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Truncated human papilloma virus 18 type L1 protein

A protein and protein technology, applied in the direction of viruses, viral peptides, viral antigen components, etc., can solve the problems of low yield, many types of miscellaneous proteins, and difficult to apply in large-scale production.

Active Publication Date: 2009-11-04
XIAMEN INNOVAX BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because most of the HPV L1 protein expressed by E. coli loses its natural conformation, it cannot produce protective antibodies against HPV.
Or the above-mentioned protein can also obtain HPV VLP (Kelsall, S.R. and J.K.Kulski (1995). JVirol Methods 53 (1): 75-90) through inclusion body purification, renaturation and other steps, but the amount of protein loss in the renaturation process Large, 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 can help the purification of the target protein (Li, M., T.P.Cripe, etal. (1997). J Virol 71 (4): 2988-95.), but the cleavage of fusion proteins often requires expensive enzymes, which still cannot be applied to large-scale production

Method used

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  • Truncated human papilloma virus 18 type L1 protein
  • Truncated human papilloma virus 18 type L1 protein
  • Truncated human papilloma virus 18 type L1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Example 1: Expression of truncated HPV18 L1 protein with sequence 1

[0152] Preparation of HPV18 L1 gene fragment used as template

[0153] Using DNA extracted from vaginal secretions of cervical cancer patients in Xiamen, Fujian Province as a template, 18H 5430F: 5'-CCT CTT GGG ATG TGC CTG TAT AC-3' (SEQ IDNO: 10) as a forward primer, 18H7190R: 5'-TAC AAA CAC AAC AAT AGA TGT ATATA-3' (SEQ ID NO: 11) is a reverse primer, and the PCR reaction is carried out in a PCR instrument (Biometra, T3) according to the following conditions:

[0154] Denaturation at 94°C for 5 minutes

[0155]

[0156] Extend at 72°C for 10min

[0157] A specific product with a size of about 1.6 kb is obtained through amplification, which is a template for preparing a DNA fragment encoding the truncated HPV-18L protein of the present invention.

[0158] Construction of non-fusion expression vector with truncated HPV18 L1 gene

[0159]The 1.6 kb DNA fragment obtained in the previous step ...

Embodiment 2

[0183] Example 2: Obtaining HPV18N65C-L1 with a purity of about 70%

[0184] The bacteria were resuspended at a ratio of 1 g of bacteria to 10 mL of lysate (20 mM Tris buffer pH 7.2, 300 mM NaCl), and the bacteria were crushed 5 times with an APV homogenizer (product of An Invensys Group) at 600 bar pressure. The JA-14 rotor was centrifuged at 13500 rpm (30000 g) for 15 min, and the supernatant was collected and detected by 10% SDS-polyacrylamide gel electrophoresis. At this time, the purity of HPV18N65C-L1 in the supernatant was about 10%. The supernatant was dialyzed by CENTRASETTE 5 tangential flow device (PALL product), the molecular weight cut-off of the membrane bag used was 30kDa, the dialysate was 10mM phosphate buffer pH 6.0 buffer solution, the dialyzed volume was three times the supernatant volume, and the pressure during operation was 0.5psi, flow rate is 500mL / min, tangential flow rate is 200mL / min. After sufficient dialysis, centrifuge with JA-10 rotor (Beckman ...

Embodiment 3

[0185] Example 3: Chromatographic purification of HPV18N65C-L1

[0186] Purification of HPV18N65C-L1 by Cation Exchange Chromatography

[0187] Instrument system: AKTA explorer 100 preparative liquid chromatography system produced by GE Healthcare former Amershan Pharmacia company.

[0188] Chromatography medium: SP Sepharose 4 Fast Flow.

[0189] Column volume: 5.5 cm x 20 cm.

[0190] Buffer: 20mM phosphate buffer pH7.0, 10mM DTT

[0191] 20mM phosphate buffer pH7.0 10mM DTT 2M NaCl.

[0192] Flow rate: 25mL / min

[0193] Detector wavelength: 280nm

[0194] The sample is 3L of HPV18N65C-L1 solution with a purity of about 70%

[0195] The elution procedure was as follows: 300mM NaCl eluted the impurity protein, 500mM NaCl eluted the target protein, collected the 500mM NaCl eluted product, and obtained a total of 1000mL HPV18N65C-L1 purified sample.

[0196] CHT-II (Hydroxyapatite Chromatography) Purification of HPV18N65C-L1

[0197] Instrument system: AKTAexplorer ...

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Abstract

The invention relates to a truncated human papilloma virus 18 type L1 protein, and viruslike particles consisting of the truncated human papilloma virus 18 type L1 protein, vaccine containing the viruslike particles and the application thereof in preventing cervical cancer.

Description

technical field [0001] The invention relates to a truncated human papillomavirus type 18 L1 protein, a virus-like particle composed of it, a vaccine containing the virus-like particle and its use in preventing cervical cancer. 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, ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/025C12N15/37C12N15/63C12N7/00C12N15/70C12N5/10C12N1/15C12N1/19C12N1/21A61K39/12A61P35/00
CPCC12N2710/20023A61K39/12C12N2710/20034A61K2039/5258C12N2710/20022C07K14/005C12N7/00A61K2039/55566A61K2039/70A61P35/00A61P37/04C12N2710/20011
Inventor 李少伟沈文通李仲艺谢明辉潘晖榕夏宁邵
Owner XIAMEN INNOVAX BIOTECH
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