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Type 45 recombinant human papillomavirus virus-like particles and preparation method thereof

A technology of human papillomavirus and HPV45L1, applied in botany equipment and methods, biochemical equipment and methods, antiviral agents, etc., can solve problems such as difficult to apply in large-scale production, large protein loss, uneven particles, etc. , to achieve good immunogenicity and prevent infection

Active Publication Date: 2021-09-17
BEIJING HEALTH GUARD BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in these purification processes, the protein loss is large and the yield is low, which makes it difficult to apply in large-scale production.
[0006] In terms of the uniformity of the HPV vaccine antigen protein VLP, the particle size dispersion of the HPV L1 VLP assembled in the prior art is represented by the polyd value, and the polyd value <15% indicates that the particles have good uniformity, and 15% to 30 % means that the particles have greater heterogeneity, and greater than 30% means that the particles are not uniform at all
Most HPV L1 VLPs prepared in the prior art are greater than 15%

Method used

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  • Type 45 recombinant human papillomavirus virus-like particles and preparation method thereof
  • Type 45 recombinant human papillomavirus virus-like particles and preparation method thereof
  • Type 45 recombinant human papillomavirus virus-like particles and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment l

[0067] Example 1: Design and synthesis of codon-optimized HPV L1 gene

[0068] The gene sequences are derived from the sequences of various types of HPV that have been published on PUBMED. All HPV DNA sequences were synthesized after codon optimization of selected HPV DNA sequences with reference to the codon preference of E. coli for gene transcription. Primers were designed according to the synthetic DNA sequence, and the synthetic gene was used as a template for PCR amplification. The resulting codon-optimized sequences were verified by DNA sequencing.

[0069] HPV DNA sequences before and after optimization:

[0070] SEQ NO.1: DNA sequence of HPV45 type L1 before optimization

[0071] SEQ NO.2: DNA sequence of optimized HPV45 type L1

Embodiment 2

[0072] Example 2: Construction and identification of recombinant vector pGEX-6P-1-GST-HPV45 L1:

[0073] Primers for amplifying the DNA fragment of HPV45 L1: (the restriction sites are BamHI and XhoI, respectively)

[0074] Forward-HPV45 L1-ApaI: 5' ACTTCAGGATCC ATGGCTCTGTGGCGTCCGTCTG

[0075] Reverse-HPV45 L1-XhoI: 5' ATCTCACTCGAGCTA TTTTTTAGAACGGATACGAAC

[0076] PCR amplification reaction system: 10 x Pfu buffer 20 μL, Pfu enzyme 4 μL, 10 mM dNTP 2.5 μL, 5’ Primer (5 μM) 10 μL, 3’ Primer (5 μM) 10 μL, template DNA 50 ng, plus d 2 H 2 0 to 200 μL.

[0077] Gene PCR amplification conditions: 95°C for 3 min; 95°C for 30 sec, 58°C for 30 sec, 72°C for 4 min; cycle 32 times; 72°C for 10 min.

[0078] The L1 gene fragment containing BamH I and XhoI restriction sites and the vector pGEX-6P-1 were subjected to BamH I / XhoI double restriction digestion treatment, and then the recovered gene fragment was combined with pGEX- 6P-1 was ligated at 16 °C for 10-15 h.

[0079] After t...

Embodiment 3

[0081] Example 3: Construction of recombinant vector pGEX-6P-1m-GST-SUMO-HPV45 L1 vector

[0082] Construction of pGEX-6p-1m vector: In order to make the ApaI restriction site (GGGCCC) near the multiple restriction site as the only ApaI restriction site of the vector, without changing the protein expression sequence of the laCl gene, point mutation ApaI (3890) can be eliminated by changing the Gly codon GGC in another ApaI recognition sequence GGGCCC of the commercially available pGEX-6p-1 vector to its synonymous codon GGT. This modification makes ApaI a site for insertion of expressed genes.

[0083] Amplify the DNA fragment primers of SUMO: (the restriction sites are ApaI and BamHI respectively)

[0084] Forward -SUMO-ApaI: ACTTCAGGGCCCCTCTGACCAGGAAGCTAAACCGTC

[0085] Reverse-SUMO-BamHI: CGCGGATCCACCGGTCTGTTCCTGGTAAAC

[0086] Primers for amplifying the DNA fragment of HPV45 L1: (the restriction sites are BamHI and XhoI, respectively)

[0087] Forward-HPV45 L1-ApaI: 5'...

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Abstract

The invention relates to type 45 recombinant human papillomavirus virus-like particles and a preparation method thereof. The specific technical points are to provide a new polynucleotide gene segment encoding a recombinant HPV45 L1 protein, a vector containing the gene segment, and a carrier including the vector. The host cell, and the HPV45 L1 fusion protein, pentamer and VLP composed of the pentamer translated and expressed by the gene fragment, the present invention also discloses the pentamer, VLP protein and the vaccine composition composed thereof in the preparation of the prophylaxis Drug application for HPV45 infection.

Description

technical field [0001] The present invention relates to virus-like particles of human papillomavirus and a preparation method thereof. More specifically, the present invention relates to a recombinant human papillomavirus L1 protein pentamer and virus-like particle (Virus-like PartiCle, VLP) and a preparation method thereof, and a vaccine composition containing the virus-like particle. Use in the prevention of human papillomavirus infection. Background technique [0002] Human papillomavirus (Human Papillomavirus, referred to as HPV) mainly through close human contact, such as sexually transmitted viruses, can cause a variety of human proliferative epithelial lesions, including papilloma (warts) and tumor-like lesions. Specifically, HPV-induced diseases mainly include three categories, category 1: cancers of the cervix, vagina, female vulva, penis and anus, and certain types of malignant lesions such as head and neck tumors. 100% of cervical cancers are caused by HPV infec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/37C12N15/70C12N1/21C12N7/04C07K14/025C07K19/00A61K39/12A61K38/16A61P31/20
Inventor 刘永江银飞伍树明高文双陈晓王雅君姜绪林张瑞霞高俊张海江李闯刘玉莹陈丹沈迩萃夏丽
Owner BEIJING HEALTH GUARD BIOTECH
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