CN117797255A

CN117797255A - Composition, nano particles, external preparation and application for treating HPV infection

Info

Publication number: CN117797255A
Application number: CN202311763484.0A
Authority: CN
Inventors: 包晟
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-12-20
Filing date: 2023-12-20
Publication date: 2024-04-02

Abstract

The invention provides a composition, nano particles, external preparation and application for treating HPV infection; the anti-HPV infection composition comprises lactoglobulin coupled to a combination antibody and anti-cervicitis pathogen IgY; the nanometer particles for resisting HPV infection comprise the composition for resisting HPV infection, which is wrapped by liposome serving as a carrier. The external preparation comprises an HPV infection resistant composition and/or HPV infection resistant nano particles and auxiliary materials. The composition for resisting HPV infection can obviously improve the curative effect of immunotherapy based on the three-in-one antibody combination, and the three antibodies cooperate to greatly improve the actual curative effect of the combined preparation and effectively improve the curative effect of HPV infection.

Description

Composition, nano particles, external preparation and application for treating HPV infection

Technical Field

The invention belongs to the field of biotechnology and pharmaceutical preparations; in particular to a composition, nano particles, external preparation and application for treating HPV infection.

Background

Human Papillomavirus (HPV), belonging to the genus papillomavirus of the family papillomavirus, is a circular double-stranded DNA virus. HPV mainly infects the skin and the stratified squamous epithelium of mucous membrane of specific parts of human body, sexual contact is the main infection path, and other paths such as contact transmission or mother and infant direct transmission are adopted. Women are reported to be repeatedly infected with HPV, and also to be simultaneously infected with multiple different types of HPV.

A research report of the analysis of the detection result of 3381 female genital tract human papillomavirus in China shows that the HPV positive rate in 3381 female is as high as 18.75 percent. At the peak of genital tract infection of fourteenth China women in 2021, students point out that the average infection rate of HPV of normal women in global cytology is estimated to be about 11.7%, and the overall infection rate of high-risk HPV of China women is about 15%. The vast array of data shows that HPV infection is most common among young people between 18 and 28 years of age, and that up to 80% of women have HPV infection before age 50, a number that is a surprise.

Cervical cancer is a disease that severely jeopardizes women's health and life, and is by far the most common HPV-related disease. Cervical cancer occurs in the cervix (the entrance from the vagina into the uterus) of women, the fourth most common cancer in women. Modern medicine has demonstrated that cervical cancer is derived from an HPV infection, which is the only cancer of clear etiology among all cancers in humans at present.

In addition, cervical inflammation is also common lower genital tract inflammation of gynaecology, accounting for 40-50% of the total number of the outpatient departments of gynaecology. The cervical canal epithelium is a single-layer columnar epithelium, has more mucous membrane folds, is easy to infect and is easy to persist. The pathogen of chronic cervicitis can ascend to cause endometritis, and can spread through the ligament lymphatic vessel beside uterus to cause chronic pelvic inflammatory disease, and leucorrhea abnormality occurs; when inflammation involves the trigonometric region of the urinary bladder, it may cause diseases of the urinary system, causing painful urination or irritative symptoms such as difficult urination. If not diagnosed and correctly treated in time, serious diseases such as infertility, ectopic pregnancy, abortion, premature birth, fetal death, intrauterine complications and the like can be caused, cervical HPV infection and related lesions can also be caused, and the sexual life quality and family harmony of patients can be influenced.

To date, the medical community has not developed effective antiviral drugs for the treatment of HPV infections, and traditional antibiotics and bactericidal drugs, disinfectants, have no effect at all on HPV. The HPV vaccines currently proposed are also generally only prophylactic, and the prophylactic bivalent, tetravalent and nine vaccines that have been popularized internationally are, respectively, preventing 2, 4 and 9 types of Human Papillomavirus (HPV) infection, but have no therapeutic effect. Injection of such vaccines is ineffective for people who have been infected with HPV. More plagued are Human Papillomaviruses (HPV) of more than 100 types, different types causing different clinical manifestations, and different people's papillomaviruses (HPV) circulating in different countries and regions. Even vaccinators are still likely to infect other species of human papillomavirus not contained in the vaccinated vaccine, and simply increasing the vaccine target antigen species for the purpose of completely preventing different Human Papillomavirus (HPV) infections throughout are not at all available.

At present, besides oral administration and injection of various chemical drugs, traditional Chinese medicines and biological agents, various modes are generally adopted to perfuse or fill the drugs and biological agents into the vagina so as to achieve the purposes of preventing and treating HPV infection and treating cervical cancer and cervicitis. Through deep research, the cervical is the main part for aggregation and reproduction of Human Papilloma Virus (HPV) and cervicitis pathogenic bacteria, and is also the primary part of cervical cytopathy and cervical cancer. To turn positive patients infected with HPV to negative, the human papillomavirus hidden in the cervix and the infected cells in the cervix must be cleared. However, due to the physiological characteristics of females, the cervical orifice is always in a closed state, and the drug delivery mode affecting vaginal infusion or filling cannot deliver drugs and biological agents to the cervical site, which results in the fact that the drugs cannot reach the focus to act, thus resulting in poor actual prevention and treatment effects and poor actual curative effects of various drugs turning positive to negative. Although the medicine can be put into the cervix by adopting the uterus dilating operation, the operation needs to be carried out by a special medical institution, which is time-consuming and labor-consuming, increases the uterus dilating operation cost and operation pain, and ensures the privacy, thus reducing the compliance of patients and being difficult to popularize and apply. In addition, chemical drugs and traditional Chinese medicines which are widely used at present have poor therapeutic effects on the diseases, and other physical therapeutic principles such as laser and freezing can cause postoperative bleeding, cervical stenosis, adhesion, infertility, infection and the like and induce various complications.

Therefore, the development of a more effective external preparation for treating HPV infection, which can be directly applied to the cervical region, and which achieves precise administration and efficient preventive and therapeutic effects, is desired in the art.

Disclosure of Invention

The invention aims to provide a composition, nanoparticles, an external preparation and application for treating HPV infection.

The invention is realized by the following technical scheme:

the present invention relates to a composition for the treatment of HPV infection comprising lactoglobulin conjugated to a combination antibody and IgY against cervicitis pathogen; the mass ratio of the lactoglobulin of the coupling combined antibody to the anti-cervicitis pathogen IgY is (1-10): (1-10); the anti-HPV infection composition can effectively prevent and treat HPV infection, cervicitis and other diseases;

the invention also relates to nano particles for resisting HPV infection, which are prepared by wrapping the HPV infection resisting composition by taking liposome as a carrier; the anti-HPV infection composition comprises lactoglobulin conjugated with a combination antibody and anti-cervicitis pathogen IgY.

The beta-lactoglobulin in the lactoglobulin of the conjugated combined antibody in the anti-HPV infection composition is combined with positively charged HPV based on negatively charged anhydrated beta-lactoglobulin to form a protein complex, so that the HPV disease load is reduced;

The anhydrated beta-lactoglobulin coupling combined antibody can effectively increase the capability of inhibiting E6/E7 oncogene by enzyme anhydration beta-lactoglobulin, endows the anhydration beta-lactoglobulin with new pharmacological action, and utilizes the respective advantages of the anhydration beta-lactoglobulin and the combined antibody to generate superposition effect and improve the treatment effect.

The anti-HPV infection nano-particles are obtained by wrapping advanced carriers such as lipid liquid crystal nano-particles (lipid liquid crystalline nanoparticles, LLCN), lipid nano-particles (Lipid Nanoparticle, LNP), solid lipid nano-particles (Solid lipid nanoparticle, SLN) and the like, can obviously improve the penetration capacity of a combined antibody into a cervical crypt and a mucous membrane gap, and simultaneously endow tissue targeting characteristics, so that the combined antibody is accurately targeted to be gathered on the cervical mucous membrane layer and slowly releases medicines; thereby fully playing the efficacy of the nanometer particles prepared by the composition of the lactoglobulin of the carrier-coated conjugate combined antibody and the anti-cervicitis pathogen IgY, and further improving the treatment effect.

The invention also relates to an external preparation for resisting HPV infection, which comprises an HPV infection resisting composition and/or HPV infection resisting nano particles and auxiliary materials; the anti-HPV infection composition comprises lactoglobulin coupled to a combination antibody and anti-cervicitis pathogen IgY; the nanometer particles for resisting HPV infection are prepared by taking liposome as a carrier to encapsulate the composition for resisting HPV infection.

Preferably, the combined antibody in the lactoglobulin of the coupled combined antibody comprises a mixture of broad-spectrum anti-HPV-IgY and small-molecule antibody Fab thereof, composite anti-E6/E7-IgY and small-molecule antibody Fab thereof, anti-PD-1/L1-IgY and small-molecule antibody Fab thereof.

Preferably, the mass ratio of the broad-spectrum anti-HPV-IgY, the composite anti-E6/E7-IgY and the anti-PD-1/L1-IgY is (1-10): (1-10): (1-10); the mass ratio of the broad spectrum anti-HPV-IgY, the broad spectrum anti-HPV-IgY small molecule antibody Fab, the composite anti-E6/E7-IgY small molecule antibody Fab, the anti-PD-1/L1-IgY and the anti-PD-1/L1-IgY small molecule antibody Fab is (1-10): (1-10): (1-10): (1-10): (1-10): (1-10).

