, housing aluminum-plastic composite membrane plastic bag sealing, mounted box, vanning are put in storage after the assay was approved.
Embodiment 8：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab tablet
Formula：
Technique
(1) it is spare twice to cross 60 meshes for D-sorbite；
(2) carboxymethyl cellulose, which is scattered in 30% ethyl alcohol, is made 1% ethanol solution；
(3) by appropriate (2) the item softwood of (1) item, the granulation of 14 mesh screens, 60 DEG C of aeration-dryings, 18 mesh sieves；With 40
Mesh sifts out appropriate fine powder and resists a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV58) E6/E7 small molecular antibody
Fab is mixed well；
(4) magnesium stearate is admixed again, is uniformly mixed together with particle by the gross, closed 4 hours or more；
(5) tabletting machine, every 600mg；
(6) after the assay was approved, it packs, tests factory entirely.
Embodiment 9：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab hard capsule.
Formula：
Technique：
(1) will resist a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV58) E6/E7 small molecular antibody Fab and
Medicinal glucose is by weight 1:The mixing of 1 ratio, stirs；
(2) small molecular antibody Fab and medicinal glucose mixture quantitative separating are entered into capsule for medicine with capsule filler, often
Grain dress 300mg；
(3) capsule is bottled, examines factory.
Embodiment 10：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab soft capsule
Formula：
Technique：
(1) tween is heated to 50 DEG C, be added dropwise to soybean oil in tween slowly while stirring, high speed
(3000r/min) stirs 15min, is cooled to room temperature, obtains solution A；
(2) formula ratio distilled water is used into autoclaving, is heated to 121-126 DEG C, pressure rises to 102.9kPa
(1.05kg/cm2) stops 60min, is then cooled to room temperature；
(3) formula ratio is resisted into a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV58) E6/E7 small molecular antibody
Fab is first dissolved with the distilled water for being cooled to room temperature after high-temperature sterilization, stirs, antibody aqueous solution B is made；
(4) a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV58) E6/ will be resisted by slowlying while stirring
E7 small molecular antibody Fab aqueous solution B is added in solution A, and high speed (3000r/min) stirs 60min or more, obtains solution C；Through
It detects, 80% partial size is in 2~50 μ ms in solution C, and average yield is up to 91.4%；
(5) the cutting molding of encapsulating machine rolling forging die, every 300mg is added in made nano-microcapsule solution C；
(6) by molded soft capsule dryness finalization；
(7) it is packed using Aluminium-coating Packer；
(8) carton package examines qualified factory.
Embodiment 11：Produce anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab injection
A kind of novel injection agent is made in obtained anti-human papilloma virus (anti-HPV) E6/E7 small molecular antibody Fab, for " two cancers
Screening " detection is injected for positive by human papilloma virus (HPV) the infected (being generally used for CIN2-CIN3 moderate and severe infection),
Or recurrence is prevented after assisting in the treatment of and perform the operation for cervical cancer patient.Specific formula and production technology are as follows：
Formula：
Technique：
(1) formula ratio water for injection is added into 0.1% needle-use activated carbon, 60 DEG C of stirring 15min；Decarbonization filtering；Filtrate is closed
100 DEG C of 30min are heated, are cooled to room temperature spare；
(2) distinguish formula ratio biphosphate sodium-hydrate and sodium phosphate dibasic heptahydrate while stirring and
In the sterilized water for injection that the charcoal treated that sodium chloride addition accounts for formula ratio 90% is crossed, dissolve by heating；Separately take polysorbate
Ester -80 is added PEG400 and mixes；It is added in above-mentioned mixed liquor under stiring；Again plus 0.1% needle-use activated carbon, 60 DEG C of stirrings
15min, decarbonization filtering；100 DEG C of 30min of filtrate airtight heating, are cooled to room temperature spare, obtain mixed liquor A；
(3) will resist while stirring small point of E6/E7 of a variety of human papilloma virus (HPV16, HPV18, HPV31, HPV58)
Sub- monoclonal antibody addition accounts for the sterilized water for injection that the charcoal treated of formula ratio 10% is crossed, and sufficiently dissolves, obtains antibody-solutions B.
(4) antibody-solutions B is added in mixed liquor A while stirring with thread, sufficiently dissolves, obtains solution C.
