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Recombinant human papilloma virus 52 virus-like particle and preparation method thereof

A human papillomavirus, tag-hpv52l1 technology, applied in the field of 52 recombinant human papillomavirus virus-like particles and its preparation, can solve the problems of large protein loss, difficult to apply in large-scale production, and insufficient uniformity of particles

Active Publication Date: 2017-05-24
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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  • Recombinant human papilloma virus 52 virus-like particle and preparation method thereof
  • Recombinant human papilloma virus 52 virus-like particle and preparation method thereof
  • Recombinant human papilloma virus 52 virus-like particle and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0067] Embodiment 1: the design and synthesis of the HPV L1 gene of codon optimization

[0068] The gene sequences were derived from the various types of HPV sequences published on PUBMED. All HPV DNA sequences were synthesized after codon optimization of the selected HPV DNA sequences according to the codon preference of Escherichia 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] DNA sequences of various types of HPV before and after optimization:

[0070] SEQ NO.1: DNA sequence of HPV52 type L1 before optimization

[0071] SEQ NO.2: DNA sequence of optimized HPV52 type L1

Embodiment 2

[0072] Example 2: Construction and identification of recombinant vector pGEX-6P-1-GST-HPV52 L1:

[0073] Primers for amplifying the DNA fragment of HPV52 L1: (restriction sites are BamHI and XhoI, respectively)

[0074] Forward-HPV52 L1-ApaI: 5'ACTTCA GGATCC ATGTCTGTTTGGCGTCCGTCTG

[0075] Reverse-HPV52 L1-XhoI: 5'ATCTCA CTCGAG CTA ACGTTTAACTTTTTTTTTTTTT

[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, add d 2 h 2 0 to 200 μL.

[0077] Gene PCR Amplification conditions: 3 min at 95°C; 30 sec at 95°C, 30 sec at 58°C, 4 min at 72°C; 32 cycles; 10 min at 72°C.

[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 digestion treatment, and then the recovered gene fragment was combined with pGEX-6P-1 containing the corresponding cohesive end using T4 DNA ligase. ...

Embodiment 3

[0081] Example 3: Construction of recombinant vector pGEX-6P-1m-GST-SUMO-HPV52 L1 vector

[0082] Construction of pGEX-6p-1m vector: In order to make the ApaI restriction site (GGGCCC) near the multi-restriction site the only ApaI restriction site of the vector, a point mutation was carried out without changing the protein expression sequence of the laCl gene 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. Through such modification, ApaI can be used to insert and express genes.

[0083] Primers for amplifying the DNA fragment of SUMO: (restriction sites are ApaI and BamHI respectively)

[0084] Forward-SUMO-ApaI: ACTTCA GGGCCC TCTGACCAGGAAGCTAAACCGTC

[0085] Reverse-SUMO-BamHI: CGC GGATCC ACCGGTCTGTTCCTGGTAAAC

[0086] Primers for amplifying the DNA fragment of HPV52 L1: (restriction sites are BamHI and XhoI, respectively)

[0087] Forwar...

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Abstract

The invention relates to a recombinant human papilloma virus 52 virus-like particle and a preparation method of the recombinant human papilloma virus 52 virus-like particle. The specific technical point is that the invention provides a polynucleotide gene segment of a novel coded recombinant HPV52 L1 protein, a carrier comprising the gene segment, a host cell comprising the carrier, an HPV52 L1 fusion protein translated and expressed by the gene segment, a pentamer and a VLP comprising the pentamer. The invention further discloses application of the pentamer, the VLP protein and a vaccine composition comprising the pentamer and the VLP protein to the preparation of a medicine used for preventing HPV52 infection.

Description

technical field [0001] The present invention relates to human papillomavirus virus-like particle and its preparation method. More specifically, the present invention relates to a pentamer of recombinant human papillomavirus L1 protein and a virus-like particle (Virus-like PartiCle, VLP) and its preparation method, and the virus-like particles Use of a vaccine composition for the prevention of human papillomavirus infection. Background technique [0002] Human papillomavirus (HPV) mainly through close contact with human body, such as sexually transmitted virus, can cause a variety of proliferative epithelial lesions in humans, including papilloma (wart) and tumor-like lesions. Specifically, HPV-induced diseases mainly include 3 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 cancer patients are caused by HPV infection, 90% of anal cancer, 40% of vulva...

Claims

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Application Information

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IPC IPC(8): C12N15/37C12N15/70C12N1/21C12N7/04C07K14/025C07K19/00A61K39/12A61K38/16A61P31/20
Inventor 刘永江伍树明高文双陈晓张海江沈迩翠王雅君姜绪林张瑞霞高俊张庆峰陈健平银飞刘玉莹夏丽
Owner BEIJING HEALTH GUARD BIOTECH
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