Wherein, the broad spectrum anti-HPV-IgY and the small molecule antibody Fab thereof can bind to various subtype HPVs and the binding force to the HPVs is far beyond that of mammal antibody IgG; anti-HPV-E6/E7-IgY is most effective in inhibiting the oncogene protein E6/E7, and only chicken antibodies (IgY) are able to specifically bind and inhibit HPV-E7 oncoproteins in positive cells and within diseased squamous epithelium; the anti-PD-1/L1-IgY is an innovative restart human immune mechanism based on the international Nobel medical prize technology and activates T cells to produce a growth effect. The composition for resisting HPV infection can obviously improve the curative effect of immunotherapy based on the three-in-one antibody combination, and the synergistic effect of the three antibodies can greatly improve the actual curative effect of the combined preparation compared with the curative effect generated by injecting PD-1 inhibitors (actizumab, divalizumab, avermectin and the like) and conveying the antibodies to cervical canal by blood circulation.

Preferably, the preparation method of the lactoglobulin of the conjugated combined antibody comprises the following steps:

(a) Preparing the required antigen according to the selected antibody types;

(b) Preparing corresponding immune eggs by using the prepared antigens respectively;

(c) Preparing the antibody-IgY of the required kind and the small molecule antibody Fab thereof respectively, and obtaining the required combined antibody;

(d) And (3) coupling the combined antibody by using the anhydrated lactoglobulin to obtain the antibody.

Preferably, the pathogens against cervicitis pathogen IgY comprise a major pathogen type causing vaginitis, in particular comprising a mixture of staphylococcus aureus, neisseria gonorrhoeae, escherichia coli, klebsiella pneumoniae, streptococcus agalactiae.

Preferably, the preparation method of the anti-cervicitis pathogen IgY comprises the following steps:

(a) Culturing selected pathogen and preparing corresponding composite antigen;

(b) Preparing an immune egg by using the composite antigen;

(c) The immune egg is used for preparing the anti-cervicitis pathogen IgY.

Preferably, the liposome comprises at least one of a lipid liquid crystal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

Preferably, the external preparation for resisting HPV infection comprises: gel, dressing, spray, gynecological lotion, male lotion, hand cleanser, powder, tablet, toothpaste, oral paste, mouthwash, buccal tablet, oral liquid, oral preparation or capsule.

The invention also relates to the application of the external preparation for resisting HPV infection, the application of the external preparation for preparing medicines for preventing and treating HPV infection, cervical infectious diseases or cervical cancer, the application of the external preparation for preparing non-diagnostic detection reagents or detection products or the application of the external preparation for preparing medical appliances for resisting HPV infection.

The invention has the following advantages:

(1) The interaction among the components of the invention is as follows: the related broad-spectrum anti-HPV-IgY and small molecule antibody Fab thereof can bind to various subtype HPVs and the binding force to the HPVs is far beyond that of mammal antibody IgG; anti-HPV-E6/E7-IgY is most effective in inhibiting the oncogene protein E6/E7, and only chicken antibodies (IgY) are able to specifically bind and inhibit HPV-E7 oncoproteins in positive cells and within diseased squamous epithelium; the anti-PD-1/L1-IgY is an innovative restart human immune mechanism based on the international Nobel medical prize technology and activates T cells to produce a growth effect.

(2) The invention can obviously improve the curative effect of immunotherapy based on the three-in-one antibody combination, and the synergistic effect of the three antibodies can greatly improve the actual curative effect of the combined preparation. Because of the small distribution of cervical capillaries, the combined antibody is directly applied to cervical lesions, and compared with the treatment effect generated by injecting PD-1 inhibitors (actizumab, divalizumab, avermectin and the like) and conveying the antibody to cervical canal by blood circulation, the treatment effect is greatly improved.

(3) The invention is based on the combination of negatively charged anhydrated beta-lactoglobulin and positively charged HPV to form a protein complex, so that the HPV disease load is reduced; the anhydrated beta-lactoglobulin coupling combination antibody can effectively increase the capability of enzyme anhydration beta-lactoglobulin to inhibit E6/E7 oncogene, endow the anhydration beta-lactoglobulin with new pharmacological action, and utilize the respective advantages of the anhydration beta-lactoglobulin and the combination antibody to generate superposition effect, thereby improving treatment effect.

(4) The anti-HPV infection nanoparticle is prepared by wrapping advanced carriers such as lipid liquid crystal nanoparticles (lipid liquid crystalline nanoparticles, LLCN), lipid nanoparticles (Lipid Nanoparticle, LNP), solid lipid nanoparticles (Solid lipid nanoparticle, SLN) and the like, can remarkably improve the penetration capacity of a combined antibody into a cervical crypt and a mucous membrane gap, and simultaneously endows a tissue targeting characteristic, enables the combined antibody to be accurately targeted and aggregated on the cervical mucous membrane layer, and slowly releases medicines; thereby fully playing the efficacy of the nanometer particles of the lactoglobulin coupled with the combined antibody and the anti-cervicitis pathogen IgY composition and improving the treatment effect.

Detailed Description

The present invention will be described in detail with reference to specific examples. It should be noted that the following examples are only further illustrative of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

This example was used to prepare high purity broad spectrum anti-HPV-IgY and its small molecule Fab dry powder.

1. Preparation of broad-spectrum HPV antigens

The broad-spectrum HPV antigen described in this example is a common epitope polypeptide protein shared by all types of HPV in gene recombination.

Although more than 200 types of HPVs exist, according to the deep research, the internal structures of all HPV viruses are analyzed by means of modern instruments and equipment and high-end algorithm technology, and all types of HPVs are found to have a group of common epitope gene sequences, and the common and conserved epitope gene sequence proteins can induce animals to generate broad-spectrum antibodies covering all serotypes of anti-HPVs. The invention screens out L1 protein common antigen epitope of high-risk types 16, 58, 18, 52, 33 and the like by using a biological information technology, adopts Blast software in NCBI to compare the amino acid sequences of the five high-risk HPV L1 proteins to obtain a common polypeptide fragment sequence, and synthesizes the common antigen epitope gene sequence protein polypeptide by using a polypeptide synthesis technology.

Compared with the prior method for screening out common epitope of partial high-risk HPV such as 16, 58, 18, 52 and 33 type L1 proteins, the method for preparing the coupled KLH common antigen synthetic epitope polypeptide antigen component has great technical progress. The common antigen epitope gene sequence protein polypeptide studied by the method of the invention is used as an antigen component to prepare anti-HPV-E6/E7-IgY and small molecule Fab thereof, and the obtained antibody can be used for not only aiming at the limited five HPV subtype L1 protein antigen epitopes of 16, 58, 18, 52, 33 types and the like, but also aiming at common antigen epitopes of all serotypes HPV-L1 and L2. The method has very important practical significance in finding and aiming at the common antigen epitope of all types of HPV and preparing the corresponding antibody, and not only realizes the broad-spectrum resistance in the true sense, so that the finally prepared HPV infection prevention product can prevent HPV infection of various subtypes, but also greatly simplifies the antigen preparation process flow and saves a lot of cost for practical application.

The specific preparation method is as follows:

(a) Amino acid sequence of HPV 16L 1 protein and single parameter setting

The amino acid sequence of the HPV 16L 1 protein is shown in SEQ ID No. 1: mslwlpseat vylppvpvsk vvstdeyvar tniyyhagts rllavghpyf pikkpnnnki lvpkvsglqy rvfrihlpdp nkfgfpdtsf ynpdtqrlvw acvgvevgrg qplgvgisgh pllnklddte nasayaanag vdnrecismd ykqtqlclig ckppigehwg kgspctnvav npgdcpplel intviqdgdm vdtgfgamdf ttlqanksev pldictsick ypdyikmvse pygdslffyl rreqmfvrhl fnragavgen vpddlyikgs gstanlassn yfptpsgsmv tsdaqifnkp ywlqraqghn ngicwgnqlf vtvvdttrst nmslcaaist settykntnf keylrhgeey dlqfifqlck itltadvmty ihsmnstile dwnfglqppp ggtledtyrf vtsqaiacqk htppapkedp lkkytfwevn lkekfsadld qfplgrkfll qaglkakpkf tlgkrkatpt tsststtakr kkr.

Four main parameters were selected using the ExPASy-ProtScale and Antibody epitope prediction epitope prediction website: hydrophilicity Prediction (Hopp & Woods Hydrophilicity Prediction), flexibility Prediction (Karplus & Schulz Flexibility Prediction), beta Turn Prediction (Chou & Fasman Beta-Turn Prediction), epitope index Prediction (Kolaskar & Tongaonkar Antigenicity Prediction). The amino acid sequence of HPV 16L 1 coat protein was entered into the working region and analyzed for its individual parameters.

(b) High-risk HPV common antigen epitope comparison and spatial structure prediction to obtain common polypeptide fragment sequence

By using UniProtKB protein library and taking HPV 16L 1 as a standard, the highest homology of 58, 18, 52 and 33 type L1 proteins is 78%, 64%, 77% and 79% respectively. And (3) comparing the amino acid sequences of the five high-risk HPV L1 proteins by Blast software in NCBI to obtain a common polypeptide fragment sequence. This epitope polypeptide is then synthesized by polypeptide synthesis techniques.

(c) In order to improve the immunogenicity of the common epitope gene sequence protein polypeptide, the common epitope polypeptide is coupled with KLH (or carrier proteins with high immunogenicity such as BSA, OVA and the like) to prepare the common antigen synthetic epitope polypeptide antigen component coupled with the KLH (or carrier proteins with high immunogenicity such as BSA, OVA and the like).

