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Method for purifying human papilloma virus advanced protein from prokaryote

A papilloma and virus technology, applied in the field of purifying human papilloma virus late protein L1 from prokaryotes, can solve the problems of large protein loss, inability to apply large-scale production, and high price

Active Publication Date: 2008-04-02
XIAMEN UNIV +1
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  • Description
  • Claims
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Problems solved by technology

However, most of the HPV L1 proteins expressed by Escherichia coli lose their natural conformation and mainly exist in the form of inclusion bodies.
Although HPV VLP can also be obtained through steps such as inclusion body purification and renaturation (Kelsall, S.R. and J.K.Kulski (1995). J Virol Methods 53(1):75-90), the amount of protein loss is large during the renaturation process. Low yield, difficult to apply in large-scale production
Although HPV L1 protein can also be solublely expressed in the correct conformation in E. coli and dissolved in the lysed supernatant of the bacteria, the expression level is low, and there are many types and large amounts of foreign proteins in the supernatant, so it is necessary to purify the target protein from it. Protein is quite difficult
Although there are also reports in the literature that the expression of L1 protein in the supernatant can be increased by means of GST fusion expression, and it is helpful for the purification of the target protein (Li, M., T.P.Cripe, et al. (1997). J Virol71 (4): 2988-95.), but the cleavage of fusion proteins often requires expensive enzymes, which still cannot be applied to large-scale production

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  • Method for purifying human papilloma virus advanced protein from prokaryote
  • Method for purifying human papilloma virus advanced protein from prokaryote
  • Method for purifying human papilloma virus advanced protein from prokaryote

Examples

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preparation example Construction

[0159] The expression systems currently used in the preparation of HPV VLPs can be divided into eukaryotic expression systems and prokaryotic expression systems.

[0160] The natural conformation of the HPVL1 protein expressed in the eukaryotic expression system is less disrupted, and can spontaneously form VLPs, often requiring only a simple purification process to obtain VLPs with the correct conformation. However, the baculovirus expression system and yeast expression system currently used in eukaryotic expression systems have defects such as low expression levels and high culture costs, which have brought great difficulties to large-scale industrial production.

[0161] Among the prokaryotic expression systems, the Escherichia coli expression system has the advantages of low culture cost and large expression amount. However, the HPVL1 protein expressed in the E. coli expression system often loses its correct natural conformation and is expressed in the precipitate in the f...

Embodiment 1

[0165]Example 1: Construction of recombinant HPV16L1 gene expression vector and expression of HPV16L1 protein

[0166] Construction of pTO-T7-HPV16L1 non-fusion expression vector

[0167] Using DNA extracted from vaginal secretions of cervical cancer patients in Xiamen, Fujian Province as a template, 16H5521F: 5'-TAT AgT TCC Agg gTC TCC AC-3' (SEQ ID NO: 1) as a forward primer, 16H7190R: 5 '-ACA ACA AAC AAC ACT AAT TCA A-3' (SEQID NO: 2) is a reverse primer, and the PCR reaction is carried out in a PCR thermal cycler (T3 type PCR instrument produced by Biometra Company) according to the following conditions: 94°C for 5 minutes ; followed by 25 cycles of 94°C for 50 seconds, 57°C for 50 seconds, and 72°C for 2 minutes and 30 seconds; and finally an extension at 72°C for 10 minutes. A specific product with a size of about 1.6kb was obtained, which was used as a template for another PCR reaction. c16HL5653F: 5'- ggA TCC CAT ATg CTT CCT AgT gAg gCC ACT gTC-3' (SEQ ID NO: 3)...

Embodiment 2

[0175] Example 2 Study on Salt-Free Precipitation and Reconstitution Conditions of HPV16L1 Protein

[0176] Massive expression of HPV-16L1 protein

[0177] Each 1L Erlenmeyer flask was filled with 500mL LB liquid medium (kanamycin resistant) to inoculate 100 μL of seed bacteria, shake the flask at 37°C for 8 hours, add IPTG to a final concentration of 0.3mM, and induce at 25°C for 6 hours. The expressed HPV-16L1 protein is expressed in the cytoplasm in two forms of inclusion body and soluble protein, and the protein expressed in the soluble form can be released into the cell lysate supernatant after the cells are broken. Although the relative content of soluble protein is low, it can still be purified by the specific salt-free precipitation method adopted in the present invention.

[0178] Determination of cell lysate

[0179] Centrifuge at 8500rpm for 10min to collect the bacteria. The precipitate obtained by centrifuging 1000mL of bacterial liquid was resuspended with...

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Abstract

The present invention provides a purification method of human papillomavirus late protein L1 from escherichia coli. Virus-like Particles; the virus-like Particles with elctrophoresis purity more than 98 percent can be produced in a large scale through salt free precipitation, re-dissolving, ion exchange chromatography, hydrophobic interaction chromatography and renaturation of the L1 protein in the lysate supernatant of escherichia coli. With good immunogenicity, the virus particles can induce neutralizing antibodies towards homologous HPV virus, and can be used as a vaccine for preventing the infection of HPV.

Description

technical field [0001] The invention relates to a method for expressing and purifying human papillomavirus L1 protein and obtaining virus-like particles using a prokaryote (especially Escherichia coli) expression system. Background technique [0002] Human papillomavirus HPV (Human Papillomavirus) is a non-enveloped DNA virus belonging to the family Papovaviridae. The viral genome is a double-stranded closed circular DNA with a size of about 7.2-8 kb and 8 open frames. The genome can be divided into three regions according to different functions: ① early region (E), about 4.5kb, encoding E1, E2, E4-E76 non-structural proteins related to virus replication, transcription and transformation; ② late region (L ), about 2.5kb, encoding major capsid protein L1 and minor capsid protein L2; ③Long regulatory region (LCR), located between the end of the L region and the beginning of the E region, about 800-900bp long, does not encode any protein , containing DNA replication, expressi...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N1/21C07K1/14C07K14/025C12N7/01A61K39/12
Inventor 李少伟沈文通潘晖榕鲜阳凌张军夏宁邵
Owner XIAMEN UNIV
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