Preparation and application of a neutralizing monoclonal antibody against hepatitis C virus

Abstract

The invention discloses preparation of a neutralizing monoclonal antibody of an anti-hepatitis C virus and application thereof. E2 protein is utilized to strengthen immunity after mice are immunized by pVAX-CpG-N2N8; spleen of the mice with the highest valence aiming at the E2 antibody is taken as an antigen-sensitized B cell and fused with a myeloma cell SP2 / 0 strain, and the fused cell is screened in HAT culture medium, so as to obtain the fused cell, and the fused grows and is cloned to obtain the hybridoma cell strain generating the monoclonal antibody disclosed by the invention. The neutralizing monoclonal antibody of the anti-hepatitis C virus E2 disclosed by the invention is generated by the hybridoma cell strain; epitope combined with the monoclonal antibody is at F550GCTWMNSTGFTKVCGAPPCVIG572 part of E2 glycoprotein. The neutralizing monoclonal antibody of the anti-hepatitis C virus disclosed by the invention has good capability of neutralizing HCV infection, can be used as a hepatitis C virus therapeutic antibody, and has a great application prospect in hepatitis C diagnosis and treatment.

Description

technical field

[0001] The invention belongs to the fields of molecular biology and infection immunity, and specifically relates to a neutralizing monoclonal antibody against hepatitis C virus (HCV), a hybridoma cell line producing the antibody and applications thereof. Background technique

[0002] my country is one of the countries with a high infection rate of HCV (hepatitis C virus, hepatitis C virus). HCV infection is one of the main factors leading to chronic hepatitis, liver cancer, and cirrhosis, which seriously affects human health. Currently there is no effective therapeutic and preventive HCV vaccine, nor effective neutralizing therapeutic antibody.

[0003] Ultimate control of HCV will depend on vaccine prophylaxis. Specific neutralizing antibodies play an important role in the process of resisting virus invasion and immune resistance to HCV attacking human cells. HCV envelope (envelope) glycoproteins (E1 and E2) can induce the body to produce neutralizing anti...

Examples

Embodiment 1

[0043] The preparation of embodiment 1 hybridoma cell line and monoclonal antibody

[0044] 1. Immunization of mice with DNA vaccine and detection of antiserum antibody titer

[0045] Using the plasmid DNA extraction kit from OMEGABiotek, pVAX-1-CpG-N2N8 was extracted, purified, and endotoxin was removed, and then different plasmids were introduced into Balb through a gene importer with an amount of 70 μg and 500 UIL-2 (immune adjuvant). / c mice (female, 6-8 weeks) were immunized in the thigh muscles on the 1st day, 10th day and 25th day respectively, and were immunized three times in total, and E2 protein (LiP, etal.Vaccine, 2007) was boosted on the 32nd day Immunization once, 60 μg / mouse, mouse serum was taken 10 days after the last booster immunization, and the antibody titer against E2 protein was detected by enzyme-linked immunosorbent assay. The mouse with the highest antibody titer was selected, and about 300 μg of E2 protein was injected intraperitoneally for 3 consec...

Embodiment 2

[0077] Example 2 Monoclonal Antibody Antibody Titer and Subtype Detection

[0078] The concentration of the purified monoclonal antibody 1C2 was determined to be 1.57 mg / mL, and the concentration of the control antibody was adjusted to be the same as that of 1C2. The control antibody was a monoclonal antibody that had no HCV neutralizing effect during the antibody screening process. Start with 1:200 and dilute the two antibodies (1:200, 1:400, 1:800, ... 1:25600) to detect the antibody titer of 1C2. The specific method is as follows:

[0079] (1) Coat ELISA plate with 10 μg / mL LE2 protein, 100 μL / well, overnight at 4°C.

[0080] (2) Block with 1% BSA, 37°C for 1 hour.

[0081] (3) Wash three times with PBST, 2 minutes each time. Then add the diluted 1C2 antibody and control antibody (the monoclonal antibody without HCV neutralizing effect during the antibody screening process), 100 μL / well, at 37°C for 1 hour.

[0082](4) Wash three times with PBST, 2 minutes each time. Th...

Embodiment 3

[0086] Embodiment 3 The detection of monoclonal antibody antibody specificity

[0087] In order to detect whether the monoclonal antibody 1C2 can specifically bind to the E2 protein, the ELISA method was used for the experiment, and the specific method is as follows:

[0088] (1) 10 μg / mL LE2 protein was coated on the ELISA plate, 100 μL / well, overnight at 4 °C; meanwhile, 10 μg / mL BSA and GST proteins were used as control proteins, and the ELISA plate was also coated, 100 μL / well.