The common epitope polypeptide is connected to KLH (or BSA, OVA and other carrier proteins with high immunogenicity) through chemical linking or gene recombination technology. Among them, various methods are applicable for chemical linking, such as carbodiimide method, glutaraldehyde method, periodic acid oxidation method, succinic anhydride method, carboxymethyl hydroxylamine method, diazotized p-aminobenzoic acid method, sodium monochloroacetate method, active ester method, anhydride method, and mixed anhydride method, and the like, and also coupling can be performed by using heterobifunctional reagents such as SPDP as a crosslinking agent.

The prepared common antigen synthesis epitope polypeptide antigen component of the coupled (KLH) is prepared according to the following steps (1-10): mixing the mixture of (1-10) with Freund's adjuvant, and stirring thoroughly to obtain broad-spectrum HPV antigen.

It should be noted that this example only uses the common antigen synthetic epitope polypeptide as a broad-spectrum HPV antigen component, but is not limited to this antigen component and the implementation steps. In practical application, common epitope gene sequences of other representative HPV subtypes (such as one, 2-6 or more than six types of HPV16, 18, 31, 33, 45, 58 and the like) can be selected, L1 protein genes of the HPV subtypes are cloned, a codon optimization strategy is adopted for transformation, an optimized gene fragment is inserted into a pFastBac Dual vector to obtain a recombinant vector, E.coli DH10Bac competent cells are transformed to obtain recombinant Bacmid, insect Sf9 cells are transfected, and Western blotting and SDS-PAGE are used for detecting the expression of the recombinant protein.

2. Preparation of immune egg

Egg laying birds (chickens, ducks, geese, turkeys, ostriches and the like) with high immune response capability are selected, an immune activation method is applied, and the prepared broad-spectrum HPV antigen is adopted for immune injection. Injecting the broad-spectrum HPV antigen prepared by the steps into the laying hens every two weeks for 3 times, and respectively detecting the immunized eggs produced by the laying hens after 15-20 days of the first immunization to obtain the anti-broad-spectrum HPV-IgY immunized eggs.

3. Preparation of broad-spectrum anti-HPV-IgY and anti-HPV small molecule antibody Fab

The method for preparing the HPV-IgY adopts a pure water extraction method, a chloroform extraction method, a cold ethanol precipitation method or an ammonium sulfate precipitation method to prepare the broad-spectrum HPV-IgY crude extract.

In this example, the pure water extraction method is used to prepare a broad-spectrum HPV-IgY resistant crude extract, and the specific procedures include: washing the prepared broad-spectrum anti-HPV-IgY immune egg with flowing water, scrubbing and sterilizing with alcohol, breaking the broad-spectrum anti-HPV-IgY immune egg with an eggbeater, filtering off egg white with a yolk sieve, leaving yolk, and stirring uniformly; adding distilled water 3-8 times of the yolk liquid volume, diluting, mixing, and adjusting pH to 5.5-6.5 with 1.0N HCl solution; fully and uniformly stirring the diluted solution with the adjusted pH value, cooling to 2-6 ℃, and standing for 12-24 hours; centrifuging the diluted solution at a high speed; placing the separated supernatant into an ultrafilter for ultrafiltration concentration for 10-20 times; then adding sodium alginate solution with the concentration of 1.0-3.0%, slowly adding sodium alginate solution to the final concentration of 0.1-0.2%, and stirring until turbidity appears; adding 1.0-3.0% CaCl ₂ The solution is stirred uniformly until the final concentration is 0.1-0.2%, and is kept stand for 8-12 hours at 3-4 ℃; centrifuging at high speed and collecting supernatant to obtain broad-spectrum HPV-IgY resisting crude extract. Then, the crude extract of the anti-broad-spectrum HPV-IgY is subjected to ion exchange column and affinity chromatography column chromatography, and the chromatography collection is dialyzed and concentrated to obtain high-purity broad-spectrum anti-HPV-IgY solution.

Continuously regulating the pH value of the broad-spectrum HPV/L1-IgY resistant crude extract solution to 3.0-5.0, and adding catalytic protease; fully stirring and dissolving to generate enzymatic reaction, then rotating and centrifuging at a low temperature and high speed, discarding precipitate to obtain supernatant, and ultrafiltering the supernatant to obtain concentrated solution; and then, sequentially passing the concentrated solution obtained after ultrafiltration through an ion exchange column and an affinity chromatography column, and dialyzing and concentrating a chromatography collection to obtain the high-purity broad-spectrum anti-HPV small-molecule antibody Fab solution.

The present example uses the bacterial virus filtration device of the virus removal filtration system manufactured by us Pall Ultrafine Filtration Company to thoroughly filter out various bacterial viruses, ensuring that the IgY and small molecule antibody Fab prepared are never any viruses and bacteria. Wherein the first bacteria filtering device is to remove bacteria such as Salmonella by using a 0.22 μm membrane sterilization filter; the second mycoplasma filtering device is to remove mycoplasma by a 0.1 μm membrane mycoplasma removing filter; the third virus filtration device was a Ultipor VFTM DV50 virus removal filter to remove a variety of viruses including influenza virus and enterovirus.

The prepared high-purity broad-spectrum anti-HPV-IgY solution and the broad-spectrum anti-HPV small-molecule antibody Fab solution are respectively freeze-dried or medium-low temperature spray-dried or fluidized bed-dried, and then added into an ultrafine grinder for grinding and crushing, so as to respectively prepare high-purity nano broad-spectrum anti-HPV-IgY dry powder and high-purity nano broad-spectrum anti-HPV small-molecule antibody Fab dry powder.

Example 2

This example is used to prepare high purity broad spectrum anti-E6/E7-IgY and its small molecule Fab nanometer dry powder.

1. Preparation of HPV-E6/E7 antigen components

The present examples may employ known methods for preparing HPV-E6/E7 antigen components, for example, recombinant bacterial vectors containing modified HPV 16E7 or recombinant viruses expressing HPV-16/18 type E6/E7, various synthetic HPV 16E7 polypeptides, the binding of the E7 proteins of HPV16 and HPV18 to specific enzymes and the use of DNA plasmids encoding HPV 16/18 type E6 and E7 as antigen components; the preparation process comprises the following conventional steps.

Since the E6/E7 antigen gene structures of HPVs of different types are also different, 15 high-risk HPVs are randomly divided into 5 groups, such as:

group A: 16. 18, 58 (Chinese female infection rate highest)

Group B: 33. 52, 31

Group C: 35. 39, 45

Group D: 51. 56, 59

Group E: 68. 73, 82

And then, the 15 high-risk HPV virus subtype E6/E7 fusion genes are expressed in eukaryotic cells or prokaryotic cells in groups and purified to prepare the E6/E7 fusion gene eukaryotic or prokaryotic expression protein antigen component.

This grouping approach is one approach; however, there are three HPV-E6 and three HPV-E7 gene proteins for each of the three types, six HPV-E6/E7 gene proteins for each of the three types, and 30 HPV-E6/E7 gene proteins for each of the 5 types. This grouping method tends to bring a huge amount of work to antigen preparation.

The method of the invention analyzes 15 high-risk type HPVs internal structure based on epidemiological investigation, screens out four high-risk type HPVs 16, 18, 31, 45 with maximum harm, highest incidence, most commonality of antigen epitope in internal structure and representativeness from 15 high-risk type HPVs, and respectively prepares 8 polypeptide protein antigen components of HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV31-E6, HPV31-E7, HPV45-E6, HPV45-E7 and the like. The method of the invention simplifies the manufacturing process flow, greatly reduces the cost of practical application and has obvious technical progress. The 8 most representative polypeptide protein antigen components and the composite antigen components thereof are adopted to prepare the HPV-E6/E7-IgY and the small molecule Fab thereof, which can inhibit the generation of HPV-E6/E7 oncogenic proteins of more than 90 percent.

This example illustrates the preparation of representative 8 polypeptide protein antigen components and their complex antigen components, but is not limited to these antigen components and the steps of the method.

This example was used to prepare representative 8 HPV-E6/E7 polypeptide protein antigen components.

(1) HPV 16-E6 polypeptide protein antigen component

According to the HPV16E6 amino acid sequence shown in SEQ ID No.2 (GenBank: AKN 79013.1): MFPDPQERPI KLPDLCTELP TTIHDIILEC VYCKQQLLRR EVYDFAFRDL CIVYRDGNPY AVCDKCLKFY SKISEYRYYC YSLYGTTLEQ QYNKPLCDLL IRCINCQKPL CPEEKQRHLD KKQRFHNIRG RWTGRCMSCC ESSRTRRETQL the number of the individual pieces of the plastic,

HPV 16-E6 polypeptide proteins were constructed by modeling through Modeller homology using MOE software.

(2) HPV 16-E7 polypeptide protein antigen component

HPV 16-E7 polypeptide proteins were constructed by Modeller homology modeling using MOE software based on the amino acid sequence of HPV16E7 shown in SEQ ID No.3 (MHGDTPTLHE YMLDLQPETT DLYCYEQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKPP).

(3) HPV18 type-E6 polypeptide protein antigen component

HPV18 type-E6 polypeptide proteins were constructed by Modeller homology modeling using MOE software based on the amino acid sequence of HPV 18E 6 shown in SEQ ID No.4 (MHGPKATLQD IVLHLEPQNE IPVDLLCHEQ LSDSEEENDE IDGVNHQHLP ARRAEPQRHT MLCMCCKCEA RIKLVVESSA DDLRAFQQLF LNTLSFVCPW CASQQ).

