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Human papillomaviruse type hybrid virus-like particles and preparation method thereof

A virus-like, hybrid technology, applied in the fields of molecular virology and immunology, which can solve safety problems and increase the cost of HPV vaccine production

Active Publication Date: 2012-10-24
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This will greatly increase the production cost of the HPV vaccine and may pose potential safety issues due to the increased dose of immunization

Method used

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  • Human papillomaviruse type hybrid virus-like particles and preparation method thereof
  • Human papillomaviruse type hybrid virus-like particles and preparation method thereof
  • Human papillomaviruse type hybrid virus-like particles and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Expression and purification of HPV L1 mutein for construction of hybrid virus-like particles

[0052] Construction of HPV L1 mutant protein expression vector

[0053] Gene cloning was carried out by site-directed mutagenesis PCR reaction, and the initial templates used included pT0-T7-HPV16L1N30C plasmid (abbreviated as 16L1 in Table 1), pT0-T7-HPV52L1N40C plasmid (abbreviated as 52L1 in Table 1), pT0-T7 - HPV6L1N5C plasmid (abbreviated as 6L1 in Table 1). The templates and primers of each PCR reaction are shown in Table 1. The amplification conditions of the PCR reaction were set as follows: denaturation at 94°C for 10 minutes, 25 cycles of "denaturation at 94°C for 50 seconds, annealing at a specified temperature for a certain time, extension at 72°C for 50 seconds", and finally Extension at 72°C for 10 minutes. The sequences of the PCR primers used are listed in Table 2.

[0054] Table 1. Each PCR reaction condition for constructing mutant HPV L1 prot...

Embodiment 2

[0072] Example 2: Renaturation and Morphological Detection of HPV L1 Mutant Protein

[0073] Refolding of mutant proteins

[0074] Get 2ml of the HPV16L1-C175A, HPV16L1-ΔC428, HPV52L1-C175A, HPV52L1-ΔC428, HPV6L1-C175S, HPV6L1-ΔC428 mutant proteins whose purity is greater than 98% in Example 1 or alone or in 2L refolding buffer (50mM PB ( Sodium phosphate buffer) pH 6.0, 2mM CaCl2, 2mM MgCl2, 0.5M NaCl) dialysis to fully exchange. After the exchange was complete, exchange was performed with 2 L of storage buffer (20 mM PB (sodium phosphate buffer) pH 6.5, 0.5 M NaCl).

[0075] Morphological detection of HPV L1 mutant protein

[0076] The HPV16L1-C175A, HPV16L1-ΔC428, HPV52L1-C175A, HPV52L1-ΔC428 proteins refolded and assembled according to the above method were observed by transmission electron microscope. The instrument used is a 100kV transmission electron microscope produced by Japan Electronics Corporation, with a magnification of 100,000 times. After renaturation,...

Embodiment 3

[0077] Example 3: HPV16 L1-C175A and HPV52L1-ΔC428 assembled hybrid VLPs with different assembly buffers and particle morphology detection

[0078] Hybrid assembly of HPV16 L1-C175A and HPV52L1-ΔC428 in different assembly buffers VLP

[0079] Measure the protein concentration of HPV16L1-C175A and HPV52L1-ΔC428, take a certain volume (about 2ml) of HPV16L1-C175A and HPV52L1-ΔC428 according to the ratio of mass ratio 1:1, mix and dialyze in 2L storage buffer (20mM PB (sodium phosphate buffer solution) pH6.5, 0.5M NaCl); 2L refolding buffer (50mM PB (sodium phosphate buffer) pH6.0, 2mM CaCl 2 , 2mM MgCl 2 , 0.5M NaCl); 20mM PB (sodium phosphate buffer) pH 7.0 with 0.5M NaCl; three buffers were exchanged for 12h.

[0080] Morphological detection of heterozygous virus-like particles

[0081] 100 μL of the samples under the above three assembly conditions were taken for transmission electron microscope observation. The instrument used is a 100kV transmission electron micro...

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Abstract

The present invention relates to human papillomavirus (HPV) type hybrid virus-like particles and a preparation method thereof. The virus-like particles can be used for preventions two or more HPV infections and diseases caused by HPV infections. The present invention further relates to uses of the protein and the virus-like particles in preparations of drug compositions or vaccines, wherein the drug compositions or the vaccines are provided for preventions of HPV infections and diseases caused by HPV infections, and the diseases comprise cervical cancer, condyloma acuminatum, and the like.

Description

technical field [0001] The present invention relates to the fields of molecular virology and immunology. Specifically, the present invention relates to human papillomavirus type heterozygous virus-like particles formed by heterozygous assembly of different types of HPV L1 proteins and a preparation method thereof. The type heterozygous virus-like particles can be used to prevent two or two More than one HPV infection and diseases caused by HPV infection. The present invention also relates to the use of the above-mentioned heterozygous virus-like particles for preparing pharmaceutical compositions or vaccines, which are used to prevent HPV infection and diseases caused by HPV infection, such as cervical cancer, condyloma acuminata, etc. . Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) mainly causes skin and mucous membrane verrucous lesions. According to its relationship with tumorigenesis, HPV can be divided into high-risk types and low-ris...

Claims

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Application Information

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IPC IPC(8): C12N7/04A61K39/12A61P31/20A61P35/00
Inventor 李少伟魏旻希王大宁顾颖张军夏宁邵
Owner XIAMEN UNIV
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