CN116554280A - 18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and preparation method and application thereof - Google Patents

18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and preparation method and application thereof Download PDF

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CN116554280A
CN116554280A CN202310563435.6A CN202310563435A CN116554280A CN 116554280 A CN116554280 A CN 116554280A CN 202310563435 A CN202310563435 A CN 202310563435A CN 116554280 A CN116554280 A CN 116554280A
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hpv
parts
protein
yolk
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赵凯
赵斌安
祖刘静
赵悅琪
史斌
赵桓震
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Shanghai Full Biotechnology Co ltd
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Abstract

The invention discloses an 18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and a preparation method and application thereof, and relates to the technical field of biological medicine. The preparation method comprises the following steps: s1, respectively immunizing different laying hens by using 18 HPVL1 protein virus particles (VLP) as antigens, and obtaining 18 egg yolk from eggs produced by the immunized laying hens; s2, mixing the 18 yolk, and extracting a yolk neutralization antibody to obtain the 18-valent HPV composite yolk neutralization antibody. The 18-valent HPV composite yolk neutralizing antibody can effectively prevent HPV infection, further can be applied to the adjuvant therapy of cervical cancer, and clinical experiments conducted in three-phase hospitals show that the conversion rate of cervical HPV infection can reach 96.97% when the 18-valent HPV composite yolk neutralizing antibody is used for treating cervical HPV infection.

Description

18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicine, in particular to an 18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and a preparation method and application thereof.
Background
Cervical cancer is a disease in which pathogenic viruses are defined in human malignancies. Studies have demonstrated that more than 99% of cervical cancer tissues incorporate Human Papillomavirus (HPV) infection. HPV can proliferate specifically on human skin, mucosal epithelium, etc., resulting in benign and malignant tumors of the epithelium.
Cervical cancer is currently one of the most common malignant tumors in women worldwide. Cervical cancer is onset, and in recent years, the cervical cancer is in a younger trend, and the cervical cancer seriously endangers the health of women. The occurrence and development of the method are changed from the quantitative change to the qualitative change, and gradually changed to the abrupt change continuous evolution process, and the process is generally considered to last for more than 10 years; cervical intraepithelial lesions CIN and cervical HPV subclinical infections are believed to be closely related to cervical cancer. Investigation shows that 80% of women will be infected with high-risk HPV during their lifetime, some of them will self-heal, called "primary infection", and about 20-30% of infected patients are difficult to self-clear, called "persistent infection", and this high-risk HPV persistent infection is the high-risk population for cervical cancer. Cervical cancer is a disease that severely jeopardizes women's health and life, and every 2 minutes worldwide one dies from cervical cancer. Worldwide, there are up to 30 million infected women. To date, the medical community has not developed effective drugs for the treatment of Human Papilloma Virus (HPV) infections, antibiotics and other bactericides, which have no effect at all on Human Papilloma Virus (HPV). HPV vaccines currently on the market have only prophylactic effects, but no effect on the treatment of HPV infection. Since Human Papilloma Virus (HPV) is a special "non-enveloped virus", the envelope coat (capsid proteins L1/L2) is removed upon invasion of human cells; the surface of the infected cell does not have the capsid protein L1 target antigen for which the vaccine is aimed, so that the vaccine is invalid; thus, HPV vaccines have no prophylactic or therapeutic effect on patients who have invaded cells with HPV and were examined positive for HPV infection.
For positive patients who have entered cells with Human Papillomavirus (HPV), traditional antibodies and many chinese herbal medicines, besides being ineffective in injecting vaccines, are not able to enter infected cells to function due to the fact that they are macromolecules; small molecule chemicals and some small molecule traditional Chinese medicines have no specificity, so that strong toxic and side effects are necessarily generated on normal cells. Therefore, no medicine can be used for treatment at present, and no eradication method exists. Most thrill, those infected with high risk Human Papilloma Virus (HPV) are 20% likely to develop cancer five years later without prior intervention.
The existing prevention and treatment means of HPV mainly comprise HPV vaccine, traditional Chinese medicine, antibiotics, interferon and the like.
HPV vaccine: the current HPV vaccine is mainly used for preventing HPV infection and is suitable for vaccination of people who are not infected with HPV, and is not effective for people who are infected with HPV. The existing HPV vaccines have bivalent, tetravalent and nine valences, and at most, only 9 genotypes of HPV infection can be prevented, and 18 genotypes of HPV which leads to cervical cancer exist, so that only partial cervical cancer can be prevented by inoculating the vaccine, and partial cervical cancer can be missed.
Traditional Chinese medicine: most of the traditional Chinese medicine components have the effects of clearing heat and detoxicating, are generally used for auxiliary treatment of chronic gynecological inflammation, and cannot be used as the first choice treatment for HPV virus. Otherwise, the slow soldier is used for dispersing and developing pathogens causing gynecological inflammation, thereby delaying the illness state.
Antibiotics: the vagina of a healthy female is an acidic environment, has beneficial bacteria with self-cleaning function, kills beneficial bacteria of the vagina after long-term abuse of antibiotics and improper method for treating gynecological inflammation, damages acid-base balance of the vagina, and cultures a large amount of drug-resistant bacteria instead, so that single inflammatory patients often have multiple infections, and repeated infection of gynecological inflammation often causes high-risk HPV infection, and if no effective measures are taken, the cancer transformation rate is 1/5 after 5 years.
Interferon: interferon is currently widely used in clinic as an antiviral and antitumor agent for commonly treating HPV viruses. However, interferon production is particularly slow, so that the use period is usually 2 to 6 months. The effective rate after treatment is only about 50 percent. The toxic and side effects of interferon are common, the most common are fever and influenza-like syndrome, patient weight loss, alopecia, emotional agitation, hematopoiesis and thrombocytopenia caused by bone marrow suppression, mild anemia, occasionally nervous system injury, endocrine system function influence and interferon antibody generation.
Thus, there are several problems with the prevention of cervical cancer:
1. the injection of vaccine into HPV infection positive patients was ineffective for the following reasons: (1) HPV enters cervical columnar epithelial cells (infection) and is removed from coat (capsid protein) and re-transcribed into E6 and E7 proteins; (2) The infected cell surface loses the target antigen of HPV vaccine, capsid protein L1, so the vaccine is ineffective;
2. The anti-HPV vaccine is not broad-spectrum, HPV has more than 100 subtypes, persistent infection leads to 18 subtypes of cervical cancer high-risk HPV, and the nine-valent vaccine can only aim at nine types of viruses at present. Complete protection is not achieved by injection of nine vaccine.
3. In order to prevent cervical cancer, it is necessary to develop a composition for preventing and assisting in treating cervical cancer, but the existing composition has potential defects, contains more antibiotics, has side effects and can destroy the living environment of normal flora of vagina.