(4) HPV18 type-E7 polypeptide protein antigen component

HPV18 type-E6 polypeptide proteins were constructed by Modeller homology modeling using MOE software based on the amino acid sequence of HPV 18E 7 shown in SEQ ID No.5 (MHGPKATLQD IVLHLEPQNE PVDLLCHEQ LSDSEEENDE IDGVNHQHLP ARRAEPQRHT MLCMCCKCEA RIELVVESSA DDLRAFQQLF LNTLSFVCPW CASQQ).

(5) HPV31 type-E6 polypeptide protein antigen component

HPV type 31-E6 polypeptide proteins were constructed by Modeller homology modeling using MOE software based on the amino acid sequence of HPV31E 6 shown in SEQ ID No.6 (MFKNPAERPRKLHELSSALEIPYDELRLNCVYCKGQLTETEVLDFAFTDLTIVYRDDTPHGVCTKCLRFYSKV SEFRWYRYSVYGTTLEKLTNKGICDLLIRCITCQRPLCPEEKQRHLDKKKRFHNIGGRWTGRCIACWRRPRTETQ V).

(6) HPV31 type-E7 polypeptide protein antigen component

HPV 31-E6 antigen polypeptide proteins were constructed by Modeller homology modeling using MOE software based on the amino acid sequence of HPV31E7 shown in SEQ ID No.7 (MRGETPTLQDYVLDLQPEATDLHCYEQLPDSSDEEDVIDSPAGQAEPDTSNYNIVTFCCQCKSTLRLCVQSTQ VDIRILQELLMGSFGIVCPNCSTRL).

(7) HPV 45-E6 polypeptide protein antigen component

HPV 45-E6 polypeptide proteins were constructed by Modeller homology modeling using MOE software based on the amino acid sequence of HPV45E6 shown in SEQ ID No.8 (MARFDDPKQRPYKLPDLCTELNTSLQDVSIACVYCKATLERTEVYQFAFKDLCIVYRDCIAYAACHKCIDFYS RIRELRYYSNSVYGETLEKITNTELYNLLIRCLRCQKPLNPAEKRRHLKDKRRFHSIAGQYRGQCNTCCDQARQE RLRRRRETQV). (8) HPV 45-E7 polypeptide protein antigen component

HPV 45-E7 polypeptide proteins were constructed by Modeller homology modeling using MOE software based on the amino acid sequence of HPV45E7 shown in SEQ ID No.9 (MHGPRETLQEIVLHLEPQNELDPVDLLCYEQLSESEEENDEADGVSHAQLPARRAEPQRHKILCVCCKCDGRI ELTVESSAEDLRTLQQLFLSTLSFVCPWCATNQ).

As will be appreciated by those skilled in the art, the polypeptide proteins constructed using the above methods have too small molecular weights and poor immunogenicity, and in order to enhance their immunogenicity, they must be coupled to a macromolecular protein having high immunogenicity, and specific coupling procedures and methods can be accomplished using methods known in the art.

The construction and coupling method of HPV16 type E7 polypeptide protein are taken as an example for the embodiment, and are described as follows:

(1) Construction of plasmids

The E7 gene was amplified by PCR using primers that encode the E7-HPV of the strand (5 "-GATGCATCACAACATGGAGATACACCTACATTGCAT-3") and the E7-HPV of the complementary strand (5 "-GGAGCTGTTATGGTTTCTGAGAACAGATGG-3").

The PCR product was cloned into pGEM-T Easy Vector to generate pGEM-E7 plasmid.

(2) Plasmid expression

In the specific procedure of this example, plasmid constructs were first established in E.coli and then transferred to lactococcus lactis by electrotransformation. The E7 gene was expressed in lactococcus lactis using the nisin induction system. The encoding gene is connected with an expression vector and transferred into a receptor cell, the cell is lysed after induced expression, and HPV16 type E7 polypeptide protein is obtained through collection and purification.

(3) Coupled high immunogenicity macromolecular proteins

In order to improve the immunogenicity of the HPV16 type E7 polypeptide protein, the E7 polypeptide protein is coupled with KLH (or carrier proteins with high immunogenicity such as BSA, OVA and the like) to prepare polypeptide protein synthetic antigen components coupled with the KLH (or carrier proteins with high immunogenicity such as BSA, OVA and the like). The specific preparation method of the feasibility of the embodiment is as follows:

A. SMCC ((N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester, N-Maleimidomethyl cyclohexanecarboxylicacid Hydroxysuccinimide ester) was dissolved in 2ml DMF (N, N-Dimethylformamide);

B. KLH (keyhole limpet hemocyanin ) was added to a triangular flask, supplemented with 1 x PBS (pH 7.2) to the desired final carrier protein concentration;

C. slowly dripping the dissolved SMCC solution into KLH, and stirring at room temperature for reaction for 1h;

D. dialysis with PBS (pH 7.4) solution at 4deg.C for 6 hours, to remove free SMCC;

E. pouring the dialyzed KLH protein into a centrifuge tube, determining the volume of the dialyzed KLH protein through the scale of the centrifuge tube, calculating the concentration of the dialyzed KLH protein according to the amount of the dialyzed KLH protein added before the reaction, and transferring the KLH-SMCC solution into the centrifuge tube according to the concentration of the dialyzed KLH protein;

F. Dissolving E7 antigen polypeptide protein with PBS (pH 7.2) solution;

G. detecting sulfhydryl groups in polypeptide proteins with Ellman reagent;

H. dripping the polypeptide protein into a KLH-SMCC tube, and uniformly mixing by a vertical mixer at room temperature for reaction for 4 hours;

J. detection of thiol groups in polypeptides using Ellman reagent.

2. Preparation of HPV-E6/E7 composite polypeptide protein antigen

The HPV16 type-E6 antigen polypeptide, HPV16 type-E7 antigen polypeptide, HPV18 type-E6 polypeptide protein, HPV18 type-E7 polypeptide protein, HPV6 type-E6 polypeptide protein, HPV6 type-E7 polypeptide protein, HPV11 type-E6 polypeptide protein and HPV11 type-E7 polypeptide protein prepared in the foregoing are respectively mixed according to the mass ratio (1-10): (1-10): (1-10): (1-10): (1-10): (1-10): (1-10): (1-10) and mixing (the selection ratio of the embodiment is 1:1:1:1:1:1:1:1) to prepare the HPV-E6/E7 composite polypeptide protein antigen component.

The prepared HPV-E6/E7 composite polypeptide protein antigen components are mixed according to the mass ratio of (1-10): mixing (1-10) (1:1 in this example) with Freund's adjuvant, homogenizing at 8000rpm in a high-speed homogenizer to obtain HPV-E6/E7 polypeptide protein complex antigen for immunization.

3. Preparation of immune egg

Selecting egg laying fowl (chicken, duck, goose, turkey, ostrich, etc.) with high immune response capability, applying immune activation method, and performing immune injection with the prepared HPV-E6/E7 polypeptide protein composite antigen. Injecting the laying hen once every two weeks for 3 times; after 15-20 days of the first immunization, the immune eggs produced by the laying hens are respectively picked up to obtain the anti-HPV-E6/E7-IgY immune eggs.

4. Preparation of anti-HPV-E6/E7-IgY and anti-HPV-E6/E7 small molecule antibody Fab

For the preparation of anti-HPV-E6/E7-IgY, the conventional pure water extraction method, chloroform extraction method, cold ethanol precipitation method or ammonium sulfate precipitation method can be adopted to prepare the anti-HPV-E6/E7-IgY crude extract.

In this example, the conventional pure water extraction method is taken as an example to prepare the anti-HPV-E6/E7-IgY crude extract, and the specific operations comprise: washing the prepared anti-HPV-E6/E7-IgY immune egg with flowing water, scrubbing and sterilizing with alcohol, breaking the anti-HPV-E6/E7-IgY immune egg with an eggbeater, filtering off egg white with a yolk sieve, leaving yolk, and stirring uniformly; adding distilled water 3-8 times of the yolk liquid volume, diluting, mixing, and adjusting pH to 5.5-6.5 with 1.0N HCl solution; fully and uniformly stirring the diluted solution with the adjusted pH value, cooling to 2-6 ℃, and standing for 12-24 hours; centrifuging the diluted solution at a high speed; placing the separated supernatant into an ultrafilter for ultrafiltration concentration for 10-20 times; then adding sodium alginate solution with the concentration of 1.0-3.0%, slowly adding sodium alginate solution to the final concentration of 0.1-0.2%, and stirring until turbidity appears; adding 1.0-3.0% CaCl ₂ The solution is stirred uniformly until the final concentration is 0.1-0.2%, and is kept stand for 8-12 hours at 3-4 ℃; centrifuging at high speed and collecting supernatant to obtain anti-HPV-E6/E7-IgY crude extract. Then, the anti-HPV-E6/E7-IgY crude extract is subjected to ion And (3) carrying out chromatography by using an exchange column and an affinity chromatography column, and dialyzing and concentrating a chromatography collection to obtain a high-purity anti-HPV-E6/E7-IgY solution.

Continuing to adjust the pH value of the anti-HPV-E6/E7-IgY crude extract solution to 3.0-5.0, and adding catalytic protease; fully stirring and dissolving to generate enzymatic reaction, then rotating and centrifuging at a low temperature and high speed, discarding precipitate to obtain supernatant, and ultrafiltering the supernatant to obtain concentrated solution; and then, sequentially passing the concentrated solution obtained after ultrafiltration through an ion exchange column and an affinity chromatography column, and dialyzing and concentrating a chromatography collection to obtain the high-purity anti-HPV-E6/E7 small-molecule antibody Fab solution.

