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Vaccine for chimeric virus-like particles and preparation method thereof

A chimeric virus, virus-like technology, applied in antiviral agents, chemical instruments and methods, hybrid peptides, etc., can solve problems such as low neutralization activity

Active Publication Date: 2011-08-17
SOUTH CHINA UNITED VACCINE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it has the following problem: the infectious pseudoviruses used in the research of Patent Document 1 are only types 16 and 18, and the antigen described in Patent Document 1 shows good neutralizing activity compared to these infectious pseudoviruses
However, subsequently, the inventors conducted neutralization experiments on the newly developed infectious pseudoviruses of types 31, 53, and 58 against the antigens in Patent Document 1. The neutralizing activity is not high

Method used

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  • Vaccine for chimeric virus-like particles and preparation method thereof
  • Vaccine for chimeric virus-like particles and preparation method thereof
  • Vaccine for chimeric virus-like particles and preparation method thereof

Examples

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preparation example Construction

[0106] The method for preparing the above-mentioned virus-like particle VLP specifically comprises the following steps: 1. Constructing a chimeric gene expressing the above-mentioned protein. 2. Construct the expression plasmid containing the chimeric gene. 3. Construction of recombinant bacmid. 4. Obtain the expression system. 5. Express and purify the obtained chimeric protein. 6. Aggregating the above-mentioned chimeric proteins to make virus-like particles.

[0107] The capsids of the invention are used in the production of L1-capsid vaccines (virus-like particles). Production of L1-capsid vaccines can use, for example, the baculovirus expression system.

[0108] The present invention includes capsid mixtures containing two or more of the aforementioned capsids of the present invention. The three types of capsids of the present invention each have different cross-reactivity, and by mixing two or more types of capsids having different cross-reactivity, there is an adva...

Embodiment 1

[0123] 1.1 Production of rabbit peptide antiserum

[0124] Two New Zealand rabbits (2.5kg-3.0kg) were immunized. The antigen used is obtained by combining chemically synthesized peptide with KLH (keyholelimpet hemocyanin). The first sensitization was by subcutaneous administration of 0.5 mg of antigen with Freund's complete adjuvant. 0.25 mg of antigen was administered subcutaneously with Freund's complete adjuvant 2 weeks after the first administration. Then, sensitization was performed in the same manner (0.25 mg) after 2 weeks, 4 weeks, and 6 weeks, and sensitization was performed 5 times in total. One week after the last sensitization, whole blood was collected to obtain serum.

[0125] Antisera obtained by immunizing rabbits with a synthetic peptide having the amino acid sequence of the HPV16 type E7-surface region were examined using the neutralization experiment shown below. The results are shown in Table 1. From the results shown in Table 1, it can be seen that th...

Embodiment 2

[0132] Embodiment 2 Preparation of chimeric genes and chimeric VLPs

[0133] 1. Use the known HPV16L1 gene sequence retrieved from Genebank to synthesize the full sequence to obtain the L1 gene, and design HPV16-specific primers to amplify the full-length L1 gene. In order to facilitate the cloning of the target gene into the baculovirus donor plasmid, BamHI and EcoRI restriction site recognition nucleotide sequences were respectively introduced into the upstream and downstream primers. PCR technology was used to amplify, and the recovered HPV16L1 PCR product was connected to the pGEM--T carrier, and positive clones were screened by blue and white spots, and the positive clone plasmids were extracted, and the HPV16L1 sequences in the positive clones were sequenced at the same time.

[0134] The known gene fragment of the corresponding epitope protein of HPV16 type E7 was retrieved from GeneBank, and the gene fragment of the corresponding epitope protein of HPV16 type E7 was sy...

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Abstract

The invention discloses chimeric protein. The chimeric protein is obtained by inserting an epitope E7 of a human papillomavirus type 16 at a ring position of human papillomavirus type 16 (HPV 16) protein L1, namely between the 140th amino acid and the 141st amino acid, wherein the epitope E7 of the human papillomavirus type 16 has genes of protein (a) with the following codes: the protein is formed by an amino acid sequence shown by an MLDLQPETT epitope (12-20E7 for short), a RAHYNIVTF epitope(49-57E7 for short), an LLMGTLGIV epitope (82-90E7 for short) or a TLGIVCPI epitope (86-93E7 for short). The invention also discloses virus-like particles formed by aggregating the chimeric protein, a mixture formed by the virus-like particles and application of the virus-like particles and the mixture of the virus-like particles to the preparation of the vaccine for HPV 16 L1-virus-like particles.

Description

technical field [0001] The invention specifically relates to a chimeric virus-like particle vaccine and a preparation method thereof. Background technique [0002] Human papillomavirus (HPV) (attached figure 1 shown) is a small DNA virus with more than 100 genotypes, including 15 genotypes (high-risk types: 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66, 68, 73) are the cause of cervical cancer. HPV16 is detected in 50-60% of cervical cancers. In Europe and America, 18 types are mostly used, and in Asia, 16 and 58 types are mostly used. [0003] The HPV virus capsid has a structure of 12 molecules of L2 protein on the regular icosahedral skeleton, and the regular icosahedral skeleton is formed by 72 L1 protein pentamers. Both ends of the L2 protein are located inside the viral capsid, while a part of its N-terminal side is exposed on the surface of the capsid (shown in FIG. 2 ). If only the L1 protein is expressed in large quantities using recombinant DNA technology, vi...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K39/295C12N7/04C07K14/795A61P31/20
Inventor 尹海滨安鸿
Owner SOUTH CHINA UNITED VACCINE INST
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