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Recombinant virus-like particle (VLP) of human papilloma virus type 31 (HPV31) and preparation method thereof

A technology of human papillomavirus and tag-hpv31l1, which is applied in the field of 31 recombinant human papillomavirus virus-like particles and their preparation, and can solve the problems of low yield, large protein loss, difficult application in large-scale production and the like

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 virus-like particle (VLP) of human papilloma virus type 31 (HPV31) and preparation method thereof
  • Recombinant virus-like particle (VLP) of human papilloma virus type 31 (HPV31) and preparation method thereof
  • Recombinant virus-like particle (VLP) of human papilloma virus type 31 (HPV31) and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0070] Embodiment 1: the design and synthesis of the HPV L1 gene of codon optimization

[0071] 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.

[0072] DNA sequences of various types of HPV before and after optimization:

[0073] SEQ NO.1: DNA sequence of HPV31 type L1 before optimization

[0074] SEQ NO.2: DNA sequence of optimized HPV31 type L1

Embodiment 2

[0075] Example 2: Construction and identification of recombinant vector pGEX-6P-1-GST-HPV31 L1:

[0076] Primers for amplifying the DNA fragment of HPV31 L1: (the restriction sites are BamHI and XhoI, respectively)

[0077] Forward-HPV31 L1-ApaI:5'ACTTCA GGATCC ATGTCTCTGTGGCGTCCGTCTG

[0078] Reverse-HPV31 L1-XhoI:5'ATCTCA CTCGAG CTA TTTTTTGGTTTTTTTACGTTTT

[0079] 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 d2H2O to 200 μL.

[0080] 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.

[0081] 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. 6P-1...

Embodiment 3

[0084] Example 3: Construction of recombinant vector pGEX-6P-1m-GST-SUMO-HPV31 L1 vector

[0085] 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, point mutation was carried out without changing the protein expression sequence of the lacI 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.

[0086] Primers for amplifying the DNA fragment of SUMO: (restriction sites are ApaI and BamHI respectively)

[0087] Forward-SUMO-ApaI: ACTTCA GGGCCC TCTGACCAGGAAGCTAAACCGTC

[0088] Reverse-SUMO-BamHI: CGC GGATCC ACCGGTCTGTTCCTGGTAAAC

[0089] Primers for amplifying the DNA fragment of HPV31 L1: (the restriction sites are BamHI and XhoI, respectively)

[0090] Forw...

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Abstract

The invention relates to a recombinant VLP of HPV31 and a preparation method thereof. Specifically, the invention discloses a novel polynucleotide gene segment coding recombinant HPV31 L1 protein, a vector containing the gene segment, a host cell containing the vector, HPV31 L1 fusion protein translated and expressed by the gene segment, a pentamer and a VLP composed of the pentamer; and the invention also discloses application of the pentamer, VLP protein and a vaccine composition composed of the pentamer and the VLP protein in preparation of drugs used for preventing HPV31 infection.

Description

technical field [0001] The present invention relates to human papilloma virus 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 human proliferative epithelial lesions, including papilloma (wart) and tumor-like lesions. Specifically, HPV-induced diseases mainly include three categories, the first category: 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%...

Claims

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Application Information

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IPC IPC(8): C12N15/37C12N15/70C12N1/21C07K19/00C07K14/025C12N7/04A61K39/12A61P31/20
Inventor 刘永江伍树明高文双陈晓任永峰王雅君姜绪林张瑞霞高俊张海江张庆峰薛俊莲张佳涛杜晓莉夏丽
Owner BEIJING HEALTH GUARD BIOTECH
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