The high-purity anti-HPV-E6/E7-IgY solution and the anti-HPV-E6/E7-IgY small-molecule antibody Fab solution are respectively subjected to freeze drying or medium-low temperature spray drying or fluidized bed drying or drying in other drying modes without influencing the antibody activity, and then are added into an ultrafine grinder for grinding and grinding to prepare high-purity nano anti-HPV-E6/E7-IgY dry powder and high-purity nano anti-HPV-E6/E7 small-molecule antibody Fab dry powder respectively.

Example 3

The embodiment is used for preparing high-purity anti-PD-1/L1-IgY and small molecule Fab nanometer dry powder thereof.

1. Preparation of PD-1/L1 Complex antigen

The present example uses conventional genetic engineering techniques to recombine human PD-1 and PD-L1 expressed proteins, respectively, and then according to (1-10): (1-10) mixing (1:1 in the embodiment); then, according to (1-10): (1-10) ratio (1:1 is selected in the embodiment) and Freund's adjuvant are mixed to prepare the human PD-1/L1 expression protein composite antigen.

2. Preparation of immune egg

In the embodiment, egg laying poultry (chicken, duck, goose, turkey, ostrich, etc.) with high immune response capability are selected, an immune activation method is applied, and the prepared human PD-1/L1 expression protein composite antigen is adopted for immune injection. Injecting the human PD-1/L1 expressed protein composite antigen prepared by the steps into the laying hens every two weeks for 3 times, and respectively detecting the immune eggs laid by the laying hens after 15-20 days of the first immunization to obtain the anti-PD-1/L1-IgY immune eggs.

3. Preparation of anti-PD-1/L1-IgY and anti-PD-1/L1 small molecule antibody Fab

The preparation of the anti-PD-1/L1-IgY can adopt a pure water extraction method, a chloroform extraction method, a cold ethanol precipitation method or an ammonium sulfate precipitation method which are known in the art to prepare a broad-spectrum anti-HPV-IgY crude extract.

The embodiment adopts a pure water extraction method to prepare the PD-1/L1-IgY resistant crude extract, and the specific operations comprise: washing the prepared anti-PD-1/L1-IgY immune eggs with flowing water, scrubbing and sterilizing with alcohol, crushing the anti-PD-1/L1-IgY immune eggs with an eggbeater, filtering off egg white with a yolk sieve, leaving yolk, and stirring uniformly; adding distilled water 3-8 times of the yolk liquid volume, diluting, mixing, and adjusting pH to 5.5-6.5 with 1.0N HCl solution; fully and uniformly stirring the diluted solution with the adjusted pH value, cooling to 2-6 ℃, and standing for 12-24 hours; centrifuging the diluted solution at a high speed; placing the separated supernatant into an ultrafilter for ultrafiltration concentration for 10-20 times; then adding sodium alginate solution with the concentration of 1.0-3.0%, slowly adding sodium alginate solution to the final concentration of 0.1-0.2%, and stirring until turbidity appears; adding 1.0-3.0% CaCl ₂ The solution is stirred uniformly until the final concentration is 0.1-0.2%, and is kept stand for 8-12 hours at 3-4 ℃; centrifuging at high speed, and collecting supernatant to obtain anti-PD-1/L1-IgY crude extract. And (3) subjecting the anti-PD-1/L1-IgY crude extract to ion exchange column and affinity chromatography column chromatography, and dialyzing and concentrating the chromatography collection to obtain high-purity anti-PD-1/L1-IgY solution.

Adjusting the pH of the anti-PD-1/L1-IgY solution to 3.0-5.0, and adding catalytic protease; stirring and dissolving fully to generate enzymatic reaction, then rotating and centrifuging at low temperature and high speed, discarding precipitate to obtain supernatant, and ultrafiltering the supernatant to obtain concentrated solution; and then, sequentially passing the concentrated solution obtained after ultrafiltration through an ion exchange column and an affinity chromatography column, and dialyzing and concentrating a chromatography collection to obtain the high-purity anti-PD-1/L1 small molecule antibody Fab solution.

Finally, the bacterial virus filtration device of the virus removal filtration system manufactured by us Pall Ultrafine Filtration Company was used to thoroughly filter out various bacterial viruses, ensuring that the IgY and small molecule antibody Fab prepared are never any viruses and bacteria. Wherein the first bacteria filtering device is to remove bacteria such as Salmonella by using a 0.22 μm membrane sterilization filter; the second mycoplasma filtering device is to remove mycoplasma by a 0.1 μm membrane mycoplasma removing filter; the third virus filtration device was a Ultipor VFTM DV50 virus removal filter to remove a variety of viruses including influenza virus and enterovirus.

And (3) freeze-drying or medium-low temperature spray-drying or fluidized bed drying the high-purity anti-PD-1/L1 small molecular antibody Fab solution, adding the high-purity anti-PD-1/L1 small molecular antibody Fab solution into an ultrafine grinder, grinding and crushing to obtain high-purity nano anti-PD-1/L1 small molecular antibody Fab dry powder.

Example 4

This example is used to prepare a broad spectrum of anti-HPV-IgY and anti-E6/E7-IgY and anti-PD-1/L1-IgY and their small molecule antibody Fab combination antibodies.

The broad-spectrum anti-HPV-IgY and the small molecular antibody Fab thereof, the anti-E6/E7-IgY and the small molecular antibody Fab thereof prepared in the above examples 1-3 are respectively combined to form the anti-PD-1/L1-IgY and the small molecular antibody Fab thereof.

The broad-spectrum anti-HPV-IgY, the broad-spectrum anti-HPV small-molecule antibody Fab, the anti-E6/E7-IgY, the anti-E6/E7 small-molecule antibody Fab, the anti-PD-1/L1-IgY and the anti-PD-1/L1 small-molecule antibody Fab are mixed according to the following ratio (1-10): (1-10): (1-10): (1-10): (1-10): (1-10), the present example is selected as 1:1:1:1:1:1, namely the broad-spectrum anti-HPV-IgY, anti-E6/E7-IgY, anti-PD-1/L1-IgY and small molecule antibody Fab combined antibody thereof.

Example 5

In the embodiment, the anhydride lactoglobulin of the coupled combined antibody is obtained by coupling the anhydride lactoglobulin with the broad-spectrum anti-HPV-IgY, anti-E6/E7-IgY, anti-PD-1/L1-IgY and small molecule antibody Fab combined antibody thereof.

In the art, the anhydrated lactoglobulin is linked to a broad spectrum of anti-HPV-IgY and anti-E6/E7-IgY and anti-PD-1/L1-IgY and their small molecule antibody Fab combination antibodies by a chemical linkage or genetic recombination technique. The method can be used for non-site-specific coupling, site-specific coupling modified by genetic engineering, or site-specific coupling independent of genetic engineering. Meanwhile, the carrier protein (linker) of the conjugate can be Bovine Serum Albumin (BSA), ovalbumin (OVA), keyhole Limpet Hemocyanin (KLH), human Serum Albumin (HSA), artificially synthesized Polylysine (PLL), artificially synthesized polypeptide and the like. The coupling of the gene recombination technology is mainly to reform the composite antibody by using a genetic engineering technology, so that the fixed-point coupling of the anhydrated lactoglobulin and the composite antibody is realized, and the specific process is not repeated.

The methods of chemical linking that are conventional in the art also include a wide variety of methods such as carbodiimide method, glutaraldehyde method, mixed anhydride method, periodic acid oxidation method, succinic anhydride method, carboxymethyl hydroxylamine method, diazotized p-aminobenzoic acid method, sodium monochloroacetate method, and the like. Among the most commonly employed methods are mainly two: the active ester method and the anhydride method can also adopt heterobifunctional reagents such as SPDP and the like as a cross-linking agent for coupling.

In particular, acyl carrier protein (MBS) can be used for activating the connector (carrier protein) of the conjugate, so that free-SH on the connector (carrier protein) of the conjugate can be connected with side chain-SH of Cys at the tail end of the polypeptide, and the specificity and the stability are improved. EDC, 1-Ethyl group- (3-dimethyl)Aminopropyl group) Carbonyl groupDiimine (II)The method for connecting-COOH on the activated linker (carrier protein) to-NH 2 on the polypeptide to-COOH on the carrier is not limited to these methods in practical applications.

The embodiment takes special-shaped bifunctional reagent as a cross-linking agent coupling and glutaraldehyde coupling as an example, and the specific operation is as follows:

(1) Treatment of the anhydrified lactoglobulin with N-succinimidyl 3- (2-pyridyldithio) propionate: dissolving the anhydride lactoglobulin in a buffer solution to obtain an anhydride lactoglobulin solution; dissolving N-succinimidyl 3- (2-pyridyl disulfide) propionate with absolute ethanol, adding into the anhydride lactoglobulin solution, and reacting at 23-25 ℃ for 30 minutes. Passing the reaction solution through a SephadexG-25 column to remove redundant SPDP and byproducts, collecting and concentrating the anhydride lactoglobulin protein solution (marked as HRP-PDP);

(2) Treatment of broad-spectrum anti-HPV-IgY and anti-PD-1/L1-IgY and their small molecule antibody Fab complex antibodies with N-succinimidyl 3- (2-pyridyldithio) propionate: dissolving a broad-spectrum anti-HPV-IgY and anti-PD-1/L1-IgY and a small molecule antibody Fab combined antibody thereof in a buffer solution to obtain a combined antibody solution; then absolute ethanol is used for dissolving N-succinimidyl 3- (2-pyridyl disulfide) propionate, and then the N-succinimidyl 3- (2-pyridyl disulfide) propionate is added into the combined antibody solution to react for 30 minutes at the temperature of 23-25 ℃. Passing the reaction solution through a SephadexG-25 column to remove excessive SPDP and byproducts, collecting and concentrating a combined antibody protein solution (labeled as ANTI-PDP);

(3) Reduction of anhydrified lactoglobulin: taking the anhydride lactoglobulin protein liquid (marked as HRP-PDP), adding solid dimercaptosuccinic alcohol (DTT), and reacting at 23-25 ℃ for about 25 minutes; then, passing through Sephadex G-25 column, collecting and concentrating reduced anhydrated lactoglobulin protein solution (labeled HRP-SH);

(4) Coupling of anhydrified lactoglobulin and combinatorial antibodies: the reduced anhydrated lactoglobulin solution (labeled HRP-SH) and the combined antibody protein solution (labeled ANTI-PDP) obtained above were mixed, reacted at about 4℃for about 20 hours, and then concentrated to obtain the anhydrated lactoglobulin of the coupled combined antibody.

