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Broad spectrum monoclonal antibodies or antigen binding fragments thereof of anti-HPV L1 protein, and applications thereof

A technology of monoclonal antibodies and binding fragments, applied in the field of molecular virology and immunology, which can solve the problems of limited application of monoclonal antibodies and low neutralization titers

Active Publication Date: 2014-01-01
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the neutralizing titers of these three monoclonal antibodies are low, 100% lower than that of specific neutralizing monoclonal antibodies (such as: neutralizing antibody V5, see Wang et al., Journal of General irology, 78:2209-2215 (1997)). times, which greatly limits the application of these monoclonal antibodies

Method used

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  • Broad spectrum monoclonal antibodies or antigen binding fragments thereof of anti-HPV L1 protein, and applications thereof
  • Broad spectrum monoclonal antibodies or antigen binding fragments thereof of anti-HPV L1 protein, and applications thereof
  • Broad spectrum monoclonal antibodies or antigen binding fragments thereof of anti-HPV L1 protein, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0381] Example 1. Preparation of anti-HPVL1 protein monoclonal antibody and analysis of broad-spectrum reactivity

[0382] In this experiment, by cross-immunizing mice with multiple types of HPV VLPs, the splenocytes of the immunized mice were fused with myeloma cells, and screened to obtain antibodies and secreted antibodies reactive to multiple types of HPV VLPs. Antibody cell lines.

[0383] Antigen preparation

[0384] Using the E. coli expression system, the L1 protein of HPV16, 33, 52, and 58 types was prepared, and its morphology was detected by transmission electron microscopy and dynamic light scattering detection (Wei Minxi et al., Preparation of human papillomavirus type 16 virus-like particles and its Immunogenicity Research, Acta Virus, 2009, 4(25)). The results showed that the four obtained L1 proteins could form particles with a diameter of about 55 nm and a shape highly similar to natural virus particles, that is, formed virus-like particles (HPV16, HPV33, HP...

Embodiment 24B3

[0412] Example 2.4 Location, identification and homology analysis of the epitope recognized by B3

[0413] Since the epitope recognized by the antibody 4B3 is a linear epitope, the epitope can be located by segmental cloning.

[0414] First, the HPV16L1 protein was cloned and expressed segmentally in Escherichia coli, and a total of 10 subcloned peptides were prepared, each peptide was 65aa, and two adjacent peptides had an overlap of 15aa (aa represents an amino acid, when it is placed in front of the number n Indicates the nth amino acid (for example, aa130 indicates the 130th amino acid), and when it is placed after the number, it means that the length of the polypeptide is n amino acids (the same below)). These 10 subcloned peptides were respectively named: HPV16L1A-HPV16L1J, wherein, HPV16L1A corresponds to aa1-65 of HPV16L1 protein, HPV16L1B corresponds to aa 51-115 of HPV16L1 protein, and HPV16L1C corresponds to aa 101-165 of HPV16L1 protein, according to And so on.

...

Embodiment 3

[0433] Example 3. WB detection of HPV L1 protein in cervical exfoliated cells of HPV infected persons

[0434] Since monoclonal antibody 4B3 can react with various types of HPV L1 in Western blot analysis, it can be used to detect the expression level of L1 protein in cervical exfoliated cells of patients. To this end, the appropriate concentration of monoclonal antibody 4B3 used in the detection method was determined first by Western blot analysis; then the detection limit of monoclonal antibody 4B3 for 11 types of HPV L1 was tested; finally, by Western blot analysis, the monoclonal antibody 4B3 tested samples from 63 patients with cervical precancerous lesions who tested positive for HPV DNA.

[0435] WB analysis of the binding of different concentrations of 4B3 to various types of HPV L1

[0436] SDS-polyacrylamide gel electrophoresis: Dilute the L1 protein of HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 to 5 μg / ml, and perform SDS-polyacrylamide gel electrophoresis.

[043...

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Abstract

The invention relates to monoclonal antibodies and antigen binding fragments thereof, which can have a broad spectrum combination with human papilloma virus (HPV) L1 protein, coding of the sequences of the monoclonal antibodies and the antigen binding fragments thereof, generation of cell strains of the monoclonal antibodies and the antigen binding fragments thereof, and methods and applications of the monoclonal antibodies and the antigen binding fragments thereof on diagnosis, prevention or treatment.

Description

technical field [0001] The present invention relates to the fields of molecular virology and immunology. Specifically, the present invention relates to monoclonal antibodies and antigen-binding fragments thereof that can broadly bind to human papillomavirus (HPV) L1 protein, sequences encoding them, cell lines producing them, and using them for diagnosis, prevention or Methods and uses of treatment. Background technique [0002] Human papillomavirus (HPV) is a non-enveloped virus, which consists of a viral capsid and about 8kb of DNA wrapped in the capsid. The viral capsid of HPV is an icosahedral particle with a diameter of 50-55 nm composed of the major capsid protein L1 and the minor capsid protein L2. At present, more than 100 types of HPV have been discovered, among which, according to their relationship with tumorigenesis, they can be divided into two categories: low-risk HPV-including HPV6 and HPV11, which have low cancer risk and mainly cause genital warts; high-ri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13C12N15/63C12N1/21C12N1/15C12N1/19C12N5/10C12P21/08C12N5/20G01N33/577G01N33/68A61K39/42A61P31/20A61P35/00C07K7/06C07K7/08C07K19/00C12N15/37C12N15/62C12P21/02
Inventor 李少伟肖洁琼魏旻希陆剑俞海张军夏宁邵
Owner XIAMEN UNIV
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