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Mutant of HPV33 (human papilloma virus 33) L1 protein

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

Active Publication Date: 2017-06-13
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 HPV33 (human papilloma virus 33) L1 protein
  • Mutant of HPV33 (human papilloma virus 33) L1 protein
  • Mutant of HPV33 (human papilloma virus 33) L1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0150] Example 1. Expression and purification of mutated HPV33 L1 protein

[0151] Construction of expression vector

[0152] A multi-point mutation PCR reaction is used to construct an expression vector encoding a mutated HPV33 L1 protein containing a segment derived from the HPV58 L1 protein, wherein the initial template used is the pTO-T7-HPV33L1N9C plasmid (which encodes a truncated N-terminal 9 amino acid HPV33 L1 protein; abbreviated as 33L1N9 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. The specific sequences of the PCR primers used are listed in Table 3.

[0153] Add 2 μL DpnI restriction endonuclease to the amplification product (50 μL), and incubate at 37° C. for 60 min. 10 μL of the digested product was used to transform 40 μL of competent Escheric...

Embodiment 2

[0181] Example 2: Assembly of HPV virus-like particles and detection of particle morphology

[0182] Assembly of HPV virus-like particles

[0183] Take a certain volume (about 2ml) of protein H33N9-58T1, H33N9-58T2, H33N9-58T3, H33N9-58T4, H33N9-58T5, H33N9-58T5-52S1, H33N9-58T5-52S2, H33N9-58T5-52S3, H33N9-58T5 -52S4, 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.

[0184] By similar method, HPV58N35, HPV33N9 and HPV52N40 proteins were assembled into HPV58N35 VLP, HPV33N9 VLP and HPV52N40 VLP, respectively.

[0185] Molecular sieve chromatography analysis

[0186] The dialyzed samples were subjected to molecular sieve chromatography analysis with 1120 Compact LC high-performance liquid chromatography sy...

Embodiment 3

[0191] Example 3: Evaluation of thermal stability of virus-like particles

[0192] Use the differential temperature calorimeter VP Capillary DSC purchased from GE Company of the United States (formerly MicroCal Company) to evaluate H33N9-58T5, H33N9-58T5-52S1, H33N9-58T5-52S2, H33N9-58T5-52S3, H33N9-58T5-52S4 The thermal stability of the formed VLP, 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-6F 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 mutational HPV33 (human papilloma virus 33) L1 protein (or a mutant thereof), an encoding sequence and a preparation method thereof, and a virus-like particle containing the same. The protein (or the mutant thereof) and the virus-like particle can be used for inducing a neutralizing antibody for resisting at least two types of HPV (such as HPV33 and HPV58, or HPV33, HPV58 and HPV52), so as to prevent the infection of at least two types of HPV and the diseases caused by infection, such as cervical cancer and condyloma acuminatum. The invention also relates to application of the protein and the virus-like particle in preparing a medicine composition or a vaccine. The medicine composition or the vaccine can be used for preventing the infection of at least two types of HPV and the diseases caused by infection, such as cervical cancer and condyloma acuminatum.

Description

technical field [0001] The present invention relates to the fields of molecular virology and immunology. Specifically, the present invention relates to a mutated HPV33 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., HPV33 and HPV58, or HPV33, HPV58 and HPV52), 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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IPC IPC(8): C07K14/025C12N15/37C12N7/04C12P21/02A61K39/12A61P31/20A61P35/00A61P17/12C12R1/19
CPCA61K39/12C07K14/005C12N2710/20022C12N2710/20023C12N2710/20034
Inventor 李少伟宋硕李智海李韵冰俞海夏宁邵
Owner XIAMEN UNIV
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