The present example can also be based on glutaraldehyde method for coupling, the specific operation is as follows:

(1) Preparation of the coupling reaction mixture: the total amount of the whole coupling reaction system comprises the following components in percentage by mass: 10-20wt% of anhydride lactoglobulin, 30-34wt% of broad-spectrum anti-HPV-IgY and anti-PD-1/L1-IgY and small molecule antibody Fab combined antibodies thereof, 55-36wt% of 0.1M pH 7.2 phosphate buffer solution, and 5-10wt% of 0.5% glutaraldehyde;

(2) The method comprises the steps of respectively weighing the formula amount of the anhydride-modified lactoglobulin and the formula amount of the broad-spectrum anti-HPV-IgY and anti-PD-1/L1-IgY and the small molecule antibody Fab combined antibody, and putting the mixture into a powder mixer to be uniformly mixed to obtain an anhydride-modified lactoglobulin and combined antibody mixture;

adding phosphate buffer solution with the formula amount of 0.1M and the pH value of 7.2 into a liquid reaction tank, then adding the mixture of the anhydride lactoglobulin and the combined antibody, and fully and uniformly stirring;

continuously adding glutaraldehyde in the formula amount, adjusting the temperature of the liquid reaction tank to 25 ℃, fully and uniformly stirring, keeping the temperature of the liquid reaction tank at 25 ℃, and standing for 60min. Obtaining a coupling reaction mixture;

(3) Isolation and purification of the coupling reaction mixture: adding a saturated ferric sulfate solution into the coupling reaction mixture in a proportion of 20-30% while stirring; then, the mixture was left at room temperature for 30 minutes to allow complete precipitation; separating the core at 2000-3000 rpm for 10 min, and discarding supernatant; washing the precipitate obtained by centrifugation with 25% saturated ferric sulfate twice, and dissolving the precipitate with physiological saline; separating heart at 3000 rpm for 15 min, collecting supernatant, and repeating the above steps three times; finally, the resultant antibody was dialyzed against 0.15M phosphate buffer (pH 7.4) for 24 hours to obtain an anhydrified lactoglobulin of the conjugated antibody.

Example 6

This example is used to prepare IgY against cervicitis pathogens.

In order to prepare the desired IgY against cervicitis pathogens, it is necessary to screen for representative pathogens that cause cervicitis. According to epidemiological investigation, the main causative agents of cervical inflammation in the vagina are staphylococcus aureus, neisseria gonorrhoeae, escherichia coli, klebsiella pneumoniae, streptococcus agalactiae. Therefore, these five bacteria are selected as representative pathogens of cervicitis in the embodiment, and other pathogens of cervicitis of vagina can be selected for practical application.

1. Preparation of antigens for immunization

Culturing a pathogen: the staphylococcus aureus, neisseria gonorrhoeae, escherichia coli, klebsiella pneumoniae and streptococcus agalactiae are respectively cultivated by a conventional method.

Preparing a composite antigen: firstly, culturing five pathogenic bacteria according to the proportion of 1-10:1-10:1-10:1-10:1-10 (in this example, 1:1:1:1) and placing into a tissue mashing homogenizer at 8000-12000 rpm; then, the bacterial protein mixture is mixed with 1-10:1-10 (1:1 in this example) of Freund's adjuvant, homogenizing at 30000rpm in a high-speed homogenizer to obtain water-in-oil emulsion, and making into bacterial protein composite antigen.

The present invention is exemplified by, but not limited to, the five bacteria described above as representative pathogens of cervicitis.

2. Preparation of immune egg

In the embodiment, egg laying poultry (chicken, duck, goose, turkey, ostrich, etc.) with high immune response capability are selected, an immune activation method is applied, and the prepared bacterial protein composite antigen is adopted for immune injection. The same dose and method are injected for the second time at intervals of 7 days after the first injection, the 3 rd time is injected at intervals of 7 days after the second injection, and the hyperimmune eggs are picked from 15 th to 20 th days after the third injection.

3. Preparation of anti-cervicitis pathogen specific IgY

The hyperimmune egg is treated with 0.5% benzalkonium bromide solution or 0.1% KMnO ₄ Soaking immune egg in the solution or other similar disinfectant for 15-30 min, sterilizing, washing with sterile distilled water, air drying, breaking eggshell, filtering off egg white with yolk sieve, adding 4-8 times distilled water to the egg white, diluting, stirring, regulating pH with 1mol/L NaOH solution or 1mol/L HCl solution to 5.5-6.0,4-6deg.C, standing overnight, centrifuging at 8000-12000r/min for 20 min, collecting supernatant, and concentrating by ultrafiltration. Purifying with ion exchange column and gel exchange column and affinity chromatographic column to obtain specific pure IgY for resisting cervicitis pathogen.

Then, various bacterial viruses were thoroughly filtered out using a bacterial virus filtration device for a virus removal filtration system manufactured in U.S. Pall Ultrafine Filtration Company, ensuring that the IgY prepared was never any virus or bacteria.

In the embodiment, the high-purity cervicitis pathogen-resistant specific pure IgY is prepared by adopting a freeze dryer for drying or medium-low temperature spray drying or fluidized bed drying or other drying modes which do not influence the activity of the antibody.

Example 7

This example is used to prepare nanoparticles of a lactoglobulin and anti-cervicitis pathogen IgY composition of conjugated antibodies encapsulated in liposomes as a carrier.

For administration of HPV infection drugs in the conventional sense, due to the large number of branches and crypts of the cervical mucosa, pathogens hide in the crypts and mucosal interstices, which are difficult for ordinary antibodies to enter for clearance. Therefore, the invention selects a new generation of carriers such as lipid liquid crystal nanoparticles (lipid liquid crystalline nanoparticles, LLCN) and lipid nanoparticles (Lipid Nanoparticle, LNP) and solid lipid nanoparticles (Solid lipid nanoparticle, SLN) as a wrapping and drug delivery system to obtain the lipid nano carrier particle broad-spectrum anti-HPV biological protein and anti-cervicitis pathogen IgY composition. The new generation carrier is used as a wrapping and drug delivery system, can increase the membrane permeability of the composition, improves the bioavailability, and has a slow release effect; thus, the permeability and the absorptivity of the composition are obviously improved, the composition can more effectively infiltrate into the crypt of cervical canal and the mucous membrane gap, stronger targeting property, delay effect and permeability are generated, and the bioavailability and the therapeutic index are greatly improved.

The encapsulation process using lipid nanoparticles as a carrier in this embodiment may be performed by using an encapsulation method conventionally known in the art.

In this embodiment, one of the lipid liquid crystal nanoparticles (lipid liquid crystalline nanoparticles, LLCN) is taken as an example to illustrate a preparation method of the new carrier, and preparation of other new carriers is performed by referring to a conventional method, and specific processes are not repeated.

Currently, the preparation methods of lipid liquid crystal nanoparticles (lipid liquid crystalline nanoparticles, LLCN) conventional in the art include a mechanical stirring method, a high-pressure homogenizing method, a heat treatment method, a spontaneous emulsification method, a combined ultrasonic technology method of spontaneous emulsification, a low-temperature spray drying method and the like.

The present invention is illustrated by the whole encapsulation process in which Lipid Liquid Crystal Nanoparticles (LLCN) are prepared by spontaneous emulsification in combination with ultrasonic technology.

The feasible lipid liquid crystal nanoparticle preparation system known in the art comprises the following components in percentage by mass based on the total amount, wherein the oil phase and the water phase comprise the following auxiliary materials in parts by mass:

the oil phase auxiliary material comprises the following components: 1 to 20 weight percent of fatty alcohol grease, 0.5 to 8 weight percent of non-fatty alcohol grease and 1 to 10 weight percent of emulsifying agent;

the water phase auxiliary material comprises the following components: 1 to 25 weight percent of organic additive, 0.01 to 2.0 weight percent of inorganic additive and the balance of water; wherein,

The fatty alcohol grease can be selected from one or more than two of hexadecanol, octadecanol and docosyl alcohol;

the non-fatty alcohol oil can be one or more than two of mineral oil, vegetable oil, animal oil and synthetic oil;

the emulsifier can be one or more of sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty alcohol polyoxyethylene ether, polyethylene glycol fatty acid ester, ethoxylated castor oil, fatty acid monoglyceride, fatty alcohol polyglycerol ester and sucrose monostearate;

the organic additive can be selected from monohydric alcohol or polyhydric alcohol with short carbon chain; wherein the molecular formula of the short carbon chain monohydric alcohol is CnH2n+1OH, wherein n is 1-12, and the short carbon chain polyhydric alcohol is mixed alcohol composed of one or more than two of ethylene glycol, 1, 2-propylene glycol, 1, 3-butanediol, propylene glycol carbonate and glycerol;

the inorganic additive can be one or more of sodium chloride, potassium chloride, magnesium sulfate and sodium sulfate.