Therefore, research and development of a new medicine capable of making a patient positive for human papillomavirus turn positive from positive to negative and preventing and treating cervical cancer is a big thing related to hundreds of millions of women's health and life worldwide, and is an important subject in front of the international medical science interface.
Disclosure of Invention
The invention aims to provide an 18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and a preparation method and application thereof, so as to solve the problems in the prior art.
Among cervical intraepithelial high-grade lesions and cervical cancer tissues, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73 are at high risk; low risk HPV genotypes: types 6 and 11.
Since HPV is difficult to reproduce by tissue culture in vitro or infection of animals, strategies for research of HPV drugs have been mainly focused on genetic engineering techniques. Epitopes are classified into linear epitopes and steric epitopes. Only the steric epitope induces neutralizing antibodies. Molecular biology researches show that L1 and L2 of HPV virus structural proteins form capsid parts of viruses, pure L1 is enough to form virus-like particles (VLPs), and has good immune neutralization effect, and neutralizing antibodies are induced, and antigen immunogenicity is strong, and antibody titer is high.
HPV major neutralizing epitopes and type specific epitopes are concentrated on protein L1, most neutralizing epitopes of L1 are conformational epitopes, loss of correct conformation of L1 in protein expression and purification often brings about loss of neutralizing epitopes, and whether L1 or L1/L2 assembly produces virus-like particles (VLPs) with the same structure as natural viruses, which have most neutralizing epitopes identical to natural viruses, can induce production of a large amount of neutralizing protective antibodies with high titer, is an ideal vaccine form. In the HPV infection process, the L1 protein on the surface of the virus mediates cell receptor recognition and membrane fusion, can specifically recognize and bind to a human cell surface receptor, invade human cells and cause human infection. If neutralizing antibodies of HPV can be developed, HPV viruses can be neutralized, so that the viruses can not bind to cell surface receptors and block virus infection, thereby achieving the effect of preventing and treating diseases caused by HPV virus infection. The invention adopts bioengineering technology, uses insect cells to express VLP antigens of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1, immunizes the antigen into layers, generates antibody IgY (egg yolk immunoglobulin) for neutralizing HPV L1, extracts 18-valent HPV L1 antibody IgY from eggs, and prepares various preparations, thereby realizing the prevention and treatment of HPV and the auxiliary treatment of cervical cancer.
The current research proves that the prokaryotic cells have no post-translational processing system of protein (such as protein folding and correct disulfide bond formation), the generated protein is inconsistent with the natural conformation, and the protection effect is unstable and unreliable. The L1 protein expressed by E.coli cannot directly form VLPs, and although a certain amount of VLPs can be formed through complex operations such as denaturation, renaturation, re-denaturation and the like, the renaturation yield is extremely low and is only 0.02% -0.04%, so that the production and research can not be obviously satisfied, and the assembly condition, molecular biological characteristics and immunogenicity of the L1-VLPs are difficult to accurately evaluate. The eukaryotic cell expressed protein has natural conformation and biological activity, and can directly obtain a large amount of VLPs in protein preparation, so that the eukaryotic expression system, namely the baculovirus-insect cell expression system, is selected as a research tool. The gene sequences of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73L1 are searched from GeneBank, the technical route and method are designed and sent to professional companies, the professional companies perform gene synthesis and cloning according to the technical route and method, the recombinant baculovirus Bacmid-L1 of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73L1 is obtained, sf9 cells are respectively infected by the recombinant baculovirus Bacmid-L1, and a system for efficiently producing HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73L1 VLPs is constructed, L1 VLPs is expressed and purified, and HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73L1 antigens are obtained.
The present invention selects HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73L1 VLPs as antigens. HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73L1 VLP proteins were then immunized against egg-laying hens, respectively, to obtain 18-valent HPV composite egg-yolk neutralizing antibodies.
Based on this, the present invention provides the following scheme:
the invention provides a preparation method of an antigen combination for an 18-valent HPV composite yolk neutralization antibody, which comprises the following steps: the genes of HPV 6L 1 protein, HPV 11L 1 protein, HPV 16L 1 protein, HPV 18L 1 protein, HPV 31L 1 protein, HPV 33L 1 protein, HPV 35L 1 protein, HPV39L1 protein, HPV 45L 1 protein, HPV 51L 1 protein, HPV 52L 1 protein, HPV 56L 1 protein, HPV 58L 1 protein, HPV 59L 1 protein, HPV 61L 1 protein, HPV 66L 1 protein, HPV 68L 1 protein and HPV 73L1 protein with the coding amino acid sequences shown in SEQ ID NO.1-18 are selected and expressed and self-assembled by a eukaryotic expression system respectively, and then separated and purified to obtain 18 kinds of virus particles, namely the antigen combination.
Further, the eukaryotic expression system is a baculovirus-insect cell expression system;
The encoding genes of the HPV 6L 1 protein, the HPV 11L 1 protein, the HPV 16L 1 protein, the HPV 18L 1 protein, the HPV 31L 1 protein, the HPV 33L 1 protein, the HPV 35L 1 protein, the HPV 39L 1 protein, the HPV 45L 1 protein, the HPV 51L 1 protein, the HPV 52L 1 protein, the HPV 56L 1 protein, the HPV 58L 1 protein, the HPV 59L 1 protein, the HPV 61L 1 protein, the HPV 66L 1 protein, the HPV 68L 1 protein and the HPV 73L 1 protein, which are optimized by insect cell expression codons, are respectively shown in SEQ ID NO. 19-36.
Further, the eukaryotic expression system is a baculovirus-insect cell expression system.
The invention also provides an antigen combination prepared by the preparation method.
The invention also provides application of the antigen combination in preparing 18-valent HPV composite yolk neutralization antibodies.
The invention also provides a preparation method of the 18-valent HPV composite yolk neutralizing antibody, which comprises the following steps:
s1, respectively immunizing different laying hens by taking 18 kinds of virus particles in the antigen combination as antigens, and obtaining 18 egg yolk from eggs produced by the immunized laying hens;
s2, mixing the 18 yolk, and extracting a yolk neutralization antibody to obtain the 18-valent HPV composite yolk neutralization antibody. Further, in step S2, the step of extracting includes: mixing the 18 yolk, adding water, stirring uniformly to obtain yolk liquid, regulating the pH to 5.1-6.0, standing, precipitating, centrifuging to obtain supernatant, removing mixed proteins and lecithin, concentrating, and drying to obtain the 18-valent HPV composite yolk neutralizing antibody.
The invention also provides an 18-valent HPV composite yolk neutralization antibody prepared by the preparation method.
The invention also provides application of the 18-valent HPV composite yolk neutralizing antibody in preparing medicaments for preventing and treating HPV infection or assisting in treating cervical cancer.
