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Truncated L1 proteins of human papilloma virus (HPV), virus-like particles as well as preparation method and application of virus-like particles

A technology of human papillomavirus and virus-like particles, which is applied in the field of L1 protein of human papillomavirus, can solve the problems of high cost, difficult protein purification, low expression level, etc., and achieve high titer effect

Inactive Publication Date: 2015-03-18
CHANGCHUN BCHT BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many proteins secreted by Saccharomyces cerevisiae are not secreted into the medium, but remain in the periplasmic space in a way that binds to cells, which creates difficulties for the purification of proteins in large-scale production
[0006] The advantages of the baculovirus-insect cell system are high safety, large capacity for cloning foreign genes, high-efficiency expression under the control of polyhedron promoters, post-translational modification, and no endotoxin pollution. There are problems of low expression level and high culture cost, which make the cost of the final product high and limit its application

Method used

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  • Truncated L1 proteins of human papilloma virus (HPV), virus-like particles as well as preparation method and application of virus-like particles
  • Truncated L1 proteins of human papilloma virus (HPV), virus-like particles as well as preparation method and application of virus-like particles
  • Truncated L1 proteins of human papilloma virus (HPV), virus-like particles as well as preparation method and application of virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Codon optimization design and synthesis of human papillomavirus L1 gene

[0052] Design full-length HPV6, HPV11, HPV16, HPV18, and HPV58 nucleic acid sequences according to the principles of codon bias used by insect cells, mRNA secondary structure, mRNA free energy stability, and GC content (see the sequence listing corresponding sequence). The above-mentioned full-length genes were commercially synthesized by Jierui Company respectively.

Embodiment 2

[0053] Example 2 Various types of HPV L1 protein-recombinant bacmid construction

[0054] Table 1. Primer list:

[0055] Primer

sequence

6F

GGATCCGCCACCATGTGGCG

6R500

CTCGAGTCAGCGCTTGGTC

6R491

CTCGAGTCAGGGTGCGGCGC

6R479

CTCGAGTCAGACGCCTGTGC

11F

GGATCCATGTGGCGCCCAT

11R501

CTCGAGTCACTTTTTGGTC

11R492

CTCGAGTCAGGCGGTGCTGGG

11R480

CTCGAGTCATCTCTTGATGCCG

16F

GCGGCCGCGCCACCATGAGCCTGT

16R505

CTCGAGTCACACAGTTCCTCTTCTTC

16R498

CTCGAGTCAGGCGGTGGTGCTGG

16R483

CTCGAGTCACAAGTTCACCCTGGGC

18F

GGATCCGCCACCATGAGCGT

18R507

CTCGAGTCACTTCTTTCACCTTCTTCC

18R498

CTCGAGTCAGCTGCTGGTGGTTGCGC

18R483

CTCGAGTCACAGCCTGGGCTTGGC

[0056] 58F

GGATCCGCCACCATGGCC

58R498

CTCGAGTCACTTCTTTCACCTTCTTCC

58R491

CTCGAGTCAGGTGCTGGGGGC

58R479

CTCGAGTCACAGGCCGCTCTG

[0057] Tabl...

Embodiment 3

[0080] Example 3 Recombinant bacmid transfection and virus amplification

[0081] 1. Recombinant bacmid transfection

[0082] 1) Cultivate sf9 insect cells: add 9X10 5 cells / well, the cells are in the logarithmic growth phase, and the survival rate is 97%; aspirate the Grace medium (purchased from Invitrogen), add Grace medium without antibiotics and serum, and let the cells adhere to the wall for at least 1h (27°C or room temperature) ;

[0083] 2) Prepare solution A: pipette 5 μl of recombinant Bacmid plasmid DNA and add to 100 μl of antibiotic-free and serum-free Grace medium;

[0084] 3) Prepare solution B: add 6 μl Cell FECTIN (purchased from Invitrogen) to 100 μl Grace medium without antibiotics and serum, mix A and B, mix gently, and incubate at room temperature for 30 minutes;

[0085] 4) Wash the adherent cells twice with antibiotic-free and serum-free Grace medium, aspirate the liquid, add 800 μl of Grace medium to the mixture of A and B, mix gently, and add to a ...

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Abstract

The invention relates to truncated L1 proteins of a human papilloma virus (HPV), virus-like particles as well as a preparation method and application of virus-like particles. The invention particularly relates to HPV6-type, HPV11-type, HPV16-type, HPV18-type or HPV58-type truncated L1 proteins of the HPV, the virus-like particles as well as the preparation method and application of the virus-like particles.

Description

technical field [0001] The present invention relates to truncated L1 protein of human papillomavirus, its virus-like particles, as well as its preparation method and use. Specifically, the present invention relates to the truncated L1 protein of human papillomavirus HPV6, HPV11, HPV16, HPV18 or HPV58, its virus-like particles, and its preparation method and use. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) is a kind of double-stranded small molecule DNA virus with strict species specificity. It mainly infects human skin and mucous membrane tissues, causing proliferative lesions of epithelial tissues in corresponding parts. According to the homology of nucleic acid sequence, HPV can be divided into more than 100 types. HPV16, 18 and 58 are highly related to the occurrence of cervical cancer, among which HPV16 is the most important carcinogenic factor. HPV6 and 11 are the causative agents of more than 90% of genital warts and laryngeal papil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/025C12N15/37C12N15/866C12N5/10C12N7/04A61K39/12A61P31/20
CPCA61K39/00C07K14/005C12N2710/20022C12N2710/20023C12N2710/20034
Inventor 孔维姜春来孙博徐菲宋海鹏张喆
Owner CHANGCHUN BCHT BIOTECH
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