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Anti-HPV16 L1 protein monoclonal antibody and detection method applying same

A technology of HPV16L1 and antibody, which is applied in the field of molecular virology and immunology, can solve the problems of large individual animal differences, long cycle, unfavorable animal welfare principles, etc., and achieve good response consistency, good neutralizing activity, and immunogenicity Evaluate the significant effect

Active Publication Date: 2020-12-25
SINO CELL TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two methods to evaluate the immunogenicity of vaccines, in vivo and in vitro. The in vivo method can intuitively reflect the immunogenicity of vaccination, but the disadvantage of this method is that the experiment in animals is costly and takes a long time, and there are still individual differences in animals. However, in the in vitro activity detection method of vaccines, monoclonal antibodies are an important tool for quality control of vaccine antigens, and antibody levels can be used to evaluate vaccine effects, among which neutralizing antibodies and their epitopes and The corresponding neutralization mechanism is also an important indicator of the vaccine development process

Method used

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  • Anti-HPV16 L1 protein monoclonal antibody and detection method applying same
  • Anti-HPV16 L1 protein monoclonal antibody and detection method applying same
  • Anti-HPV16 L1 protein monoclonal antibody and detection method applying same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: Construction and screening of antibody library

[0061] 1. Construction of Antibody Library

[0062] 1.1 Animal immunity:

[0063] Get the HPV16 L1 VLP protein (SEQ ID NO: 19) expressed in the insect cell system, and emulsify it with Freund's complete adjuvant (Sigma company, product number F5881) and Freund's incomplete adjuvant (Sigma company, product number F5506) respectively to make Freund's Complete Freund's Adjuvant Immunogen and Freund's Incomplete Adjuvant Immunogen, wherein the volume ratio of HPV16 L1 (500ug / piece) to Freund's Complete Adjuvant and Freund's Incomplete Adjuvant is 1:1.

[0064]

[0065]

[0066] By way of multi-point subcutaneous injection on the back, 2-2.2 kg of Japanese big-eared white rabbits (2-2.2 kg, purchased from Beijing Baier Kangnaite Experimental Rabbit Breeding Biotechnology Development Co., Ltd.) were immunized with Freund's complete adjuvant immunogen. .

[0067] After the first immunization, the animals w...

Embodiment 2

[0087] Example 2 Construction, Production and Purification of Antibodies

[0088] Antibody construction and production:

[0089] The nucleotide sequence (SEQ ID NO:3) of the heavy chain variable region of the R001scFv antibody was spliced ​​with the heavy chain signal peptide sequence (SEQ ID NO:11) by PCR method, and then passed Hind III and KpnI (source: Fermentas ) was digested and inserted into the pSTEP2 vector with the sequence of the rabbit IgG1 heavy chain constant region (SEQ ID NO:5) to obtain the complete expression vector of the heavy chain sequence (SEQ ID NO:9). The nucleotide sequence (SEQ ID NO:4) of the light chain variable region of the R001scFv antibody was spliced ​​with the light chain signal peptide sequence (SEQ ID NO:12) by PCR method, and then the insertion band was digested by Hind III and BamHI The complete light chain sequence (SEQ ID NO: 10) expression vector was obtained from the pSTEP2 vector with the rabbit kappa light chain constant region (SE...

Embodiment 3

[0097] The biological identification of embodiment 3 antibody

[0098] 3.1 Identification of antibody specificity:

[0099] 3.1.1 Monoclonal antibody HPV 16 R001 has no cross-reaction with other types of HPV VLP

[0100] Using indirect ELISA method, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 complete VLP protein was diluted to 2 μg / mL with phosphate buffer at pH 7.2 , 100 μL / well was coated on the microtiter plate, the antibody to be tested was diluted to 10 ng / mL and added, and the color was developed by the secondary antibody labeled with horseradish peroxidase.

[0101] Table 2. HPV 16 R001 Antibody Specific Identification Results

[0102] coat protein OD450-blank HPV6 VLP 0.004 HPV11 VLP 0.001 HPV16 VLP 2.531 HPV18 VLP 0.000 HPV31 VLPs 0.002 HPV33 VLPs 0.001 HPV35 VLP 0.001 HPV39 VLP 0.004 HPV45 VLP 0.002 HPV51 VLPs 0.001 HPV52 VLPs 0.003 ...

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Abstract

The invention provides an anti-human papilloma virus (HPV) L1 protein monoclonal antibody, an antigen binding fragment and a detection method applying the antibody. The antibody has strong binding force with an HPV16 L1 antigen, has no cross reaction with HPV6, 11, 18, 31, 33, 35, 39, 45, 52, 56, 58 and 59 types, can specifically neutralize HPV16 pseudotype viruses, and has neutralizing activity.The monoclonal antibody obtained by screening of the invention shows good reaction consistency and neutralization activity on virus-like particle antigens derived from different expression systems; the recognized epitope is a dominant epitope in both immune guinea pig serum and immune human serum; and the monoclonal antibody is suitable for being used as a detection antibody in ELISA quantification to perform immunogenicity evaluation on HPV vaccines.

Description

Technical field: [0001] The invention belongs to the field of molecular virology and immunology, specifically, the invention relates to a monoclonal antibody capable of specifically binding to human papillomavirus (HPV16) L1 protein and an antigen-binding fragment thereof, sequences encoding them, and using them to treat HPV The immunogenicity of the vaccine was evaluated. Background technique: [0002] Human papilloma virus (HPV) is an epitheliophilic virus with high specificity. HPV is a non-enveloped DNA virus with a particle size of 50-60 nm. The viral capsid is composed of 72 capsomers, which are regular icosahedral particles with a diameter of 50-55 nm composed of the main capsid L1 protein and the minor capsid L2 protein. Studies have shown that the L2 protein has a high degree of variability, which is related to the polymorphism of the virus antigen; while the L1 protein is highly conserved and is the main type-specific antibody, which can induce the body to produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13G01N33/569
CPCC07K16/084G01N33/56983C07K2317/565C07K2317/33C07K2317/76G01N2333/025G01N2469/10
Inventor 罗春霞孙春昀庞琳孙玲玲胡萍
Owner SINO CELL TECH INC
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