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HCV (hepatitis C virus) core antigen and antibody thereof as well as hybridoma cell lines secreting antibody

A hybridoma cell line and hepatitis C virus technology, applied in the direction of viral peptides, antiviral immunoglobulins, analytical materials, etc., can solve the problems of difficult large-scale screening, complicated detection operations, expensive prices, etc., and achieve reliable Detection method, high purity, and high-efficiency secretion effect

Active Publication Date: 2012-10-17
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection of HCV virology is complex, easy to contaminate, and expensive, making it difficult to use for large-scale screening

Method used

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  • HCV (hepatitis C virus) core antigen and antibody thereof as well as hybridoma cell lines secreting antibody
  • HCV (hepatitis C virus) core antigen and antibody thereof as well as hybridoma cell lines secreting antibody
  • HCV (hepatitis C virus) core antigen and antibody thereof as well as hybridoma cell lines secreting antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment one immunogen

[0031] 1. Design and synthesize the primer sequence of the HCV core region gene fragment (synthesized by Shanghai Boshang Biotechnology Co., Ltd.), as shown in SEQ ID NO.3 and 4.

[0032] 2. Extract HCV infected patients (20 HCV serum samples used for HCV RNA extraction are from HCV-RNA positive but HCV antibody-negative patients collected by Qilu Hospital of Shandong University, including 16 males and 4 females, with an average age of 45 years old. The genotyping test results were 13 cases of Ib subtype, 7 cases of 2a subtype) serum RNA, reverse transcription PCR, and the reaction system was as follows:

[0033]

[0034] The PCR reaction was carried out according to the following conditions: 94°C for 30s, 55°C for 30s, 72°C for 30s, after 35 cycles, 72°C for 10min.

[0035] 3. After agarose gel electrophoresis, the target PCR product was recovered from the gel, connected to the pMD18-T vector, and transformed into DH5α ...

Embodiment 2

[0046] Embodiment 2 hybridoma preparation monoclonal antibody

[0047] 1. Animal immunity:

[0048] Select 6-week-old Balb / c female mice, subcutaneously inject the immunogen at multiple points, use Freund's complete adjuvant, mix at a ratio of 1:1, and inject subcutaneously with incomplete Freund's adjuvant 10 days after the initial immunization, once a week, three times in a row. After the last immunization, blood was collected by docking the tail, and the mouse antibody titer was initially identified by indirect ELISA method. After 10 days, a booster immunization was given.

[0049] 2. Cell fusion:

[0050] Before fusion, mouse myeloma cells SP2 / 0 were resuscitated and cultured with 8-azaguanine to ensure that the cells were in the best growth state during fusion. On the day of fusion, mouse peritoneal macrophages were taken to prepare feeder cells, and 10ml HAT medium was added to seed them in 96-well plates.

[0051] Three days after the booster immunization of the im...

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Abstract

The invention discloses an HCV (hepatitis C virus) core antigen, which is a polypeptide encoded by a DNA (deoxyribonucleic acid) of which the amino acid sequence is shown in SEQ ID NO.1 and the nucleotide sequence is shown in SEQ ID NO.2. The invention also discloses a hybridoma cell line secreting monoclonal antibodies, the hybridoma cell line is named as SC16-H6 and has been preserved in the China General Microbiological Culture Collection Center on July 14th, 2011, and the preservation number is CGMCC No. 5074. The invention also discloses another hybridoma cell line secreting monoclonal antibodies, the hybridoma cell line is named as SC23-G5 and has been preserved in the China General Microbiological Culture Collection Center on July 14th, 2011, and the preservation number is CGMCC No. 5075. The invention also discloses a monoclonal antibody of an anti-HCV core antigen, which is secreted by the hybridoma cell line (preservation number: CGMCC No. 5074) or the hybridoma cell line (preservation number: CGMCC No. 5075), and has a specificity to the polypeptide shown in SEQ ID No.1.

Description

technical field [0001] The invention relates to a core antigen aimed at the epidemic strain of hepatitis C virus infection in my country, an antibody specific to the antigen, and a hybridoma cell strain capable of secreting and producing the antibody. Background technique [0002] Hepatitis C is an infectious disease caused by hepatitis C virus (HCV) infection, and has become another viral infectious disease that has attracted global attention after hepatitis B and AIDS. At present, there are about 170 million hepatitis C virus carriers in the world, and the number is increasing at a rate of 3 to 4 million people per year; more than 60 million people are infected in my country, with an average infection rate of 3.2%, which is a medium-high prevalence area , HCV genotypes are mainly 1b and 2a, of which 1b type accounts for more than 70%. [0003] HCV infection is prone to chronic transformation, 80-85% of HCV infected patients develop chronic hepatitis C, 20% of them develop ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/18C07K16/10C12N15/51C12N5/20G01N33/577C12R1/91
Inventor 王传新王丽丽王顺
Owner SHANDONG UNIV QILU HOSPITAL
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