Antigen conformation epitope mimic peptide of hepatitis C virus F protein and application thereof

A hepatitis C virus and conformational epitope technology, applied in the field of biomedicine, can solve the problems of high preparation cost, difficulty in realization, and complexity, and achieve the effects of stable phage particles, no need for purification process, and good immunogenicity

Inactive Publication Date: 2012-07-18
CHINA PHARM UNIV +2
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0009] The current research on the antigenicity of the F protein of hepatitis C virus is mainly the complete sequence of the F gene containing the linear epitope. Some researchers used the prokaryotic expression of the 65-134 amino acid of the F protein inserted with the tropism codon of Escherichia coli to prepare a detection kit ( CN 101407813B), the key to the realization of this method is the acquisition of F protein fragments (continuous 70 amino acid residue sequences) as coated antigens. Due to the complexity of the required molecular biology experiment techniques, its realization is difficult, and the preparation The cost is high; and the antigenic conformation epitope mimic peptide of the HCV F protein screened by a phage peptide library as shown in SEQ ID NO.1 has not been reported so far

Method used

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  • Antigen conformation epitope mimic peptide of hepatitis C virus F protein and application thereof
  • Antigen conformation epitope mimic peptide of hepatitis C virus F protein and application thereof
  • Antigen conformation epitope mimic peptide of hepatitis C virus F protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, prokaryotic expression and purification of hepatitis C virus F protein

[0038] 1. Materials: E.coli TG1 was purchased from Stratagen. IPTG was purchased from Promega Corporation. Glutathione Sepharose 4B gel was purchased from Pharmacia. The PCR kit was purchased from Shenergy Gaming Company. Primers were synthesized by Shanghai Boya Company. Various restriction endonucleases, DNA ligases, vectors and protein markers were purchased from TaKaRa Company. DNA gel recovery kit was purchased from Shanghai Huashun Company.

[0039] 2. Method:

[0040] 1) Primers

[0041] Upstream primer P1: 5′-GT AGCACAAATCCTAAGCCTCAGAG-3' (SEQ ID NO. 2);

[0042] Upstream primer P2: 5'-CTAAGCCTCAGAGAAAGCCAAACGTAACACC-3' (SEQ ID NO.3);

[0043] Upstream primer P3: 5′-GT CCAAACGTAACACC-3' (SEQ ID NO. 4);

[0044] Downstream primer P4: 5′-GA GCAACCAGGCAGA-3' (SEQ ID NO. 5).

[0045] The above primers refer to the master thesis "Cloning, Expression and Preliminary ...

Embodiment 2

[0055] Example 2, Purification and Identification of F Protein Antibody

[0056] 1) Materials: HRP-labeled goat anti-mouse IgG was purchased from Nanjing Arendi Company. BALB / c mice aged 6-8 weeks were purchased from Shanghai Slack Experimental Animal Co., Ltd. and raised by the Military Medical Research Institute of Nanjing Military Region. Freund's complete adjuvant and Freund's incomplete adjuvant are both sigma products. Western blot kit was purchased from Huamei Bioengineering Company.

[0057] 2) Preparation of anti-HCV-F antibody The concentration of purified HCV-F measured by spectrophotometer is 1 μg / μL, and the antigen and adjuvant are mixed according to the volume ratio of 1:1, and repeatedly sucked with a sterile syringe for 30 minutes to make it completely emulsified . Two mice were used as blank controls, and eight mice were used for immune experiments. In the first week, the mice were subcutaneously injected with the emulsified antigen and Freund's complete ad...

Embodiment 3

[0060] Embodiment 3, phage random peptide library screening

[0061] 1) M13 phage dodecapeptide library (Ph.D.-12 TM Phage Display Peptide Library Kit) purchased from New England Biolabs company

[0062] 2) Screening method: Coat the microtiter plate with mouse anti-HCV-F antibody (100 μg / ml) diluted in coating solution (carbonic acid buffer solution, pH 9.6), overnight at 4°C, add blocking solution at 4°C for 2-3 hours, wash with TBST, and spin dry the washing solution. Add diluted phage random peptide library to each well, and the number of phage added in each round should be 2×10 10-11 CFU, incubate at room temperature for 1 hour, shake off the liquid, rinse with TBST buffer containing 0.1% Tween-20 (v / v), and finally use 100 μl pH 2.2 glycine-hydrochloric acid buffer to elute the protein specifically bound to the F protein antibody phage, and immediately neutralized with 10 μl pH 9.8 Tris buffer, 10 μl was taken for phage titer determination, and the remaining phages w...

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Abstract

The invention belongs to the field of molecular biology and immunology, and discloses antigen conformation epitope mimic peptide of hepatitis C virus F protein and the application of the antigen conformation epitope mimic peptide. The sequence of the antigen conformation epitope mimic peptide of the hepatitis C virus F protein is SEQID (SEQuence IDentifier) NO.1. The antigen conformation epitope mimic peptide adopts the phage display technology, takes anti-rat HCV-F (Hepatitis C Virus-F) antiserum as coating antibodies, screens a phage 12-mer peptide library, and then adopts methods such as ELISA (Enzyme-Linked Immuno-Sorbent Assay), DNA (Deoxyribose Nucleic Acid) sequencing, immunoblotting, bioinformatic analysis and the like to process positive clones that are obtained after screening is carried out for four times, so that the antigen conformation epitope mimic peptide of the hepatitis C virus F protein is found finally, and the antigen conformation epitope mimic peptide can be in specific binding with the anti-rat HCV-F antiserum. The antigen conformation epitope mimic peptide of the hepatitis C virus F protein can be used for preparing a hepatitis C F antibody diagnostic kit. The antigen conformation epitope mimic peptide of the hepatitis C virus F protein has important social benefits and economic benefits for preventing and controlling hepatitis C.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a hepatitis C virus F protein antigen conformation epitope mimic peptide and application thereof. Background technique [0002] According to the National Statutory Infectious Disease Epidemic Report issued by the Ministry of Health in December 2011, among the 28 infectious diseases in my country, the incidence and mortality of viral hepatitis rank first. Hepatitis C virus (HCV) is the main cause of blood transfusion or community-acquired non-A non-B hepatitis. It is the pathogen of hepatitis C. The worldwide infection rate is 3%. More than 170 million. In my country, about 75% of patients with acute hepatitis C infection will develop into chronic hepatitis, and about 20% of patients will eventually develop into end-stage liver diseases such as liver cirrhosis and hepatocellular carcinoma. HCV infection has become a severe public health problem faced by our country and even th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K19/00C12N15/51G01N33/576
Inventor 岳明邓小昭鲁卫东孔晶韦娟余晓杰徐孝东张云张锦海
Owner CHINA PHARM UNIV
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