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CN-122011168-A - Preparation method and application of anti-human papilloma virus IgY antibody

CN122011168ACN 122011168 ACN122011168 ACN 122011168ACN-122011168-A

Abstract

The invention relates to the technical field of IgY antibodies, in particular to a preparation method and application of an anti-human papillomavirus IgY antibody, comprising the following steps of S1, preparation of an immunogen, S2, step immunization, S3, collection and pretreatment of high-immunity eggs, collection of eggs laid by immunized hens from 18 to 25 days after primary immunization, surface disinfection, taking yolk liquid for standby, primary extraction of S4 and antibodies, primary concentration and purification of S5 and antibodies, fine purification of S6 and antibodies, preparation treatment of S7 and antibodies, and full play of cross protection potential of high immunogenicity of L1 VLPs by adopting an 'HPV L1 virus-like particle (VLP)' immunization strategy, and the invention is matched with Gao Xiaozuo agent systems to effectively induce birds to produce high-titer neutralizing antibodies with wide cross reactivity, thereby overcoming the defects of limited single antigen targeting type and narrow protection range.

Inventors

  • HE YUQIANG
  • LI ZHANG
  • LI JIAO
  • ZHANG MINGQI
  • LIU YINRONG

Assignees

  • 山东中大基因科技有限公司

Dates

Publication Date
20260512
Application Date
20260204

Claims (10)