[0089] (2) Block with 1% BSA, 37°C for 1 hour.

[0090] (3) Wash three times with PBST, 2 minutes each time. Then add monoclonal antibody 1C2 (1.57mg / mL) diluted 1:800, 100 μL / well, 37°C for 1 hour.

[0091] (4) Wash three times with PBST, 2 minutes each time. Then add HRP-goat anti-mouse IgG secondary antibody (1:4000) 100 μL / well, 37°C for 1 hour.

[0092] (5) Wash three times with PBST, 2 minutes each time. Add TMB for color development, 50 μL / well, then add 2M concentrated sulfuric acid t...

technical field

[0001] The invention belongs to the fields of molecular biology and infection immunity, and specifically relates to a neutralizing monoclonal antibody against hepatitis C virus (HCV), a hybridoma cell line producing the antibody and applications thereof. Background technique

[0002] my country is one of the countries with a high infection rate of HCV (hepatitis C virus, hepatitis C virus). HCV infection is one of the main factors leading to chronic hepatitis, liver cancer, and cirrhosis, which seriously affects human health. Currently there is no effective therapeutic and preventive HCV vaccine, nor effective neutralizing therapeutic antibody.

[0003] Ultimate control of HCV will depend on vaccine prophylaxis. Specific neutralizing antibodies play an important role in the process of resisting virus invasion and immune resistance to HCV attacking human cells. HCV envelope (envelope) glycoproteins (E1 and E2) can induce the body to produce neutralizing anti...

Examples

Embodiment 1

[0043] The preparation of embodiment 1 hybridoma cell line and monoclonal antibody

[0044] 1. Immunization of mice with DNA vaccine and detection of antiserum antibody titer

[0045] Using the plasmid DNA extraction kit from OMEGABiotek, pVAX-1-CpG-N2N8 was extracted, purified, and endotoxin was removed, and then different plasmids were introduced into Balb through a gene importer with an amount of 70 μg and 500 UIL-2 (immune adjuvant). / c mice (female, 6-8 weeks) were immunized in the thigh muscles on the 1st day, 10th day and 25th day respectively, and were immunized three times in total, and E2 protein (LiP, etal.Vaccine, 2007) was boosted on the 32nd day Immunization once, 60 μg / mouse, mouse serum was taken 10 days after the last booster immunization, and the antibody titer against E2 protein was detected by enzyme-linked immunosorbent assay. The mouse with the highest antibody titer was selected, and about 300 μg of E2 protein was injected intraperitoneally for 3 consec...

Embodiment 2

[0077] Example 2 Monoclonal Antibody Antibody Titer and Subtype Detection

[0078] The concentration of the purified monoclonal antibody 1C2 was determined to be 1.57 mg / mL, and the concentration of the control antibody was adjusted to be the same as that of 1C2. The control antibody was a monoclonal antibody that had no HCV neutralizing effect during the antibody screening process. Start with 1:200 and dilute the two antibodies (1:200, 1:400, 1:800, ... 1:25600) to detect the antibody titer of 1C2. The specific method is as follows:

[0079] (1) Coat ELISA plate with 10 μg / mL LE2 protein, 100 μL / well, overnight at 4°C.

[0080] (2) Block with 1% BSA, 37°C for 1 hour.

[0081] (3) Wash three times with PBST, 2 minutes each time. Then add the diluted 1C2 antibody and control antibody (the monoclonal antibody without HCV neutralizing effect during the antibody screening process), 100 μL / well, at 37°C for 1 hour.

[0082](4) Wash three times with PBST, 2 minutes each time. Th...

Embodiment 3

[0086] Embodiment 3 The detection of monoclonal antibody antibody specificity

[0087] In order to detect whether the monoclonal antibody 1C2 can specifically bind to the E2 protein, the ELISA method was used for the experiment, and the specific method is as follows:

[0088] (1) 10 μg / mL LE2 protein was coated on the ELISA plate, 100 μL / well, overnight at 4 °C; meanwhile, 10 μg / mL BSA and GST proteins were used as control proteins, and the ELISA plate was also coated, 100 μL / well.

[0089] (2) Block with 1% BSA, 37°C for 1 hour.

[0090] (3) Wash three times with PBST, 2 minutes each time. Then add monoclonal antibody 1C2 (1.57mg / mL) diluted 1:800, 100 μL / well, 37°C for 1 hour.

[0091] (4) Wash three times with PBST, 2 minutes each time. Then add HRP-goat anti-mouse IgG secondary antibody (1:4000) 100 μL / well, 37°C for 1 hour.

[0092] (5) Wash three times with PBST, 2 minutes each time. Add TMB for color development, 50 μL / well, then add 2M concentrated sulfuric acid t...

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