Specifically, the preparation method of the Lipid Liquid Crystal Nanoparticle (LLCN) coated conjugated combined antibody lactoglobulin and anti-cervicitis pathogen IgY composition known in the art can specifically comprise the following steps:

(1) Heating the oil phase component consisting of fatty alcohol grease, non-fatty alcohol grease and emulsifier to 70-90 ℃, and fully and uniformly stirring to obtain the uniformly dispersed oil phase component;

(2) Heating the water phase component consisting of the organic additive, the inorganic additive and the water to 70-90 ℃, and fully and uniformly stirring to form a uniformly dispersed water phase component; then cooling to 60-65 ℃, slowly adding the lactoglobulin coupled with the composite antibody and the IgY composition for resisting cervicitis while stirring, and continuing stirring for 60min to obtain an aqueous solution of the lactoglobulin and the IgY composition for resisting cervicitis containing the coupled composite antibody;

(3) Homogenizing the oil phase component at a high speed of 15000-25000 rpm, cooling to 60-65 ℃ after homogenizing, adding the lactoglobulin containing the coupling combined antibody and the aqueous solution of the anti-cervicitis pathogen IgY composition into the mixture, continuing homogenizing for 5-20 min, controlling the stirring speed to 300-800 rpm, and keeping the temperature at 60-65 ℃; and then cooling to room temperature to obtain the lactoglobulin of the coupled combined antibody coated by the Lipid Liquid Crystal Nanoparticle (LLCN) and the anti-cervicitis pathogen IgY composition.

The present embodiment is illustrated by only one of the specific preparation processes, and the specific processing procedure is as follows:

(1) The preparation system comprises the following components in total: the combined antibody conjugated lactoglobulin (example 6) and anti-cervicitis pathogen IgY composition (example 7) 10-20wt%, glycerol Monooleate (GMO) 30-45wt%, poloxamer 407 (F127) 3-5wt%, pH5.0 Phosphate Buffer (PBS) 6-10wt%, distilled water balance.

(2) Adding poloxamer 407 (F127) in the formula amount into distilled water in the formula amount, heating to 70-90 ℃, and fully and uniformly stirring to form a uniformly dispersed water phase component A;

adding the lactoglobulin of the formula amount of the coupling combined antibody and the anti-cervicitis pathogen IgY composition into a Phosphate Buffer Solution (PBS) of the formula amount, and fully and uniformly stirring to form a composition solution B;

adding the composition solution B to the aqueous phase component a while stirring to form an aqueous phase C;

slowly dripping Glycerol Monooleate (GMO) into the water phase, and stirring at a rotation speed of 750r/min for 3 hours at 50 ℃ to obtain a mixture D;

and (3) placing the mixture D into an ultrasonic crusher to carry out ultrasonic crushing for 5-10min (5-10 s/time, 5-25s each time) to obtain the lactoglobulin and anti-cervicitis pathogen IgY composition of the coupling combined antibody taking the lipid nanoparticles as the carrier.

Effect verification

Experiment 1

The assay was used to detect antibody binding titers of broad-spectrum anti-HPV-IgY antibodies (example 1) against 15 high-risk Human Papillomaviruses (HPV).

HPV16 type, HPV18 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV45 type, HPV51 type, HPV52 type, HPV56 type, HPV58 type, HPV59 type, HPV68 type, HPV73 type and HPV82 type are selected as detection antigens, and the antibody titers of the obtained broad-spectrum anti-HPV-IgY are detected by an ELISA method (enzyme-linked immunosorbent assay), and the results are shown in the following table 1 (binding titer detection results).

TABLE 1

Note that: the concentration of the broad-spectrum anti-HPV-IgY antibody solution in the test sample was 1mg/ml.

As can be seen from the detection results in Table 1, the broad-spectrum anti-HPV-IgY antibodies prepared by the invention have high antibody binding titers to 15 high-risk Human Papilloma Virus (HPV) antigens.

Experiment 2

The experiment is used for detecting the antibody binding titer of a broad-spectrum anti-HPV-Fab small-molecule antibody to 15 high-risk Human Papillomaviruses (HPVs).

HPV16 type, HPV18 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV45 type, HPV51 type, HPV52 type, HPV56 type, HPV58 type, HPV59 type, HPV68 type, HPV73 type and HPV82 type are selected as detection antigens, and the antibody titers of the obtained broad-spectrum anti-HPV-Fab small-molecule antibodies (example 1) are detected by an ELISA method, and the results are shown in the following Table 2 (binding titer detection results).

TABLE 2

Note that: the concentration of the broad-spectrum anti-HPV-Fab small molecule antibody solution in the test sample was 1mg/ml.

As can be seen from the detection results in the table 2, the prepared broad-spectrum anti-HPV-Fab small-molecule antibodies have high antibody binding titers to 15 high-risk Human Papilloma Virus (HPV) antigens.

Experiment 3

The experiment is used for detecting the antibody binding titer of the broad-spectrum anti-HPV-E6/E7-IgY and anti-HPV-E6/E7 small molecule antibody Fab to 15 HPV-E6/E7 oncogene proteins.

As the detection antigen, HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV31-E6, HPV31-E7, HPV33-E6, HPV33-E7, HPV35-E6, HPV35-E7, HPV39-E6, HPV39-E7, HPV45-E6, HPV45-E7, HPV51-E6, HPV51-E7, HPV52-E6, HPV52-E7, HPV56-E6, HPV56-E7, HPV58-E6, HPV58-E7, HPV59-E6, HPV59-E7, HPV68-E6, HPV68-E7, HPV73-E6, HPV73-E7, HPV82-E6 and HPV82-E7 were selected, and the antibody titers of the obtained broad-spectrum anti-HPV-E6/E7-IgY were detected by the "ELISA" method (results of binding titers are shown in Table 3).

TABLE 3 Table 3

Note that: the concentration of the broad-spectrum anti-HPV-E6/E7-IgY antibody solution in the test sample is 1mg/ml.

As can be seen from the detection results in the table 3, the broad-spectrum anti-HPV-E6/E7-IgY antibody prepared by the invention has very high antibody binding titers to 15 high-risk type HPV-E6/E7 oncogene proteins, and the broad-spectrum anti-HPV-E6/E7-IgY antibody has ideal broad-spectrum property.

Experiment 4

This experiment was used to detect antibody binding titers of anti-cervicitis pathogen IgY against representative cervicitis pathogens.

The antibody binding titers of the prepared anti-cervicitis pathogen IgY (example 6) were measured by an "ELISA" method using staphylococcus aureus, neisseria gonorrhoeae, escherichia coli, klebsiella pneumoniae, streptococcus agalactiae as detection antigens, respectively, and the results are shown in table 4 (binding titer measurement results) below.

TABLE 4 Table 4

Note that: the concentration of the anti-cervicitis pathogen IgY solution in the test sample is 1mg/ml.

From the detection results, the prepared anti-cervicitis pathogen IgY has high antibody binding titers to 5 representative cervicitis pathogen antigens.

Experiment 5

The experiment adopts the lactoglobulin of the coupling combined antibody prepared in the embodiment 7 and the nanometer microgel preparation of the anti-cervicitis pathogen IgY composition to be sprayed into the cervix of HPV infection positive patients, and the curative effect is observed.

Basic information:

selection criteria: (1) having a sexual history between 25 and 50 years of age; (2) cervical cancer screening TCT (-), HPV (-) within 3 years; (3) The cervical cancer is screened for any one of TCT (-), high risk HPV-15 types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 types) to a plurality of types (+).

Exclusion criteria: (1) a history of cervical cancer and precancerous lesions in the past; (2) conventional HPV (+); (3) The cervical cancer is screened for other HPV types (+) except for the high-risk HPV-15 types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 types); (4) a history of hysterectomy; (5) current pregnancy; (6) patients without follow-up conditions.

A total of 160 women meeting the above criteria were selected as subjects for this study. Subjects were randomized into 81 groups a (treatment group) and 79 groups B (control group). The two groups of patients have no obvious difference in common data such as age, disease course and the like.

The positive rates of the treatment group and the control group are detected and counted respectively by adopting the FQ-PCR method before treatment.

Therapeutic method

Patients in group A (treatment group) were treated with a combination antibody conjugated lactoglobulin and anti-cervicitis pathogen IgY composition nanoparticle formulation by photoelectric self-controlled cervical dosing manipulator for 30 days. After vagina is flushed before sleeping, the lactoglobulin coupled with the combined antibody and the anti-cervicitis pathogen IgY composition nanoparticle preparation are filled into a photoelectric self-control cervical drug delivery manipulator injection tube according to the specification, a miniature luminous electronic eye assembly is inserted, and a flexible cervical drug delivery manipulator finger fingertip is aligned to a cervical orifice according to the display of a luminous electronic eye visual electronic eye on a mobile phone screen; then, the flexible cervical drug delivery mechanical finger is slowly inserted into the cervical canal, and then the nanoparticle gel is injected into the cervical canal to finish cervical drug delivery.

Group B (control) patients were treated with recombinant human interferon alpha 2a plugs for 30 days. After vaginal irrigation before sleeping, 1 recombinant human interferon alpha 2a suppository is injected into the fornix behind the vagina or the vaginal stump by pushing, 1 suppository is used every night, and 30 days is a 1 treatment course.

It should be noted that hip bath and sexual life are prohibited during the administration period, and after 30 days, the positive rates of the treatment group and the control group are detected and counted by using the above FQ-PCR method, respectively.