  1. 1. The preparation method and application of the anti-human papilloma virus IgY antibody are characterized by comprising the following steps: S1, preparing an immunogen, namely performing first emulsification treatment on a human papilloma virus antigen and Freund 'S complete adjuvant to obtain a primary immunogen, and performing second emulsification treatment on the HPV antigen and Freund' S incomplete adjuvant to obtain a booster immunogen; S2, step type immunization: primary immunization, namely inoculating the primary immunogen to egg laying hens in a multipoint injection mode, wherein the injection dose of each hen is 0.8-1.2ml, the number of injection points is 20-80, and the injection points comprise subcutaneous tissues and muscle tissues; The first boosting, namely inoculating the boosting immunogen to the same egg laying hen in a multipoint injection mode on the 12 th to 16 th days after the first boosting, wherein the injection dose of each hen is 0.8 to 1.2ml, and the injection point number is 15 to 60 points; Serial boosting, repeating the boosting every 12-16 days for 2-4 times; maintaining immunity, namely performing boosting immunity on the egg laying hen every 6-10 weeks after finishing series boosting immunity, and maintaining the immune period to be not less than 24 weeks; S3, collecting and preprocessing hyperimmune eggs, namely collecting eggs laid by immunized hens from the 18 th to the 25 th days after primary immunization, and taking yolk liquid for later use after surface sterilization; S4, preliminary extraction of the antibody, namely mixing the yolk liquid and the diluent according to the volume ratio of 1:4 to 1:15, uniformly stirring, regulating the pH value of the mixed liquid to 4.8-5.5 by using acid liquor, standing for 6-24 hours at 2-8 ℃, centrifuging for 10-30 minutes at the temperature of 2-8 ℃ and the temperature of 8000-15000 Xg, and collecting a first supernatant; s5, carrying out preliminary concentration and purification on the antibody, namely carrying out ultrafiltration treatment on the first supernatant through an ultrafiltration membrane with the molecular weight cutoff of 30kDa to 300kDa, concentrating by 5-30 times, and replacing the concentrated supernatant into a neutral buffer solution to obtain concentrated antibody liquid; S6, fine purification of the antibody, namely purifying the concentrated antibody liquid sequentially through at least one chromatographic column, wherein the chromatographic column is selected from an affinity chromatographic column, an ion exchange chromatographic column and a size exclusion chromatographic column; and S7, preparing an antibody, namely sequentially passing the finely purified antibody solution through a filter membrane with the diameter of 0.45 mu m and a filter membrane with the diameter of 0.22 mu m for sterilization and filtration, then adding a stabilizer and a preservative, and adjusting the pH value to 6.5-7.8.
  2. 2. The method for preparing anti-human papillomavirus IgY antibody and application of the anti-human papillomavirus IgY antibody of claim 1, wherein HPV antigen in the step S1 is selected from HPV virus-like particles, recombinant expression HPVL1 protein, recombinant expression HPVE6 protein and recombinant expression HPVE7 protein, and the first emulsification treatment and the second emulsification treatment adopt a double-syringe butt-joint injection method until stable emulsion which is not diffused in water is formed.
  3. 3. The method for preparing anti-human papillomavirus IgY antibody and the application thereof according to claim 1, wherein the step S2 further comprises an immunization monitoring step of collecting serum or yolk samples of immunized chickens before and after each immunization for 7-10 days, measuring the anti-HPV specific antibody titer by an ELISA method, dynamically adjusting the time of subsequent boosting according to an antibody titer change curve, and executing the next boosting when the antibody titer is increased into a plateau phase.
  4. 4. The preparation method and application of the anti-human papillomavirus IgY antibody according to claim 1, wherein the diluent in the step S4 is deionized water, phosphate buffer, tris-HCl buffer or acetic acid-sodium acetate buffer, and the acid solution is 0.5-2.0N hydrochloric acid solution or acetic acid solution.
  5. 5. The preparation method and application of the anti-human papillomavirus IgY antibody of claim 1, wherein the neutral buffer solution in the step S5 is phosphate buffer solution or Tris-HCl buffer solution with pH of 7.0-7.5 and the concentration is 10-100mM, and the ultrafiltration treatment is carried out in an environment of 2-8 ℃ and adopts a tangential flow filtration mode.
  6. 6. The method for preparing anti-human papillomavirus IgY antibody and the application of the anti-human papillomavirus IgY antibody according to claim 1, wherein the fine purification in the step S6 adopts one of the following three chromatographic paths: the concentrated antibody liquid sequentially flows through an affinity chromatographic column and a size exclusion chromatographic column; the concentrated antibody liquid sequentially flows through an ion exchange chromatographic column and a molecular exclusion chromatographic column; and a third path, wherein the concentrated antibody liquid sequentially flows through an affinity chromatographic column, an ion exchange chromatographic column and a size exclusion chromatographic column.
  7. 7. The preparation process and application of one kind of anti-human papillomavirus IgY antibody of claim 6, wherein the affinity chromatographic column has ligand of recombinant protein G and recombinant protein L, and the affinity elution adopts glycine-hydrochloric acid buffer solution of pH2.5-3.5, and the affinity elution is neutralized with Tris buffer solution of pH 8.0-9.0.
  8. 8. The preparation process and application of one kind of anti-human papillomavirus IgY antibody of claim 6, wherein the ion exchange chromatographic column is one kind of anion exchange chromatographic column with quaternary ammonium salt as medium and the elution is one linear buffer solution of pH7.0-8.5 containing 0-1.0M sodium chloride.
  9. 9. The preparation process and application of one kind of anti-human papillomavirus IgY antibody of claim 6, characterized in that the molecular exclusion chromatographic column has separation range of 10-1000kDa and the eluting solution is phosphate of pH6.5-7.5 containing 100-200mM sodium chloride and flow rate of 0.5-1.5ml/min.
  10. 10. The method for preparing anti-human papillomavirus IgY antibody and the application of the anti-human papillomavirus IgY antibody according to claim 1, wherein the stabilizer in the step S7 is one or more selected from trehalose, sucrose, mannitol, glycerol and human serum albumin, the mass volume concentration of the stabilizer in a final antibody solution is 0.5-10%, and the preservative is one or more selected from thimerosal, phenol, m-cresol and chlorhexidine gluconate, and the mass volume concentration of the stabilizer in the final antibody solution is 0.01-0.5%.