(5) with the pH value of the NaOH solution adjusting solution C of 0.1mol to 6.0~7.5.
(6) use sterilized 0.45 μm 0.22 μm of miillpore filter of series connection by solution C filtration sterilization.In sterile filling and sealing machine
Encapsulating is in sterile chamber；Lamp inspection, leak detection, lettering, packaging factory.
The information that the present invention is included, under the spirit and scope without departing from following the claims, the present invention is various
Deviate accurate description, is obvious for those skilled in the art related to the present invention.The present invention does not recognize
In the range of being limited in defined program, property or composition, because preferred embodiment and other descriptions are served only for illustrating
The particular aspects of invention are provided at present.For in chemistry, biochemistry or those skilled in the relevant art, the present invention is realized
In the description mode of various modifications, should all belong to the protection domain of appended claims of the present invention interior.
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description,
Within all these improvement or transformation should all belong to the protection domain of appended claims of the present invention.
Claims (11)
1. a kind of anti-human papilloma virus (anti-HPV) small molecular antibody, which is characterized in that the anti-human papilloma virus (anti-HPV) small molecular antibody by
Following method is prepared：
A, antigen is prepared：Freund's adjuvant is added in human papillomavirus polypeptides, is placed in high speed homogenizer high speed and homogenizes, obtain
To immune human papillomavirus polypeptides antigen；
B, immune egg is prepared：Every two weeks to the human papillomavirus polypeptides antigen of laying hen injection primary immunization, 2-5 is injected
It is secondary, it takes laying hen to produce immune egg behind at least the 12nd day after last time injection, and carry out coded markings, obtains anti-human nipple
Egg is immunized in tumor virus-IgY；
C, anti-human papilloma virus (anti-HPV)-IgY crude extract is prepared；
D, anti-human papilloma virus (anti-HPV)-IgY sterling solution is prepared；
E, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is prepared：By anti-human papilloma virus (anti-HPV)-IgY sterling solution adjust pH to
Catalytic proteins enzyme is added in 3.0-5.0；Dissolution is sufficiently stirred, generates enzymatic reaction, then low-temperature and high-speed rotating centrifugal, discards
Supernatant is precipitated to obtain, ultrafiltration is carried out to supernatant, obtains concentrate；Concentrate obtained after ultrafiltration is subjected to gel chromatography again, it will
Gleanings dialysis concentration is chromatographed, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is obtained.
2. anti-human papilloma virus (anti-HPV) small molecular antibody according to claim 1, which is characterized in that step E is prepared
Anti-human papilloma virus (anti-HPV) small molecular antibody Fab also utilizes polyethylene glycol, dextran or the modification of polyaminoacid long-actingization, is grown
The anti-human papilloma virus (anti-HPV) small molecular antibody Fab of effectization.
3. anti-human papilloma virus (anti-HPV) small molecular antibody according to claim 2, which is characterized in that step E is prepared
Anti-human papilloma virus (anti-HPV) small molecular antibody Fab carries out long-actingization using polyethylene glycol and modifies, and is repaired using polyethylene glycol long-actingization
The process conditions of decorations are as follows：
Borate buffer pH value is adjusted to 6.5-10.0, is added in anti-human papilloma virus (anti-HPV) small molecular antibody Fab after adjusting pH
Borate buffer, make the concentration 0.1-0.5mg/mL of anti-human papilloma virus (anti-HPV) small molecular antibody Fab, be added polyethylene glycol into
Row reaction, and the ratio of the molar ratio of anti-human papilloma virus (anti-HPV) small molecular antibody Fab and polyethylene glycol is 1/10~1/30, reaction
Temperature is 15 DEG C~30 DEG C, and the reaction time controls in 1h~3h.
