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Mutant of human papillomavirus type 51 L1 protein

A technology of L1 protein and protein, applied in the field of molecular virology and immunology, can solve the problems of safety and HPV vaccine production cost increase

Pending Publication Date: 2020-04-03
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 papillomavirus type 51 L1 protein
  • Mutant of human papillomavirus type 51 L1 protein
  • Mutant of human papillomavirus type 51 L1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0262] Example 1. Expression and purification of mutated HPV51 L1 protein

[0263] Construction of expression vector

[0264] Using Gibson assembly (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009; 6:343–5. doi:10.1038 / nmeth.1318 .) to construct an expression vector encoding a mutated HPV51 L1 protein, which contains a specific segment derived from HPV69 L1 and / or a specific segment derived from HPV26L1. In short, PCR reaction is first used to obtain a short fragment containing a mutation and a long fragment not containing a mutation, and then the Gibson assembly system is used to link the two fragments into a circle.

[0265] The initial templates used include pTO-T7-HPV51N9C plasmid (the HPV51L1 protein with 9 amino acid truncated at its coding N-terminal; abbreviated as 51L1N9 in Table 2), pTO-T7-HPV69N0C plasmid (its coding full-length HPV69 L1 protein; Abbreviated ...

Embodiment 2

[0309] Example 2: Assembly of HPV virus-like particles and detection of particle morphology

[0310] Assembly of HPV virus-like particles

[0311] Take a certain volume (about 2ml) of protein H51N9-69T1, H51N9-69T2, H51N9-69T3, H51N9-69T4, H51N9-69T5, H51N9-69T1-26S2, H51N9-69T1-26S3, H51N9-69T1-26S4, H51N9-69T1 -26S5, H51N9-69T2-26S1, H51N9-69T2-26S3, H51N9-69T2-26S4, H51N9-69T2-26S5, H51N9-69T3-26S1, H51N9-69T3-26S2, H51N9-69T3-26S4, H51N9-69T , H51N9-69T4-26S1, H51N9-69T4-26S2, H51N9-69T4-26S3 and H51N9-69T4-26S5, respectively dialyzed to (1) 2L storage buffer (20mM sodium phosphate buffer pH 6.5, 0.5M NaCl); (2) 2L refolding buffer (50mM sodium phosphate buffer pH 6.0, 2mM CaCl 2 , 2mM MgCl 2 , 0.5M NaCl); and (3) 20 mM sodium phosphate buffer pH 7.0, 0.5M NaCl. Dialysis was performed for 12 h in each of the three buffers.

[0312] By similar method, HPV51N9, HPV69N0 and HPV26N0 proteins were assembled into HPV51N9 VLP, HPV69N0 VLP and HPV26N0 VLP, respectively.

...

Embodiment 3

[0319] Example 3: Evaluation of neutralizing antibody titers in mouse serum after immunization with virus-like particles 1

[0320] In this experiment, the virus-like particles used were: H51N9-69T1 VLP, H51N9-69T2 VLP, H51N9-69T3 VLP, H51N9-69T4 VLP and H51N9-69T5 VLP.

[0321] In this experiment, the immunization scheme is shown in Table 4. All mice (6-week-old BalB / c female mice) were divided into 2 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). Each group was further subdivided into 8 subgroups, control subgroups 1 and 2 were immunized with HPV51N9 VLP alone and HPV69N0 VLP alone, and control subgroup 3 was immunized with mixed HPV51 / HPV69 VLP (i.e., HPV51N9 VLP and HPV69N0 VLP mixture, wherein each VLP is administered at the specified immunization dose), experimental subgroups 1, 2, 3, 4, and 5 were immunized with H51N9-69T1 VLP, H51N9-69T2 VLP, H51...

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Abstract

The invention relates to a mutated HPV51 L1 protein (or a variant thereof), a coding sequence and preparation method thereof, and a virus-like particle containing the same. The protein (or the variantthereof) and the virus-like particle can induce a neutralizing antibody resistant to at least two types of HPV (for example, HPV51 and HPV69, or HPV51, HPV69 and HPV26), and thereby can be used to prevent the at least two types of HPV infection, and diseases such as cervical cancer and condyloma acuminatum caused by the infection. The invention also relates to a use of the above-mentioned proteinand virus-like particle for preparation of a pharmaceutical composition or a vaccine, the pharmaceutical composition or the vaccine can be used to prevent the at least two types of HPV infection, anddiseases such as cervical cancer and condyloma acuminatum caused by the infection.

Description

technical field [0001] The present invention relates to the fields of molecular virology and immunology. Specifically, the present invention relates to a mutated HPV51 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 Neutralizing antibodies against at least two types of HPV (e.g., HPV51 and HPV69, or HPV51, HPV69 and HPV26), useful for preventing infection by said at least two types of HPV and diseases resulting from said 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 pharmaceutical composition or a vaccine, which 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. Background technique [0002] Human papilloma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/025C12N15/37C12N15/70A61K39/12A61P31/20A61P35/00
CPCC07K14/005C12N15/70A61K39/12A61P31/20A61P35/00C12N2710/20022C12N2710/20023C12N2710/20034A61K2039/5258A61K2039/55505A61K2039/585C07K14/015C12N7/00
Inventor 李少伟王大宁陈洁顾颖张军夏宁邵
Owner XIAMEN UNIV
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