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Mutant of human papilloma virus-18 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: 2019-12-10
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 papilloma virus-18 L1 protein
  • Mutant of human papilloma virus-18 L1 protein
  • Mutant of human papilloma virus-18 L1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0213] Example 1. Expression and purification of mutated HPV18 L1 protein

[0214] Construction of expression vector

[0215] 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 the expression vector of the HPV18 L1 protein containing the mutation of the specific segment derived from the HPV45 L1 protein and / or the specific segment derived from the HPV59 L1. Briefly, a PCR reaction was first used to obtain a short fragment containing the mutation and a long fragment not containing the mutation, and then the two fragments were ligated into a circle using the Gibson assembly system.

[0216]The initial templates used include the pTO-T7-HPV18N65L1 plasmid (the HPV18 L1 protein with 65 amino acids truncated at the N-terminal of its encoding, which is named HPV18N65; abbreviated as 18L1N65 in Table 2), pTO-T7-...

Embodiment 2

[0247] Example 2: Assembly of HPV virus-like particles and detection of particle morphology

[0248] Assembly of HPV virus-like particles

[0249] Take a certain volume (about 2ml) of protein H18N65-45T1, H18N65-45T2, H18N65-45T3, H18N65-45T4, H18N65-45T5, H18N65-45T3-59S1, H18N65-45T3-59S2, H18N65-45T3-59S4, H18N36 -59S5, H18N65-45T4-59S1, H18N65-45T4-59S2, H18N65-45T4-59S3, H18N65-45T4-59S5, H18N65-45T1-59S5, H18N65-45T2-59S5, H18N65-45T1T3-59S5, 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.

[0250] By similar method, HPV18N65, HPV45N27 and HPV59 L1 proteins were assembled into HPV18N65 VLP, HPV45N27 VLP and HPV59 VLP, respectively.

[0251] Molecular sieve chromatography analysis

[0252] A 1120Compact LC high-performance l...

Embodiment 3

[0257] Example 3: Evaluation of thermal stability of virus-like particles

[0258] Use the differential temperature calorimeter VP Capillary DSC purchased from GE Company of the United States (formerly MicroCal Company) to evaluate proteins HPV18N65, HPV45N27, HPV59, H18N65-45T3, H18N65-45T4, H18N65-45T3-59S1, H18N65-45T4-59S1, H18N65 - thermal stability of the VLP formed by 45T1T3-59S5, wherein the storage buffer of the protein was used as a control, and each protein was scanned at a heating rate of 1.5°C / min in the range of 10°C-90°C. Test results such as Figures 6A-6H shown. The results showed that the VLPs formed by each protein had extremely high thermal stability.

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Abstract

The invention relates to a mutational HPV18 (human papilloma virus-18) L1 protein (or a variant thereof), a coding sequence and a preparation method of the protein or the variant, and a virus-like particle comprising the protein or the variant. The protein (or the variant thereof) and the virus-like particle can induce a neutralizing antibody capable of resisting at least two categories of HPV (such as HPV18 and HPV45, or HPV18, HPV45 and HPV59) so as to prevent the infection of at least two categories of HPV and diseases, including cervical cancer and pointed condyloma, caused by the infection. The invention also relates to a purpose of the above protein and the virus-like particle for preparing a medicine composition or a vaccine. The medicine composition or the vaccine can be used for preventing infection of at least two categories of HPV and diseases, including cervical cancer and pointed condyloma, caused by the infection.

Description

[0001] This application is based on the application with CN application number 201810566208.8 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 HPV18 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., HPV18 and HPV45, or HPV18, HPV45 and HPV59), thereby being 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/025C12N15/37A61K39/12A61P31/20A61P35/00
CPCC07K14/005A61K39/12A61P31/20A61P35/00C12N2710/20022C12N2710/20023C12N2710/20034C12N2710/20071A61K2039/5258A61K2039/53A61K2039/55505C12N7/00
Inventor 李少伟宋硕何茂洲史晶洁顾颖夏宁邵
Owner XIAMEN UNIV
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