Description

Preparation method and application of anti-human papilloma virus IgY antibody Technical Field The invention relates to the technical field of IgY antibodies, in particular to a preparation method and application of an anti-human papillomavirus IgY antibody. Background Human Papillomavirus (HPV) infection is a main cause of various diseases such as cervical cancer, genital warts and the like, and the incidence rate is continuously high in the global scope, so that public health safety is seriously threatened; the prior main prevention and control means comprise preventive vaccines and local physical/chemical treatment, but the prior preventive vaccines cannot cover all high-risk types and have limited actions on infected people, and local administration of interferon and the like has the defects of unstable effect, potential side effect, higher cost and the like, in recent years, the prior art lacks a multi-target immunogen design and screening system capable of efficiently inducing a broad spectrum, high neutralizing antibodies by using specific antigens to immunize egg laying birds, extracts high-titer specific antibodies (IgY) from yolk thereof as an emerging passive immunization strategy, shows high safety in the field of virus prevention and control, is easy to realize large-scale production, can be designed against specific pathogens and the like, however, the technology still faces a series of key bottlenecks in the transformation process of HPV prevention and control, firstly, the prior art lacks a multi-target immunogen design and screening system capable of efficiently inducing the broad spectrum, high neutralizing antibodies by using L1 virus-like particles (VLP) can induce the specific antibodies, but is difficult to generate wide cross protection covering various types, secondly, the prior art lacks a traditional bird immunization scheme has strong random performance, is based on animal immune response step-like, has scientific screening capability and has high-titer, has high toxicity, high purity, high toxicity, high activity, high purity, high activity and high purity, can not be extracted by the antibodies, and has relatively high toxicity, and high toxicity, can be extracted by the method has relatively high toxicity, and high toxicity, seriously affecting the safety and effectiveness of subsequent preparation development and clinical application. Therefore, we make improvements to this, and put forward a preparation method and application of anti-human papillomavirus IgY antibody. Disclosure of Invention The invention aims to provide a preparation method and application of an anti-human papillomavirus IgY antibody, so as to solve the problems in the background technology. In order to achieve the above purpose, the present invention provides the following technical solutions: The method comprises the following steps: S1, preparing an immunogen, namely performing first emulsification treatment on a Human Papilloma Virus (HPV) antigen and Freund' S complete adjuvant to obtain a primary immunogen; S2, step type immunization: primary immunization, namely inoculating the primary immunogen to egg laying hens in a multipoint injection mode, wherein the injection dose of each hen is 0.8-1.2ml, the number of injection points is 20-80, and the injection points comprise subcutaneous tissues and muscle tissues; The first boosting, namely inoculating the boosting immunogen to the same egg laying hen in a multipoint injection mode on the 12 th to 16 th days after the first boosting, wherein the injection dose of each hen is 0.8 to 1.2ml, and the injection point number is 15 to 60 points; Serial boosting, repeating the boosting every 12-16 days for 2-4 times; maintaining immunity, namely performing boosting immunity on the egg laying hen every 6-10 weeks after finishing series boosting immunity, and maintaining the immune period to be not less than 24 weeks; S3, collecting and preprocessing hyperimmune eggs, namely collecting eggs laid by immunized hens from the 18 th to the 25 th days after primary immunization, and taking yolk liquid for later use after surface sterilization; S4, preliminary extraction of the antibody, namely mixing the yolk liquid and the diluent according to the volume ratio of 1:4 to 1:15, uniformly stirring, regulating the pH value of the mixed liquid to 4.8-5.5 by using acid liquor, standing for 6-24 hours at 2-8 ℃, centrifuging for 10-30 minutes at the temperature of 2-8 ℃ and the temperature of 8000-15000 Xg, and collecting a first supernatant; s5, carrying out preliminary concentration and purification on the antibody, namely carrying out ultrafiltration treatment on the first supernatant through an ultrafiltration membrane with the molecular weight cutoff of 30kDa to 300kDa, concentrating by 5-30 times, and replacing the concentrated supernatant into a neutral buffer solution to obtain concentrated antibody liquid; S6, fine purification of the antibody, namely purifying the con