The invention also provides a medicine for preventing and treating HPV infection or assisting in treating cervical cancer, and the medicine comprises the 18-valent HPV composite yolk neutralizing antibody.
Further, the dosage forms of the medicine comprise spray, oral lozenge, liposome liquid crystal microcapsule, vaginal liposome gel, external lotion, suppository, freeze-dried powder or effervescent tablet.
Further, when the medicine is a spray, the raw materials comprise the following components in parts by weight: 0.1-0.8 part of 18-valent HPV composite yolk neutralizing antibody, 0.003-0.04 part of bacteriostat, 0.1-12 parts of glycerol, 0.04-0.25 part of preservative, 0.05-1 part of lysozyme and 0.01-0.16 part of plant essence;
when the medicine is an oral lozenge, the raw materials comprise the following components in parts by weight: 0.15-0.8 part of 18-valent HPV composite yolk neutralizing antibody, 0.8-7 parts of citric acid, 4-38 parts of xylitol, 38-52 parts of starch, 0.03-0.7 part of preservative and 0.2-0.6 part of water-soluble plant essence;
When the medicine is liposome liquid crystal microcapsule, the raw materials comprise the following components in parts by weight: 0.5-2.5 parts of 18-valent HPV composite yolk neutralizing antibody, 3-7.5 parts of lecithin and 12-30 parts of cholesterol;
when the medicine is vaginal liposome gel, the raw materials comprise the following components in parts by weight: 3-15 parts of liposome liquid crystal microcapsule, 0.08-1 part of edetate disodium, 0.2-2 parts of carbomer 974P, 10.03-0.6 parts of polycarbophil AA-12 parts of glycerol, 0.04-0.25 part of preservative, 0.4-10 parts of benzyl alcohol, 0.3-1.4 parts of triethanolamine and 0.01-0.05 part of plant essence;
when the medicine is an external washing lotion, the raw materials comprise the following components in parts by weight: 0.1-0.8 part of 18-valent HPV composite yolk neutralizing antibody, 28-37 parts of fatty alcohol polyoxyethylene ether sulfosuccinate monoester disodium, 16-25 parts of fatty alcohol polyoxyethylene ether ammonium sulfate, 1.0-8 parts of glycerol, 0.04-0.25 part of preservative and 0.016-0.13 part of plant essence;
when the medicine is suppository, the raw materials comprise the following components in parts by weight: 0.1-0.8 part of 18-valent HPV composite yolk neutralizing antibody, 8-12 parts of gelatin, 20-37 parts of glycerol, 25-41 parts of mannitol, 0.05-0.2 part of preservative and 0.02-0.1 part of plant essence;
when the medicine is a freeze-dried powder preparation, the liquid raw materials of the freeze-dried powder preparation comprise the following components in parts by weight: 0.1-0.8 part of 18-valent HPV composite yolk neutralizing antibody, 0.009-0.18 part of bovine serum albumin, 1.5-13 parts of trehalose, 0.04-0.2 part of preservative and 0.016-0.12 part of plant essence;
When the medicine is an effervescent tablet, the raw materials comprise the following components in parts by weight: 0.4-0.6 part of 18-valent HPV composite yolk neutralizing antibody, 30-34 parts of hydroxypropyl-beta-cyclodextrin, 13-16 parts of citric acid, 2-4 parts of magnesium stearate, 35-39 parts of sodium bicarbonate, 60003.5-4.5 parts of polyethylene glycol, 0.05-0.1 part of preservative and 0.025-0.06 part of plant essence.
In the preparation of the 18-valent HPV composite yolk neutralization antibody preparation, the bacteriostat is nisin, and the preservative is one or more of potassium sorbate, chlorhexidine gluconate and polyhexamethylene biguanide, wherein:
(1) NISIN (NISIN) acts like a surfactant and acts on both vegetative cells and spores of bacteria. The main action point of NISIN on nutrient cells is cytoplasmic membrane, which inhibits the biosynthesis of peptidoglycan in cell wall, thus preventing the synthesis of cytoplasmic membrane and phospholipid compound, and causing the leakage of cell membrane content, which seriously causes cell lysis, and has obvious antibacterial effect at a concentration of three to five parts per million. The effect on spores is to inhibit their germination during the initial stages of spore expansion. NISIN primarily inhibits most gram positive bacteria, particularly bacterial spores. NISIN also inhibits certain species of staphylococci, streptococci, micrococcus and lactobacilli, and inhibits most spores of clostridium and bacillus.
(2) Polyhexamethylene guanidine is a polyguanidine polymer, can generate ionization in aqueous solution, and the hydrophilic part of the polyhexamethylene guanidine contains strong electropositivity, adsorbs various bacteria and viruses which are usually electronegative, enters cell membranes, inhibits the synthesis of liposome in the membranes, causes the apoptosis of thalli, and achieves the optimal sterilization effect. Is stronger than quaternary ammonium salt cationic surfactant, is also effective to fungi, has no irritation to tissues, and is widely used for skin disinfection, wound washing, and soaking and disinfection of surgical instruments.
(3) The chlorhexidine gluconate has stronger antibacterial effect on gram-positive bacteria than gram-negative bacteria, is stronger than quaternary ammonium salt cationic surfactant, is also effective on fungi, has no irritation to tissues, and is widely used for skin disinfection, wound irrigation and surgical instrument soaking and disinfection.
The invention discloses the following technical effects:
the invention prepares the 18-valent HPV composite yolk neutralizing antibody, which has the following functions:
(1) Blocking genital tract HPV infection, reducing HPV load and preventing cervical lesion; promoting HPV positive to turn negative; auxiliary treatment of cervical cancer;
(2) Blocking HPV infection, and can be used for treating dermatoses caused by HPV infection, and reducing condyloma acuminatum recurrence rate after physical treatment.
The 18-valent HPV composite yolk neutralizing antibody can be prepared into spray, lotion, suppository, gel, effervescent tablet, freeze-dried powder, lozenge and the like, can realize HPV prevention and treatment and auxiliary treatment of cervical cancer, and creates a simple, safe, effective, sanitary and economic personalized prevention and treatment mode of cervical cancer.
According to the invention, clinical trials of people are carried out in three-dimensional hospitals, cervical HPV infection is treated by using the 18-valent HPV composite yolk neutralizing antibody, the negative conversion rate can reach 96.97%, and the 18-valent HPV composite yolk neutralizing antibody can effectively neutralize HPV viruses and further can effectively prevent and treat cervical cancer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of electrophoresis identification of HPV 11L 1 SDS-PAGE;
FIG. 2 is HPV 11L 1 VLP morphology;
FIG. 3 shows the result of SDS-PAGE analysis of the isolated and purified IgY in step (2) of example 3.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1. Gene synthesis, cloning
Only specific amino acid sequences can be expressed and self-assembled to form virus-like particles, so that HPV L1 genes are selected to carry out codon optimization of an insect expression system, after experimental technical routes and methods are designed, recombinant baculovirus Bacmid-L1 of HPV6, 11, 16, 18, 33, 39, 61, 66, 68 and 73L1 is obtained by sending HPV6, 11, 16, 18, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1 insect expression codon optimization gene sequences to Anhui gene technology Co-Ltd according to the designed technical routes and methods, cloning and sequencing and confirming.
HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73L1 protein sequences and insect expression codon optimization sequences are shown in table 1:
TABLE 1
2. Construction of a System for efficient production of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1 VLPs
Sf9 cells were infected with recombinant baculoviruses Bacmid-L1 of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1, respectively. Systems were constructed that efficiently produced HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73l1 VLPs, respectively.
Expression and identification of HPV L1 protein in Sf9 cells:
a. virus infection of Sf9 cells: respectively at 175cm 2 Inoculating 2×10 culture flask 7 And (3) adding 20mL of culture solution into Sf9 cells in logarithmic growth phase, culturing at 27 ℃ for 2 hours to enable the cells to be completely adhered, removing the culture solution, adding 9mL of new culture solution, respectively adding 1mL of recombinant baculovirus Bacmid-L1 virus solution of L1 (MOI is about 0.01-0.1), culturing on a horizontal shaking table at 27 ℃ for 1 hour, and addingCulture medium 20mL,27 ℃ continued to cultivate to 72h.
b. Cell recovery and isolation of nuclei
Cells fuse and shed 72 hours after infection. The mixture was recovered in a centrifuge tube, and centrifuged at 5000g for 5 minutes at 4 ℃. The pelleted cells were washed 2 times with PBS buffer, 5% NP-40 PBS (disruption of cell membranes) was added, and the cells were spun down and ice-washed for 15 minutes. Centrifuge at 4℃for 30 min with 5000 g. And (5) depositing cell nuclei for detection.
c. Protein polyacrylamide gel electrophoresis (SDS-PAGE) identifies L1 expression.
Taking a sample of the cell nucleus to be tested, and identifying the expression conditions of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1 according to conventional SDS-PAGE electrophoresis.
The SDS-PAGE electrophoresis identification of HPV 11L 1 is shown in FIG. 1.
3. Expressing and purifying VLPs to obtain HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1 VLP antigens
A. Sucrose density gradient centrifugation purification of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73l1 VLPs
Respectively at 175cm 2 Inoculating 2×10 culture flask 7 And (3) adding 20mL of culture solution into Sf9 cells in the logarithmic growth phase, culturing at 27 ℃ for 2 hours, enabling the cells to be completely adhered, removing the culture solution, adding 9mL of new culture solution, adding 1mL of recombinant Bacmid-L1 virus solution (MOI is about 0.01-0.1) of HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73 respectively, culturing on a horizontal shaker at 27 ℃ for 1 hour, adding 20mL of culture solution, and continuously culturing at 27 ℃ for 72 hours to obtain the cells. The adherent cells were blown into suspension with 1mL of 1% Triton-X100, transferred to a 1.5mL centrifuge tube, sonicated at 4℃with 50% power for 1s intervals of 5s, and co-disrupted for 1min. After crushing, centrifuging for 10min at the temperature of 1500g and 4 ℃, separating supernatant and sediment, wherein the supernatant is an ultracentrifuge sample. To prevent protein degradation, the disruption process must be performed on ice.
The sucrose solution with the mass concentration of 60%,50%,40%,30% and 20% is added into a 13.2mL ultracentrifuge tube in sequence by taking 1mL each, so as to form a sucrose cushion. The disrupted cell supernatant was added to the uppermost layer of sucrose pad and ultracentrifuged with the parameters: 100 Centrifuge at 000g for 5h. After centrifugation, the sucrose pad was discarded, and 300. Mu.L of the sucrose pad was dispensed from the top to the bottom into a 1.5mL centrifuge tube. 15. Mu.L of the protein preparation was taken and the purification effect of sucrose density gradient centrifugation was examined by SDS-PAGE.
B. Virus-like particle assembly morphology detection
2% preparation of phosphotungstic acid: 0.2g of phosphotungstic acid powder was weighed and dissolved in 10mL of phosphate buffer solution, and 1mM NaOH was added dropwise to adjust the pH to 6.4-7.0.
And carrying out negative dyeing sample preparation on the virus-like particles obtained by purification through phosphotungstic acid dye liquor, and then observing the assembly morphology under a transmission electron microscope. And (3) clamping the copper mesh on a silica gel plate with the right side upwards, dripping a drop of sample to cover the copper mesh, placing the copper mesh in a ventilation kitchen for ventilation for 10min, and sucking the redundant residual liquid by using filter paper when the copper mesh is in a semi-dry state. And (3) dropwise adding a drop of 2% phosphotungstic acid to cover the copper mesh, carrying out negative dyeing for 2min, and sucking the excessive dyeing liquid attached to the copper mesh by using filter paper. Finally, the mixture is washed three times by distilled water, and can be observed by an electron microscope after being dried.
The morphological detection results of HPV 11L 1 VLPs are shown in figure 2.
Example 2
HPV L1 VLP protein immunization:
viroid VLPs can mimic the natural state of a virus, inducing neutralizing antibodies. The HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73l1 VLP antigens prepared in example 1 were used to immunize egg-laying hens, respectively, by: individual VLP antigens were taken 50 μg each time, emulsified by adding adjuvant ISA 70 (antigen: adjuvant=30:70 by volume ratio), and injected intramuscularly into egg-laying hens, 1mL each, at 3 spots. The initial immunization was performed 3 times at 28 days intervals, and eggs were collected 14 days after the 3 rd immunization to extract IgY antibodies. The immunization was then boosted once every 2 months, and the antigen dose and immunization program were the same as the initial immunization.
Example 3
The steps of IgY antibody extraction and purification are as follows:
(1) Mixing eggs of egg laying hens immunized by HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1 VLP antigens respectively according to equal proportion, separating by a yolk sieve to obtain yolk, adding distilled water 4 times (4-8 times can achieve the same effect), stirring and mixing uniformly to obtain yolk liquid, regulating pH to 5.2 (5.1-6.0 can achieve the same effect) by using 1M HCl, and standing and precipitating at 4 ℃ overnight.
(2) The yolk liquid was centrifuged at 8000rpm for 20 minutes and the pellet was discarded. Collecting supernatant, slowly dripping n-octanoic acid into the supernatant until the n-octanoic acid content is 1wt% (up to 2 wt%) and stirring and mixing uniformly. Standing at 4deg.C for 2 hr, centrifuging at 4deg.C at 8,000 rpm (up to 12000 rpm) for 30min, and collecting supernatant for ultrafiltration concentration.
