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Preparation and application of hepatitis C virus recombinant protein

A virus and protein technology, applied in the direction of anti-viral immunoglobulin, application, viral peptide, etc., can solve the problem of not being broad-spectrum

Inactive Publication Date: 2016-02-17
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high variability of HCV itself, the encapsulation of lipoprotein components in serum, the shielding of neutralization sites by glycosylation, and the effect of interfering antibodies, the antibodies produced by early HCV candidate vaccines are often only for the same subtype The virus has a neutralizing effect, not broad-spectrum

Method used

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  • Preparation and application of hepatitis C virus recombinant protein
  • Preparation and application of hepatitis C virus recombinant protein
  • Preparation and application of hepatitis C virus recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0221] Expression of embodiment 1sE2 in fruit fly S2 cell

[0222] Recombinant plasmids used to express HCVsE2 protein in Drosophila S2 cells such as figure 1 As shown in A, the sE2 wild-type coding gene or the codon-optimized gene encodes the target protein in the extracellular region of the E2 protein (including amino acids 384-661). Drosophila S2 cells were transiently transfected with vectors expressing the wild-type sE2 coding gene or the codon-optimized gene, and 2×10 cells were removed 48 hours after transfection. 6 Cells were added chromium chloride with a final concentration of 5 μM to induce expression, and the supernatant was collected after 72 hours to detect that the optimized sE2 expression was significantly enhanced ( figure 1 B). Therefore, the inventors selected codon-optimized expression vectors for subsequent experiments. Drosophila S2 cells were co-transfected with pMT-Bip / sE2opti / His and pCoBlast, and the monoclonal cell line sE2A was obtained after bla...

Embodiment 2

[0224] The characteristic analysis of embodiment 2sE2 protein

[0225] The purified sE2 protein was detected by Western blot with His monoclonal antibody and E2 monoclonal antibody (AP33) respectively, and both produced about 46kDa positive bands, indicating that the protein was indeed the target sE2 protein ( figure 2 A). After deglycosylation of sE2 protein was treated with PNGaseF, it was detected with the same antibody, and the positive band position was found to be about 34kDa ( figure 2 A), suggesting that the sE2 protein expressed in S2 insect cells has glycosylation modification.

[0226] Through ELISA detection, sE2 can well bind to the monoclonal antibody AR3A that recognizes discontinuous conformational epitopes, and its ELISA reading value is positively correlated with the monoclonal antibody concentration; although sE2 has a certain binding to the monoclonal antibody AP33 that recognizes linear epitopes, the reaction is not strong ( figure 2 B). This result ...

Embodiment 3

[0230] Embodiment 3sE2 can specifically detect HCV infection patient's serum

[0231] In order to determine whether sE2 can specifically bind to HCV-specific antibodies in HCV-infected persons, the inventors coated the ELISA plate with sE2 protein, and then used diluted healthy human serum and two subtypes (1b and 2a) hepatitis C patient serum respectively The incubation is followed by detection of specific IgG antibodies binding to sE2.

[0232] The results showed that compared with healthy human serum, patient serum produced a high binding activity, and the average OD value was still as high as 0.5-1.0 when diluted at 1:20000 ( image 3 ), indicating that sE2 can efficiently, specifically, and broadly bind the anti-E2 antibody in the serum of infected patients. Therefore, sE2 can be used to develop a simple and rapid diagnostic kit for HCV infection.

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Abstract

The present invention discloses preparation and application of a hepatitis C virus (HCV) recombinant protein, specifically discloses a separated HCV antigen peptide, which is derived from an E2 protein of HCV virus; the antigenic peptide can bind to an anti-HCV antibody; and the HCV is a Con1 strain of HCV 1b genotype. The invention provides a composition containing the above antigen peptide and a preparation method thereof. The HCV antigen peptide of the invention can effectively and specifically combine with anti-E2 antibody in serum of an infected patient in a broad-spectrum way, and can be used to develop a diagnostic kit for the detection of HCV infection.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to the preparation and application of a hepatitis C virus recombinant protein. Background technique [0002] Hepatitis C virus (HCV) infection is one of the main causes of human liver cirrhosis and liver cancer. There are about 170,000,000 people infected worldwide, and the infection rate is as high as 3%, which greatly threatens human health. Currently, there is no preventive or therapeutic vaccine against hepatitis C virus. [0003] With the deepening of clinical research, more and more evidences show that neutralizing antibodies play an important role in the control and clearance of HCV infection. In patients, the rapid induction of neutralizing antibodies, the titer and breadth of neutralizing antibodies are directly related to the clearance of HCV infection. Both chimpanzee experiments and patient clinical sample analysis have shown that during acute HCV infe...

Claims

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Application Information

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IPC IPC(8): C07K14/18C07K19/00C12N15/51C12N15/62A61K39/29A61K48/00A61P31/14C07K16/10G01N33/68G01N33/569
Inventor 黄忠钟劲李大鹏王雪松李莉
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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