Instrument and reagent

A Hema3200 type Polymerase Chain Reaction (PCR) amplifier, a DA800 type full-automatic nucleic acid molecule hybridization instrument and an HPV genotyping detection kit.

Detection method

Sample collection: exposing the cervix with a speculum, wiping cervical secretions with a cotton swab, placing a special cervical brush at the cervical orifice, and rotating the cervical brush for 4-5 circles to obtain a sufficient cervical epithelial cell specimen; taking out the cervical brush, putting the cervical brush into a special tube filled with cell preservation solution, breaking the brush handle along the crease of the brush handle, screwing the tube cover, and then checking in time.

The detection method comprises the following steps: extracting DNA by a conventional method; adding DNA and reagent according to the requirement of the instruction book of the kit, and circulating for 40 times according to 93-3 min, 93-40 s, 55-40 s, 72-40 s; amplifying for 72-7 min; the amplified product is denatured for 98-8 min and then immediately placed in ice water; hybridization was performed on a fully automated nucleic acid molecule hybridization instrument. The results are interpreted according to the instruction book of the kit. When any type of HPV-DNA exceeds a threshold, the detection result is positive. Patients who detected 1 or more HPVs whose HPV-DNA exceeded the threshold value were all judged as positive patients, and the positive proportion was calculated based on this.

Statistical method

SPPS17.0 statistical software was used to statistically analyze the collected data, the counting data was tested using t-test, and the metering data material X2. When P < 0.05, the difference is statistically different.

Experimental results

The positive rates of group A (treatment group) and group B (control group) were calculated by the above method, respectively, and the details are shown in Table 5 (clinical test results).

TABLE 5

As can be seen from the data in Table 5, the patients were re-diagnosed 90 days after the end of a treatment course (during which sexual life was disabled), the treatment group was re-examined, and the number of HPV-DNA patients exceeding the threshold was reduced from 81 to 7, and the negative conversion rate was 91.4%; in comparison, the number of patients with HPV-DNA exceeding the threshold is reduced from 79 to 51, the negative conversion rate is 35.4%, and P is less than 0.05, which are obviously different.

The experiment shows that the administration of the nanometer particles of the lactoglobulin and the anti-cervicitis pathogen IgY composition of the coupling combined antibody has better therapeutic effect on HPV infected patients.

Experiment 6

In this experiment, the nanoparticle gel preparation of the carrier-coated conjugate antibody-conjugated lactoglobulin and the anti-cervicitis pathogen IgY composition prepared in example 7 was sprayed into the cervix of cervicitis patients, and the curative effect was observed.

Basic information

120 outpatients diagnosed with mild-moderate cervical erosion were selected and randomly divided into 58 control and 62 treatment groups. 58 cases of control group aged 18-45 years, average (35.1+ -1.7) years; 62 treatment groups were aged 19-47 years, with an average (35.7.+ -. 1.3) years. The age, course and disease comparison difference of the two groups of patients are not statistically significant (P is more than 0.05), and the patients are comparable.

Diagnostic criteria

The erosion area is classified into light, medium and heavy according to the erosion area. The erosion area is less than 1/3 of the total cervical area, the erosion area is mild (I DEG), 1/3-1/2 is moderate (II DEG), and the erosion area is greater than 1/2 is heavy (III DEG).

Cervical erosion can be represented as 3 types, depending on the extent of inflammation and the rate of columnar epithelial growth: (1) simplex: in the early stage of inflammation, the erosion surface is covered by a single-layer columnar epithelium, the surface is flat, and the appearance is smooth; (2) particle type: the erosion surface is uneven and granular due to the hyperplasia of cervical gland epithelium and interstitial hyperplasia; (3) mastoid process: the hyperplasia of glandular epithelium and interstitium is obvious, the surface roughness is more obvious, and mastoid process is formed.

Therapeutic method

Treatment group: the nanoparticle gel preparation of the IgY composition of the lactoglobulin and the anti-cervicitis pathogen coupled to the combined antibody of the embodiment 7 of the invention is used for injecting the nanoparticle gel into the cervix through the photoelectric automatic control cervix administration manipulator according to the operation mode of the photoelectric automatic control cervix administration manipulator after the vulva is cleaned before sleeping. 1 branch (3 g/branch) is used every night, and 15 days is a 1 treatment course.

Control group: treatment with recombinant human interferon alpha 2a plugs. After vaginal irrigation prior to sleep, 1 recombinant human interferon alpha 2a plug was injected into the posterior fornix or vaginal stump. 1 dose per night, 1 dose per time, 15 days as 1 course of treatment.

It should be noted that during both treatment periods, sexual life and bath were prohibited and menstrual period was stopped.

Therapeutic effect judgment criterion

And (3) healing: the cervical is smooth, the erosion surface disappears, and the clinical symptoms completely disappear;

the effect is shown: clinical symptoms are obviously relieved, cervical erosion area is reduced, and depth is shallow;

invalidation: clinical symptoms are not relieved, and cervical erosion surface and depth are not obviously changed.

Effective rate = cure rate + significant efficiency.

Statistical treatment

The data were analyzed using SPSS11.0 statistical software. The comparison of two groups of clinical data adopts t test, and the comparison of the effective rate and cure rate of two groups of medicaments adopts r test.

Experimental results

The total effective rate of the treatment group is 90.4%, and the total effective rate of the control group is 53.5%. The efficacy of the treatment group was significantly better than that of the control group, the differences were significant (P < 0.05), as detailed in Table 6 below (comparison of clinical effects of the two groups of patients).

TABLE 6

Group of	n	Healing of the wound	Has obvious effect	Improvement of	Invalidation of	Cure rate (%)	Total effective rate (%)
								Treatment group	62	56	5	1	0	90.3	90.4
Control group	58	31	3	8	16	53.4	53.5

From the data in Table 6, it can be seen that the administration of the nanoparticles of the lactoglobulin and the anti-cervicitis pathogen IgY composition of the conjugated antibodies of the present invention to cervicitis patients has a better therapeutic effect on cervicitis patients.

It will be apparent that the above embodiments are merely examples for clarity of illustration and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims

1. A composition for treating HPV infection comprising: lactoglobulin coupled to the combined antibody and IgY against cervicitis pathogen; the mass ratio of the lactoglobulin of the coupling combined antibody to the anti-cervicitis pathogen IgY is (1-10): (1-10).

2. A nanoparticle for resisting HPV infection, which is characterized by being prepared by wrapping the HPV infection-resisting composition by taking liposome as a carrier; the anti-HPV infection composition comprises lactoglobulin conjugated with a combination antibody and anti-cervicitis pathogen IgY.

3. An external preparation for resisting HPV infection, comprising: an anti-HPV infection composition and/or anti-HPV infection nanoparticle and adjuvant; the anti-HPV infection composition comprises lactoglobulin coupled to a combination antibody and anti-cervicitis pathogen IgY; the nanometer particles for resisting HPV infection are prepared by taking liposome as a carrier to encapsulate the composition for resisting HPV infection.

4. The composition for treating HPV infection of claim 1, wherein the conjugate antibody in lactoglobulin of the conjugate antibody comprises: broad spectrum anti-HPV-IgY and its small molecule antibody Fab, composite anti-E6/E7-IgY and its small molecule antibody Fab, anti-PD-1/L1-IgY and its small molecule antibody Fab mixture.

5. The composition for treating HPV infection of claim 4, wherein the mass ratio of the broad-spectrum anti-HPV-IgY, the complex anti-E6/E7-IgY and the anti-PD-1/L1-IgY is (1-10): (1-10): (1-10); the mass ratio of the broad spectrum anti-HPV-IgY, the broad spectrum anti-HPV-IgY small molecule antibody Fab, the composite anti-E6/E7-IgY small molecule antibody Fab, the anti-PD-1/L1-IgY and the anti-PD-1/L1-IgY small molecule antibody Fab is (1-10): (1-10): (1-10): (1-10): (1-10): (1-10).

6. The composition for treating HPV infection of claim 1, wherein the method for preparing the lactoglobulin of the conjugated combination antibody comprises:

(a) Preparing the required antigen according to the selected antibody types;

7. The composition for the treatment of HPV infection according to claim 1, wherein the pathogen against cervicitis pathogen IgY comprises a major pathogen type causing vaginitis, in particular comprising a mixture of staphylococcus aureus, neisseria gonorrhoeae, escherichia coli, klebsiella pneumoniae, streptococcus agalactiae.

8. The composition for treating HPV infection of claim 1, wherein the anti-cervicitis pathogen IgY is prepared by the process of:

(a) Culturing selected pathogen and preparing corresponding composite antigen;

(b) Preparing an immune egg by using the composite antigen;

(c) The immune egg is used for preparing the anti-cervicitis pathogen IgY.

9. The nanoparticle for combating HPV infection according to claim 2, wherein said liposome comprises at least one of a lipid liquid crystal nanoparticle, a lipid nanoparticle or a solid lipid nanoparticle.

10. An external preparation for the treatment of HPV infection according to claim 3, comprising: gel, dressing, spray, gynecological lotion, male lotion, hand cleanser, powder, tablet, toothpaste, oral paste, mouthwash, buccal tablet, oral liquid, oral preparation or capsule.

11. The use of an external preparation for resisting HPV infection, which is characterized by the use for preparing a medicament for preventing and treating HPV infection, cervical infectious diseases or cervical cancer, the use for preparing a non-diagnostic detection reagent or detection product or the use for preparing an HPV infection-resisting medical device.