Ultrafiltration concentration: diatomite with a final concentration of 3wt% was added as a filter aid, and vacuum filtration (0.09 MPa) was performed using a 0.45 μm mixed cellulose ester film to obtain a filtrate. 5 ten-thousandth of ultrafiltration membrane with retention amount is selected, the materials are fed at room temperature, and the extract is subjected to ultrafiltration concentration in a single-stage intermittent operation mode, so that the concentrated IgY is obtained. Pasteurization, 0.22 μm membrane filtration sterilization, the isolated purified IgY.
(3) And (3) freeze-drying: and (3) freeze-drying the separated and purified IgY obtained in the step (2) by adopting 2% of sucrose, 3% of glycine, 0.5% of Tween 80 and 0.2% of gelatin as protective agents.
Lyophilization operation:
a) Pre-freezing: firstly, placing a container bottle filled with IgY antibody solution on a partition board of a freeze dryer, pre-freezing at a set temperature of 5 ℃ for 30min to reach the set temperature, maintaining for 1h, cooling to-60 ℃ for 10min to reach the set temperature, and maintaining for 6h for freezing;
b) Sublimation drying: heating the set temperature of the baffle system to-25 ℃ for 10 hours to reach the preset temperature, maintaining for 25 hours, and simultaneously maintaining the vacuum degree of 0.2m Bar to finish sublimation drying;
c) And (5) drying: and (3) heating the temperature of the separator system to 30 ℃, setting the temperature to be the preset temperature for 100min, maintaining the temperature for 10h for drying, and simultaneously maintaining the vacuum degree of 0.2m Bar, and ending the freeze-drying. Namely, isolated and purified HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1 egg yolk neutralization antibody Igys freeze-dried powder.
Detection of Igys lyophilized powder: antibody IgY titers were measured by ELISA (Shanghai Bowmember Biotechnology Co., ltd.). Measuring IgY concentration: total protein content= (OD 280 1.4); igY content = total protein content x IgY purity.
Through detection, the IgYs freeze-dried powder prepared in the embodiment has the content of 500mg IgY/g freeze-dried powder and the purity of 50 percent.
SDS-PAGE electrophoretic analysis of IgY antibodies:
SDS-PAGE analysis is carried out on the IgY separated and purified in the step (2).
Sample preparation: 3% concentrated gel, 8% separation gel, 8. Mu.L spot, 80V for 60 min. The electrophoresis was stopped when the indicator reached 1-2cm in front of the separation gel. Coomassie brilliant blue staining, decolorizing. The results are shown in FIG. 3, where the heavy chain (H) is 67-70KD and the light chain (L) is 22-30KD.
ELISA titers analysis of IgY antibodies:
HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73L1 egg yolk neutralization antibody IgY titers were determined by ELISA.
ELASA method:
(1) The antigen was diluted to 5. Mu.g/mL with ELISACoating Buffer. 100 μl was added to each well and coated overnight at 4deg.C or for two hours at room temperature.
(2) 3% BSA/TBS was blocked, 200. Mu.L per well was added, and the reaction was allowed to stand at room temperature for 1 hour.
(3) TBST was washed three times.
(4) Except for row H, 100 mu L of 1% BSA/TBST is added into each hole, igY samples are diluted according to the recommended concentration, then the diluted samples are diluted in half, 200 mu L of sample diluent is added into each hole of row H, 100 mu L of diluent is taken from each hole of row H to row G, the mixture is uniformly mixed and then sequentially diluted to row B, and 100 mu L of diluent is sucked after the mixture of row B is uniformly mixed. Row a served as negative control. Incubate for two hours at room temperature.
(5) TBST washing three times
(6) The secondary antibody was diluted with 1% BSA/TBST and 100. Mu.L per well. Incubate for one hour at room temperature.
(7) TBST was washed five times.
mu.L of substrate solution (10mL Citrate Buffer,0.4mLTMB,33. Mu.L 3%H) was added to each well 2 O 2 ) Mixing.
(8) Adding stop solution (1M HCl or 1M H) into each hole when it changes color 2 SO 4 ) 50. Mu.L. The time taken for the discoloration to occur is 2-5 minutes.
(9) OD was measured and recorded at a wavelength of 450 nm.
The samples OD/negative control average OD >2.1 was positive, and the highest dilution when the samples were positive was ELISA titer.
Results: the IgY titers of the HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73 egg yolk neutralizing antibodies are respectively: 2 12 ，2 14 ，2 13 ，2 12 ，2 13 ，2 14 ，2 13 ，2 14 ，2 14 ，2 12 ，2 13 ，2 13 ，2 14 .2 14 ，2 12 ，2 13 ，2 12 ，2 14 。
Example 4
Antibody neutralization activity assay
The neutralizing activity of the antibodies was tested by a pseudovirus-cell neutralization model.
The monogenotype HPV6 IgY antibodies were diluted to 40. Mu.g/mL with PBS, then the antibodies were subjected to a 2-fold gradient dilution to 0.09765625. Mu.g/mL, 50. Mu.L of each concentration of antibody was incubated with 50. Mu.L of HPV6 pseudovirus (100 TCID 50/50. Mu.L) in 96-well plates for one hour at 4℃and negative, positive, cell and pseudovirus controls were set. Each mixture was then individually placed in 96-well plates pre-plated with 293FT cells and cultured in a cell incubator for 72 hours. And then observing the fluorescence condition, collecting cells, detecting fluorescence by using a flow cytometry, and if the cells have an inhibition effect, calculating the fluorescence inhibition rate according to the fluorescence inhibition rate= (1-experimental group/control group) ×100%, and taking the fluorescence inhibition rate which is more than 90% as the neutralization titer of the IgY on pseudoviruses. The result shows that the antibody has an inhibition effect on HPV6 pseudovirus, and the 90% inhibition rate concentration is 10 mug/mL; none of the HPV11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73 pseudoviruses was inhibited. The HPV 6L 1 IgY antibody is shown to be an HPV6 virus neutralizing antibody with high titer and good specificity.
Simultaneously carrying out neutralization experiments of HPV11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68 and 73HPV11, HPV16, HPV18, HPV30, HPV31, HPV32, HPV35, HPV42, HPV43, HPV44, HPV51, HPV52, HPV53, HPV54 and HPV 55L 1 egg yolk neutralization antibodies IgY respectively, the concentration of the inhibition rate of 90 percent is 10 percent respectively 10, 10 10, 20, 10, 20 μg/mL.