4. anti-human papilloma virus (anti-HPV) small molecular antibody according to claim 1, which is characterized in that step E is obtained anti-human
Papillomavirus small molecular antibody Fab is also by being dried to obtain powdered anti-human papilloma virus (anti-HPV) small molecular antibody Fab；
Powdered anti-human papilloma virus (anti-HPV) small molecular antibody Fab is also coupled by natural CPPs or artificial synthesized CPPs
The anti-human papilloma virus (anti-HPV) small molecular antibody Fab of the strong coupling of permeation cell film ability is obtained, wherein the method being coupled includes：
Take 1-3 parts powdered of anti-human papilloma virus (anti-HPV) small molecular antibody Fab dry powder, the CPPs-EGFP1-3 of following mass fraction
Part, 5-10 parts of BSA-NS protein microsphere, are dissolved in the PBS of respective pH7.5,0.01mol/L respectively, and obtaining concentration is 0.5-
The anti-human papilloma virus (anti-HPV) small molecular antibody Fab solution of 1.5g/L, concentration are the CPPs-EGFP solution of 0.5-1.5g/L, concentration
For the protein microsphere solution of 2.5-5g/L, then respectively with volume ratio 1:10~1:30 ratio is slowly added to the second of 10-25mol/L
Reaction 10-30 minutes is stirred at room temperature, by the reaction mixture and CPPs- of anti-human papilloma virus (anti-HPV) small molecular antibody Fab in alcoholic solution
The reaction mixture of EGFP is separately added into ultra-filtration centrifuge tube, respectively with the acetate buffer of pH4.5,0.01mol/L and
The PBS of pH7.5,0.01mol/L are cleaning solution centrifuge washing under the conditions of 4 DEG C；BSA-NS protein microsphere reaction mixture is added
Enter ultra-filtration centrifuge tube, and centrifuge washing is distinguished under the conditions of 4 DEG C to remove remaining SPDP with the PBS of pH7.5,0.01mol/L
Obtain the SPDP derivative of anti-human papilloma virus (anti-HPV) small molecular antibody Fab-PDP, CPPs-EGFP-PDP, BSA-NS-PDP, 4 DEG C
It saves backup；Appropriate DTT, which is added, in obtained anti-human papilloma virus (anti-HPV) small molecular antibody Fab-PDP Acetate Solution makes to react
The final concentration of 50mmol/L of DTT in system, is gently stirred 30 minutes at room temperature, is added in ultra-filtration centrifuge tube, with pH7.5,0.01mol/L
PBS be cleaning solution centrifuge washing under the conditions of 4 DEG C, remove residue DTT, obtain anti-human papilloma virus (anti-HPV) small molecular antibody Fab-
PDP-SH solution, and mixed immediately with CPPs-EG-FP-PDP, BSA-NS-PDP solution, 4 DEG C of stirring 15-20h are centrifuged with PBS
Washing, precipitating, finally obtains CPPs-EG-FP-BSA-NS- anti-human papilloma virus (anti-HPV) small molecular antibody Fab protein conjugate.
5. anti-human papilloma virus (anti-HPV) small molecular antibody according to claim 1, which is characterized in that use pure water in step C
Extraction method, chloroform extraction method, cold ethanol precipitation method or ammonium sulfate precipitation method prepare anti-human papilloma virus (anti-HPV)-IgY crude extract.
6. anti-human papilloma virus (anti-HPV) small molecular antibody according to claim 5, which is characterized in that use pure water in step C
Extraction method prepares anti-human papilloma virus (anti-HPV)-IgY crude extract, specifically includes：Prepared anti-human papilloma virus (anti-HPV)-IgY is immunized
Egg is cleaned with circulating water, wipes of alcohol wash disinfection, then smashes anti-human papilloma virus (anti-HPV)-IgY with eggbeater and egg is immunized, and is sieved using yolk
Egg white is removed in sieving, leaves yolk, stirs evenly；Distilled water is added by 3-8 times of egg yolk liquid volume again, be diluted and mixes
It is even, with 1.0N HCI solution tune pH to 5.5-6.5；The dilution for mixing up pH value is further stirred, then by it
It is cooled to 2-6 DEG C, stands -24 hours 12 hours；By dilution in high speed centrifugation；Separating obtained supernatant is taken to set in ultrafilter
It carries out being concentrated by ultrafiltration 10-20 times；Then the sodium alginate solution that concentration is 1.0-3.0% is added, is slowly added sodium alginate solution extremely
Final concentration of 0.1-0.2%, stirring are muddy to occurring；Add 1.0-3.0%CaCl2Liquid, until final concentration of 0.1-0.2%,
Stir evenly, 3-4 DEG C standing 8-12 hours；High speed centrifugation simultaneously takes supernatant, obtains anti-human papilloma virus (anti-HPV)-IgY crude extract；
Step D is specifically included：Obtained anti-human papilloma virus (anti-HPV)-IgY crude extract is dissolved in the M PB phosphorus of pH7.0,0.01mol/L
In phthalate buffer, then ion exchange column and gel chromatography column chromatography are successively crossed respectively, it is pure to obtain anti-human papilloma virus (anti-HPV)-IgY
Product solution；
Human papillomavirus polypeptides in step A are HPV-E6, HPV-E7 or HPV-E6-E7；Human papillomavirus polypeptides with not
The ratio of the mass ratio of family name's adjuvant is (1-10):(10-1)；
The ratio that mass ratio is added in catalytic protein enzyme in step E is 0.01%-0.1%；Then, low-temperature and high-speed is centrifuged.
