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Mutant of human papilloma virus 68 type L1 protein

A protein and variant technology, applied in the fields of molecular virology and immunology, can solve problems such as safety issues and increased production costs of HPV vaccines

Pending Publication Date: 2019-12-10
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this approach will lead to a substantial increase in the production cost of the HPV vaccine, and may cause potential safety issues due to the increased dose of immunization

Method used

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  • Mutant of human papilloma virus 68 type L1 protein
  • Mutant of human papilloma virus 68 type L1 protein
  • Mutant of human papilloma virus 68 type L1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1. Expression and purification of mutated HPV68 L1 protein

[0117] Construction of expression vector

[0118] Multiple-point mutagenesis PCR reaction is used to construct the expression vector of the HPV68 L1 protein (H68N0-39T1) encoding the mutation containing the specific segment derived from the HPV39 L1 protein, wherein the initial template used is the pTO-T7-HPV68N0C plasmid (its It encodes the full-length HPV68 L1 protein, which is abbreviated as 68L1N in Table 2). The templates and primers used in each PCR reaction are shown in Table 2, and the amplification conditions of the PCR reaction are set as follows: denaturation at 94°C for 10 minutes; 7 minutes 30 seconds); a final extension of 10 minutes at 72°C. Annealing temperature and time are listed in Table 2. The specific sequences of the PCR primers used are listed in Table 3.

[0119] 2 μL of DpnI restriction endonuclease (Fermentas (MBI), catalog number: FD1704, 2500 U / tube) was added to the a...

Embodiment 2

[0146] Example 2: Assembly of HPV virus-like particles and detection of particle morphology

[0147] Assembly of HPV virus-like particles

[0148]Take a certain volume (about 2ml) of protein H68N0-39T1, H68N0-39T2, H68N0-39T3, H68N0-39T4 and H68N0-39T5, respectively dialyze into (1) 2L storage buffer (20mM sodium phosphate buffer pH 6.5, 0.5 M NaCl); (2) 2L refolding buffer (50mM sodium phosphate buffer pH 6.0, 2mM CaCl2, 2mM MgCl2, 0.5M NaCl); and (3) 20mM sodium phosphate buffer pH 7.0, 0.5M NaCl. Dialysis was performed for 12 h in each of the three buffers.

[0149] By a similar method, HPV68N0 and HPV39N15 proteins were assembled into HPV68N0 VLPs and HPV39N15 VLPs, respectively.

[0150] Molecular sieve chromatography analysis

[0151] A 1120Compact LC high-performance liquid chromatography system from Agilent Corporation of the United States was used to carry out molecular sieve chromatography analysis on the dialyzed samples, wherein the analytical column used wa...

Embodiment 3

[0156] Example 3: Evaluation of neutralizing antibody titers in mouse sera after immunization with VLPs

[0157] In this experiment, the immunization scheme is shown in Table 4. All mice (6-week-old BalB / c female mice) were divided into 3 groups: aluminum adjuvant group 1 (immunization dose was 5 μg, using aluminum adjuvant), aluminum adjuvant group 2 (immunization dose was 1 μg, using aluminum adjuvant), and aluminum adjuvant group 3 (the immunization dose was 0.2 μg, using aluminum adjuvant). Each group was further subdivided into 8 subgroups, and control subgroups 1-3 were treated with separate HPV68N0 VLPs, separate HPV39N15 VLPs and mixed HPV68 / HPV39 VLPs (ie, a mixture of HPV68N0 VLPs and HPV39N15 VLPs, wherein each VLP All were administered at the specified immunization dose) for immunization, and experimental subgroups 1-5 were immunized with H68N0-39T1 VLP, H68N0-39T2 VLP, H68N0-39T3 VLP, H68N0-39T4 VLP and H68N0-39T5 respectively.

[0158] Five mice / subgroup were i...

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Abstract

The invention relates to mutated HPV68L1 protein (or a variant thereof), and a coding sequence and preparation method of the mutated HPV68L1 protein (or the variant thereof), as well as virus-like particles containing the mutated HPV68L1 protein (or the variant thereof). The protein (or the variant thereof) and the virus-like particles can induce neutralizing antibodies which can resist at least two types of HPVs (such as HPV68 and HPV39), so that the protein (or the variant thereof) and the virus-like particles can be used for preventing infection with at least two types of HPVs, and diseasescaused by the infection, such as cervical cancer and condyloma acuminatum. The invention also relates to the purpose of the protein and the virus-like particles for preparation of a medical composition or a vaccine. The medical composition or the vaccine can be used for preventing the infection with at least two types of HPVs, and the diseases caused by the infection, such as the cervical cancerand the condyloma acuminatum.

Description

[0001] This application is based on the application with CN application number 201810566210.5 and the application date is June 4, 2018, and claims its priority. The disclosure content of this CN application is hereby incorporated into this application as a whole. technical field [0002] The present invention relates to the fields of molecular virology and immunology. Specifically, the present invention relates to a mutated HPV68 L1 protein (or its variant), its coding sequence and preparation method, and a virus-like particle comprising it, and the protein (or its variant) and the virus-like particle are capable of inducing The neutralizing antibody against at least two types of HPV (for example, HPV39 and HPV68) can be used to prevent the at least two types of HPV infection and the diseases caused by the infection, such as cervical cancer and genital warts. The present invention also relates to the use of the above-mentioned protein and virus-like particles for preparing a p...

Claims

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Application Information

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IPC IPC(8): C07K14/025C12N15/37C12P21/02A61K39/12A61P31/20A61P35/00A61P17/12C12R1/19
CPCC07K14/005A61K39/12A61P31/20A61P35/00A61P17/12C12N2710/20022C12N2710/20023C12N2710/20034A61K2039/5258
Inventor 顾颖杨与柔王大宁王颖彬李少伟夏宁邵
Owner XIAMEN UNIV
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