Example 5
Preparation of an IgY spray of an 18-valent HPV virus yolk neutralization antibody:
TABLE 2
The raw material formula is shown in table 2, and the preparation process is as follows:
(1) Sterilizing pharmaceutical glycerol, herba Menthae essence and flos Rosae Rugosae essence with ultraviolet light for 24 hr, and sealing;
(2) Heating the formula amount of distilled water to 90 ℃ and staying for 15 minutes; cooling to 60 ℃, adding pharmaceutical grade glycerol and chlorhexidine gluconate while stirring, stirring at low speed for 30 minutes until the components are completely dissolved, and cooling to room temperature to obtain solution A;
(3) Adding essence (flos Rosae Rugosae essence) into the solution A under stirring, stirring at low speed for 60min until completely dissolving to obtain solution B;
(5) Slowly dripping herba Menthae essence into solution B under stirring, and stirring at low speed for 60min;
(8) Slowly adding Igys freeze-dried powder, lysozyme and NISIN into the solution B while stirring, and stirring at low speed for 60min; obtaining a solution C;
(9) Measuring the pH value of the solution C by a pH meter, regulating the pH value to 4.5+/-0.1 by using citric acid (or disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4.5), packaging in a small portable spray bottle after cleaning and disinfection, labeling and leaving a factory.
The IgY spray of the 18-valent HPV virus yolk neutralization antibody prepared in the embodiment is sprayed on genitals and after sexual intercourse, and is sprayed on male and female genitals for preventing HPV infection and cervical cancer.
Example 6
Preparation of an IgY oral lozenge of an 18-valent HPV virus yolk neutralization antibody:
TABLE 3 Table 3
The raw material formulation is shown in table 3, and the preparation process is as follows:
the preparation method comprises the steps of carrying out microwave sterilization on raw materials except IgYs freeze-dried powder, mixing the raw materials subjected to microwave sterilization and the IgYs freeze-dried powder according to a proportion, and carrying out dry granulation, tabletting and microwave sterilization to obtain an 18-valent HPV virus yolk neutralizing antibody IgY oral lozenge, wherein each tablet is 0.38 g, and the IgY oral lozenge is used for treating HPV infection in an oral cavity.
Example 7
Preparation of IgY liposome liquid crystal microcapsule of 18-valent HPV virus yolk neutralization antibody:
TABLE 4 Table 4
The raw material formulation is shown in table 4, and the preparation process is as follows:
(1) Mixing lecithin and cholesterol, and then according to the mass ratio of 1:5, dissolving in diethyl ether to obtain lecithin and cholesterol diethyl ether solution;
(2) IgYs freeze-dried powder is prepared according to the mass ratio of 1:100, adding 4mmol/L Phosphate Buffer (PBS) to prepare an antibody solution with the concentration of 1%;
(3) The lecithin and cholesterol ether solution in the step (1) and the antibody solution in the step (2) are mixed according to the volume ratio of 3:1, mixing uniformly in proportion;
(4) Ultrasonic treatment for 2min (each treatment for 0.5min, and interval for 0.5 min);
(5) Rotary evaporating in water bath under reduced pressure until gel is formed, oscillating with vortex to allow gel phase inversion, and evaporating to remove diethyl ether;
(6) Ultracentrifugation (35000 r/min,30 min) to separate out non-entrapped antibodies;
(7) Washing the precipitate with water twice, and centrifuging in a high-speed centrifuge to obtain precipitate;
(8) Diluting the obtained precipitate with 10mmol/LPBS for 5 to obtain the IgY liposome liquid crystal microcapsule of the 18-valent HPV virus yolk neutralization antibody.
Example 8
Preparation of IgY vaginal liposome gel of 18-valent HPV virus yolk neutralization antibody:
TABLE 5
The raw material formulation is shown in Table 5, and the preparation process is as follows:
adding disodium edentate, carbomer 974P, polycarbophil AA-1, chlorhexidine gluconate, rose essence and glycerin into purified water, heating, stirring uniformly and fully swelling, adding benzyl alcohol and IgY liposome liquid crystal microcapsule of the 18-valent HPV virus egg yolk neutralization antibody, fully and uniformly stirring when cooling to room temperature, finally adding triethanolamine, and stirring while adding to obtain homogeneous gel, thus obtaining the 18-valent HPV virus egg yolk neutralization antibody IgY vaginal liposome gel.
The prepared IgY vaginal liposome gel of the 18-valent HPV virus yolk neutralization antibody is filled into a disposable vaginal washer in a volume of 3 mL/branch. Is administered at cervical part for preventing HPV infection and assisting in treating cervical cancer.
Example 9
Preparation of an IgY external washing liquid of an 18-valent HPV virus yolk neutralization antibody:
TABLE 6
The raw material formulation is shown in Table 6, and the preparation process is as follows:
(1) Sterilizing pharmaceutical glycerin, peppermint essence, osmanthus essence and 403 and 503 foaming agents according to the formula amount by ultraviolet irradiation for 24 hours, and aseptically sealing for later use;
(2) Heating the formula amount of distilled water to 90 ℃ and staying for 15 minutes; then cooling to 60 ℃, adding medical grade glycerol while stirring, stirring at a low speed for 30 minutes until the medical grade glycerol is completely dissolved, and cooling to room temperature to obtain solution A;
(3) Adding osmanthus essence into the solution A while stirring, and stirring at a low speed for 60min until the osmanthus essence is completely dissolved to obtain a solution B;
(4) Heating 403 to 80deg.C, adding 503 dropwise into 403 under stirring, and stirring at low speed for 30min to obtain solution C;
(5) Maintaining the temperature of the solution C at 80 ℃, slowly adding peppermint oil into the solution C while stirring, and stirring at a low speed for 60min to obtain a solution D;
(6) Heating the solution D to 90 ℃ for high-temperature sterilization for 5min, and then cooling to room temperature;
(7) Slowly adding the solution B into the solution D while stirring, and stirring at a low speed for 60min; if the homogeneous stable emulsion solution is not formed, the stirring time is prolonged to prepare solution E;
(8) Slowly adding IgYs freeze-dried powder into the solution E while stirring, and stirring at a low speed for 60min; if a homogeneous stable emulsion solution is not formed, the stirring time is prolonged to prepare a solution F;
(9) Measuring the pH value of the solution F by a pH meter, regulating the pH value to 4.5+/-0.1 by using citric acid (or disodium hydrogen phosphate-citric acid buffer solution) and standing for one night until the upper foam is completely dissolved, then packaging in a plastic container after cleaning and sterilizing, and labeling for delivery.
Can be used for daily cleaning or vaginal douche of male and female genital organs, and for preventing and treating HPV infection and cervical cancer.