7. a kind of preparation method of anti-human papilloma virus (anti-HPV) small molecular antibody, which is characterized in that including：
A, antigen is prepared：Freund's adjuvant is added in human papillomavirus polypeptides, is placed in high speed homogenizer high speed and homogenizes, obtain
To immune human papillomavirus polypeptides antigen；
B, immune egg is prepared：Every two weeks to the human papillomavirus polypeptides antigen of laying hen injection primary immunization, 2-5 is injected
It is secondary, it takes laying hen to produce immune egg behind at least the 12nd day after last time injection, and carry out coded markings, obtains anti-human nipple
Egg is immunized in tumor virus-IgY；
C, anti-human papilloma virus (anti-HPV)-IgY crude extract is prepared；
D, anti-human papilloma virus (anti-HPV)-IgY sterling solution is prepared；
E, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is prepared：By anti-human papilloma virus (anti-HPV)-IgY sterling solution adjust pH to
Catalytic proteins enzyme is added in 3.0-5.0；Dissolution is sufficiently stirred, generates enzymatic reaction, then low-temperature and high-speed rotating centrifugal, discards
Supernatant is precipitated to obtain, ultrafiltration is carried out to supernatant, obtains concentrate；Concentrate obtained after ultrafiltration is subjected to gel chromatography again, it will
Gleanings dialysis concentration is chromatographed, anti-human papilloma virus (anti-HPV) small molecular antibody Fab is obtained.
8. anti-human papilloma virus (anti-HPV) small molecular antibody described in any one of claim 1-6 claim is as preparation for preventing and treating
Human papilloma virus (HPV) infection, and/or the drug for preventing and treating cervical carcinoma, sterile products, health care product or Medical treatment device
Application in tool.
9. a kind of composition, which is characterized in that including anti-human papilloma virus (anti-HPV) described in any one of claim 1-6 claim
Small molecular antibody and at least one other pharmaceutically acceptable component.
10. composition according to claim 9, which is characterized in that the anti-human papilloma virus (anti-HPV) small molecular antibody addition
Auxiliary material or base-material or chemical drugs, Chinese medicine, are made liquid crystal microcapsules, gel, suppository, cream, washing lotion, tablet, effervescent tablet, capsule
At least one of agent, soft capsule, spray, or anti-human papilloma virus (anti-HPV) (HPV) E6, E7, E6/E7 small molecular antibody is made
At least one of Fab water for injection injection and powder-injection.
11. composition described in claim 9 or 10 is as preparation for preventing and treating human papilloma virus (HPV) infection, and/or using
Application in drug, sterile products, health care product or the medical instrument for preventing and treating cervical carcinoma.
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| CN111690055A (en) * | 2019-03-11 | 2020-09-22 | 深圳市雅臣智能生物工程有限公司 | anti-African swine fever virus and CD polyclonal small molecule antibody, preparation method and application |
| CN113201068A (en) * | 2020-02-03 | 2021-08-03 | 深圳市雅臣智能生物工程有限公司 | Gene recombination anti 2019-nCoV and other coronavirus IgY and small molecule antibody and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111690055A (en) * | 2019-03-11 | 2020-09-22 | 深圳市雅臣智能生物工程有限公司 | anti-African swine fever virus and CD polyclonal small molecule antibody, preparation method and application |
| CN110054686A (en) * | 2019-04-17 | 2019-07-26 | 深圳市雅臣智能生物工程有限公司 | HPV-L1, E6/E7-IgY and its small molecular antibody against a broad spectrum and application |
| CN113201068A (en) * | 2020-02-03 | 2021-08-03 | 深圳市雅臣智能生物工程有限公司 | Gene recombination anti 2019-nCoV and other coronavirus IgY and small molecule antibody and application thereof |
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