Example 10
Preparation of 18-valent HPV yolk neutralization antibody IgY suppository:
TABLE 7
The raw material formulation is shown in Table 7, and the preparation process is as follows:
heating purified water to 90deg.C, adding gelatin, swelling, cooling, maintaining at 60deg.C, adding glycerol, stirring, adding mannitol, stirring to dissolve, cooling to below 40deg.C, adding IgYs lyophilized powder, adding polyhexamethylene biguanide, adding flos Osmanthi Fragrantis essence and herba Menthae essence, stirring at low speed, pouring into suppository mold, standing at conventional low temperature, taking out, and plastic packaging.
The product is used for female vagina/genital tract, and the yolk neutralizing antibody IgY of 18 HPV in suppository is specifically combined with 18 HPV viruses causing cervical cancer, so as to effectively block further infection of HPV viruses, treat HPV infection and assist in treating cervical cancer.
Example 11
Preparation of 18-valent HPV virus yolk neutralization antibody IgY freeze-dried powder preparation:
TABLE 8
The raw material formulation is shown in table 8, and the preparation process is as follows:
the IgYs freeze-dried powder, bovine serum albumin and trehalose are fully dissolved in purified water, then polyhexamethylene biguanide, osmanthus fragrans essence and peppermint essence are added, after being uniformly mixed, 3 mL/branch is put in a penicillin bottle, freeze-dried is carried out for 4 hours under the pressure of 5.0mBar, and the mixture is sealed for preservation.
The product is used for cleaning female vagina in daily home or medical institution, 3mL physiological saline is added for full dissolution, and the soft tube is injected into vagina for cleaning, wherein 18-valent HPV virus yolk neutralization antibody IgY is specifically combined with 18 induced condyloma acuminatum HPV viruses, so that further infection of HPV viruses is effectively blocked, HPV viruses are discharged out of the body, the purpose of turning negative is achieved, cervical HPV infection is treated, and cervical cancer is treated in an auxiliary way.
Example 12
Preparation of an 18-valent HPV virus yolk neutralization antibody IgY effervescent tablet:
TABLE 9
The raw material formulation is shown in Table 9, and the preparation process is as follows:
(1) And (3) ultrasonically dissolving the hydroxypropyl-beta-cyclodextrin in purified water, adding polyethylene glycol 6000, and performing ultrasonic treatment, and stopping ultrasonic treatment when the particle size distribution in the aqueous solution is within the range of 200-500 nm to obtain an auxiliary material solution.
(2) Slowly adding IgYs lyophilized powder, chlorhexidine gluconate, flos Osmanthi Fragrantis essence and herba Menthae essence into the adjuvant solution, and stirring with high-speed disperser at 15000 rpm for 30min to obtain intermediate 1.
(3) And (3) dehydrating the intermediate 1 by adopting a nanofiltration membrane with the aperture of 800Da to obtain a concentrated solution, drying at 50 ℃, adding citric acid, sodium bicarbonate and magnesium stearate according to an equivalent incremental method, sieving, mixing uniformly, and tabletting to obtain the 18-valent HPV virus yolk neutralizing antibody IgY effervescent tablet.
The product is used for female vaginal/genital tract filling, preventing and treating cervical HPV infection, is convenient to use, and the 18-valent HPV virus yolk neutralizing antibody IgY in the effervescent tablet can be specifically combined with 18 HPV viruses causing cervical cancer, effectively blocks HPV virus infection to prevent, and can also assist in treating cervical cancer.
Effect verification example 1
Clinical tests of the 18-valent HPV virus yolk neutralization antibody IgY for preventing and treating cervical HPV infection are carried out in three-kingdom 3 in China.
(1) Test subjects
262 HPV positive patients were selected, aged 23-58 years.
Inclusion criteria included: patients with transient, persistent or recurrent infections, including patients with partial mild cytologic changes (less than CIN II), with rejection of patients above CIN III, with cervical cancer, and post cervical cancer surgery.
(3) Test procedure
The subjects were divided into an experimental group and a control group, and effervescent tablets (132 cases) and interferon preparations (130 cases) prepared in example 11 were used, respectively.
Sample collection: sampling is carried out on the patient in a non-menstrual period, and intravaginal medication is avoided before sampling; sampling by using a special disposable cervical cell sampling system for obtaining the certification of a drug administration, and sampling by using a cotton swab is contraindicated; the sample required for detection is cervical exfoliated cells instead of cervical secretions, and a cotton swab is required to be used for wiping the cervical secretions clean before sampling, so that excessive blood and mucus in the sample are avoided as much as possible; sampling was performed according to manufacturer's instructions for a disposable cervical cell sampling system.
Treatment cycle: the effervescent tablet prepared in example 11 was used in the experimental group for 3 months, and the product was used for 21 days continuously, 1 tablet per day, and 0.5g each month except for the menstrual period and 1 day before and after the menstrual period. Review after 4 weeks of disablement. The control group was given a preparation of interferon a-2b for 3 months and was given a review after 4 weeks of discontinuation.
The curative effect observation index is as follows: HPV 21 typing and quantitative detection system of Jiangsu Shuoshi biotechnology Co., ltd is adopted to detect HPV positive patients before and after treatment, and the condition of HPV positive to negative is judged and recorded.
(3) Conclusion(s)
Clinical results of experimental groups show that the number of negative turning cases is 128, the number of negative turning cases is 4, and the negative turning rate is 96.97%; the control group had 45 cases of negative turning, 85 cases of negative turning, and a negative turning rate of 34.6%.
(4) Adverse reactions
Experimental group: adverse reactions are not seen, but are forbidden for the people allergic to the eggs.
Control group: a small number of female patients may develop symptoms such as vaginal discomfort, swelling, burning pain, itching, vaginal tingling, running water, and the like, and sometimes symptoms such as nausea, vomiting, and the like are accompanied.
Interferon is currently widely used in clinic as an antiviral and antitumor agent for commonly treating HPV viruses. However, interferon production is particularly slow, so that the use period is usually 2 to 6 months. The effective rate after treatment is only about 50 percent. The toxic and side effects of interferon are common, the most common are fever and influenza-like syndrome, patient weight loss, alopecia, emotional agitation, hematopoiesis and thrombocytopenia caused by bone marrow suppression, mild anemia, occasionally nervous system injury, endocrine system function influence and interferon antibody generation.
Therefore, the yolk neutralizing antibody IgY of the 18-valent HPV virus has an important effect on the positive-to-negative conversion of HPV infected patients, and can be used for preparing medicaments for treating HPV infection.
At most, the similar products on the market contain 9-valent HPV IgY antibodies, cervical cancer is infected by 18 types of HPV, the products are incompletely covered on HPV and can leak a part of HPV, the product contains 18-valent HPV IgY antibodies, completely covers 18 types of HPV which cause cervical cancer, cannot leak any genotype which causes cervical cancer, and is suitable for any crowd infected with different HPV genotypes related to cervical cancer.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (10)

1. A method for preparing an antigen combination for an 18-valent HPV composite yolk neutralizing antibody, comprising the steps of: the method comprises the steps of selecting and carrying out expression and self-assembly on genes of HPV 6L 1 protein, HPV 11L 1 protein, HPV 16L 1 protein, HPV 18L 1 protein, HPV 31L 1 protein, HPV 33L 1 protein, HPV 35L 1 protein, HPV 39L 1 protein, HPV 45L 1 protein, HPV 51L 1 protein, HPV 52L 1 protein, HPV 56L 1 protein, HPV58L1 protein, HPV 59L 1 protein, HPV 61L 1 protein, HPV 66L 1 protein, HPV 68L 1 protein and HPV 73L 1 protein with coding amino acid sequences shown as SEQ ID NO.1-18 respectively by using a eukaryotic expression system, separating and purifying to obtain 18 types of virus particles, namely the antigen combination.
2. The method of claim 1, wherein the eukaryotic expression system is a baculovirus-insect cell expression system;
the encoding genes of the HPV 6L 1 protein, the HPV 11L 1 protein, the HPV 16L 1 protein, the HPV 18L 1 protein, the HPV 31L 1 protein, the HPV 33L 1 protein, the HPV 35L 1 protein, the HPV 39L 1 protein, the HPV 45L 1 protein, the HPV 51L 1 protein, the HPV 52L 1 protein, the HPV 56L 1 protein, the HPV 58L 1 protein, the HPV 59L 1 protein, the HPV 61L 1 protein, the HPV 66L 1 protein, the HPV 68L 1 protein and the HPV 73L 1 protein, which are optimized by insect cell expression codons, are respectively shown in SEQ ID NO. 19-36.
3. An antigen combination prepared according to the preparation method of claim 1 or 2.
4. Use of the antigen combination of claim 3 for the preparation of an 18-valent HPV composite yolk neutralising antibody.
5. The preparation method of the 18-valent HPV composite yolk neutralizing antibody is characterized by comprising the following steps of:
s1, respectively immunizing different laying hens by using 18 kinds of virus particles in the antigen combination of claim 3 as antigens, and obtaining 18 egg yolk from eggs produced by the immunized laying hens;
S2, mixing the 18 yolk, and extracting a yolk neutralization antibody to obtain the 18-valent HPV composite yolk neutralization antibody.
6. The method according to claim 5, wherein in step S2, the step of extracting comprises: mixing the 18 yolk, adding water, stirring uniformly to obtain yolk liquid, regulating the pH to 5.1-6.0, standing, precipitating, centrifuging to obtain supernatant, removing mixed proteins and lecithin, concentrating, and drying to obtain the 18-valent HPV composite yolk neutralizing antibody.
7. An 18-valent HPV composite yolk neutralising antibody prepared according to the method of claim 5 or 6.
8. Use of the 18-valent HPV composite yolk neutralizing antibody of claim 7 in the manufacture of a medicament for the prevention of HPV infection or the adjuvant treatment of cervical cancer.
9. A medicament for preventing HPV infection or aiding in the treatment of cervical cancer, comprising the 18-valent HPV complex egg yolk neutralizing antibody of claim 7.
10. The medicament according to claim 9, wherein the dosage form of the medicament comprises a spray, an oral tablet, a liposome liquid crystal microcapsule, a vaginal liposome gel, a lotion for external use, a suppository, a lyophilized powder preparation or an effervescent tablet;
When the medicine is a spray, the raw materials comprise the following components in parts by weight: the 18-valent HPV composite yolk neutralizing antibody of claim 6, 0.1-0.8 parts, bacteriostat 0.003-0.04 parts, glycerin 0.1-12 parts, preservative 0.04-0.25 parts, lysozyme 0.05-1 parts and plant essence 0.01-0.16 parts;
when the medicine is an oral lozenge, the raw materials comprise the following components in parts by weight: the 18-valent HPV composite yolk neutralizing antibody of claim 6, 0.15-0.8 parts, 0.8-7 parts of citric acid, 4-38 parts of xylitol, 38-52 parts of starch, 0.03-0.7 parts of preservative and 0.2-0.6 parts of water-soluble plant essence;
when the medicine is liposome liquid crystal microcapsule, the raw materials comprise the following components in parts by weight: the 18-valent HPV composite yolk neutralizing antibody of claim 6, 0.5-2.5 parts, lecithin 3-7.5 parts, and cholesterol 12-30 parts;
when the medicine is vaginal liposome gel, the raw materials comprise the following components in parts by weight: 3-15 parts of liposome liquid crystal microcapsule, 0.08-1 part of edetate disodium, 0.2-2 parts of carbomer 974P, 10.03-0.6 parts of polycarbophil AA-12 parts of glycerol, 0.04-0.25 part of preservative, 0.4-10 parts of benzyl alcohol, 0.3-1.4 parts of triethanolamine and 0.01-0.05 part of plant essence;
When the medicine is an external washing lotion, the raw materials comprise the following components in parts by weight: the 18-valent HPV composite yolk neutralizing antibody of claim 6, 0.1-0.8 parts, 28-37 parts of fatty alcohol-polyoxyethylene ether sulfosuccinate monoester disodium, 16-25 parts of fatty alcohol-polyoxyethylene ether ammonium sulfate, 1.0-8 parts of glycerin, 0.04-0.25 parts of preservative and 0.016-0.13 parts of plant essence;
when the medicine is suppository, the raw materials comprise the following components in parts by weight: the 18-valent HPV composite yolk neutralizing antibody of claim 6, 0.1-0.8 parts, gelatin 8-12 parts, glycerin 20-37 parts, mannitol 25-41 parts, preservative 0.05-0.2 parts and plant essence 0.02-0.1 parts;
when the medicine is a freeze-dried powder preparation, the liquid raw materials of the freeze-dried powder preparation comprise the following components in parts by weight: the 18-valent HPV composite yolk neutralizing antibody of claim 6, 0.1-0.8 parts, bovine serum albumin 0.009-0.18 parts, trehalose 1.5-13 parts, preservative 0.04-0.2 parts and plant essence 0.016-0.12 parts;
when the medicine is an effervescent tablet, the raw materials comprise the following components in parts by weight: the 18-valent HPV composite yolk neutralizing antibody of claim 6, 0.4-0.6 parts, hydroxypropyl-beta-cyclodextrin 30-34 parts, citric acid 13-16 parts, magnesium stearate 2-4 parts, sodium bicarbonate 35-39 parts, polyethylene glycol 60003.5-4.5 parts, preservative 0.05-0.1 parts and plant essence 0.025-0.06 parts.
CN202310563435.6A 2023-05-18 2023-05-18 18-valent HPV composite yolk neutralizing antibody for preventing and treating cervical HPV infection, and preparation method and application thereof Pending CN116554280A (en)

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