Hepatitis B virus surface antigen monoclonal antibody and its application

Abstract

The invention provides a hepatitis B virus surface antigen monoclonal antibody and application thereof, belonging to the field of immunology. In the present invention, after the hepatitis B virus surface antigen is immunized into mice, the mouse spleen cells are fused with the mouse myeloma cells, and the hybridoma cell line SVAHBV-1 that can stably secrete the hepatitis B virus surface antigen monoclonal antibody is screened, and its deposit number is SVAHBV-1. It is CGMCC No.10320, the titer of the monoclonal antibody it secretes is high, up to 106, it can specifically distinguish a variety of common viruses, with good specificity, it can be used for the preparation of hepatitis B virus surface antigen content detection kits and antibodies The detection kit can also be used for differential diagnosis of hepatitis B virus surface antigen, and has wide application prospects and market value.

Description

technical field

[0001] The present invention relates to the field of immunology and vaccinology, in particular to an anti-hepatitis B virus surface antigen monoclonal antibody, a hybridoma cell line producing the antibody and the application of the antibody. Background technique

[0002] Hepatitis B virus (Hepatitis B Virus, HBV) is a hepatotropic virus and a DNA retrovirus of the hepatovirus family. It was discovered by Dana in 1965 and is called Dan-style particles. It mainly exists in liver cells and damages liver cells. The pathogen can infect the body through blood, mother to child and sexual transmission, causing liver cell inflammation, necrosis, fibrosis, damage to the liver, and triggering viral hepatitis B disease, referred to as hepatitis B. There are two types of hepatitis B: acute and chronic. Acute hepatitis B can be cured in 90% of adults, while chronic hepatitis B has different manifestations, including chronic hepatitis B carriers, chronic active hepatitis ...

Examples

Embodiment 1

[0030] Embodiment 1 immunogen preparation and animal immunization

[0031] (1) The p5GK3 plasmid was constructed by molecular cloning technology, and the uracil synthase (orotidine-5-phosphate decarboxylase) gene Ura3 of Hansenula was knocked out by electrotransformation to obtain the corresponding uracil deficiency After the Hansenula strains were selected, the constructed uracil-auxotrophic Hansenula strains were screened through the auxotrophic medium. The HBsAg expression plasmid pDKXMPHT was constructed by molecular cloning technology, transformed into uracil auxotrophic Hansenula, and the Hansenula strains capable of expressing recombinant HBsAg were screened.

[0032] (2) Take the recombinant Hansenula working seed batch bacterial strain expressing the surface antigen of hepatitis B virus constructed by DNA recombination technology, carry out three-stage fermentation through Erlenmeyer flask, seed tank and production tank, and harvest the bacteria.

[0033] (3) Break u...

Embodiment 2

[0039] Example 2 Cell Fusion and Strain Construction

[0040] (1) Resuscitate and culture the SP2 / 0 cell line before cell fusion, expand the culture 3 days before fusion, remove RPMI1640 cell culture medium (Gibco) 1 day before fusion, and add culture medium again to prepare SP2 / 0 cells.

[0041] (2) The immunized mice were sacrificed, and the mouse splenocyte suspension was prepared according to conventional methods.

[0042] (3) Add an appropriate amount of incomplete IMDM culture medium (Gibco) according to the counting results of splenocytes and SP2 / 0 cells, shake and mix the SP2 / 0 cells, and pipette the splenocytes evenly. Then the splenocytes and SP2 / 0 cells were combined at a ratio of 1:2 in a 50ml centrifuge tube and mixed.

[0043] (4) Add incomplete IMDM culture medium to 50ml, centrifuge for 5 minutes, and pour out the supernatant. Lightly tap the bottom of the fusion tube to loosen and evenly precipitate the cells, and place the centrifuge tube in a 37°C water ba...

Embodiment 3

[0050] Example 3 Monoclonal Antibody Cell Line Ascites Preparation and Antibody Titer Detection

[0051] Resuscitate the frozen hybridoma cells obtained in Example 2 according to conventional methods, and cultivate them. When the cells cover more than 50% of the bottom of the 25ml cell culture bottle, BALB / c mice can be inoculated intraperitoneally according to conventional methods, and the ascites can be collected regularly. HBV-1.

[0052] The hepatitis B vaccine stock solution is diluted with 0.01M PBS1:60, 100 μl / well is coated with an enzyme-labeled plate, overnight at 4°C, and the antibody titer of the hepatitis B virus surface antibody ascites HBV-1 secreted by the hybridoma cells of the present invention is detected, and the antibody is detected OD value / negative serum test OD value (calculated as 0.05 if less than 0.05) is greater than or equal to 2.1, the antibody result is positive, and the dilution factor corresponding to the antibody at this time is the antibody t...

technical field

[0001] The present invention relates to the field of immunology and vaccinology, in particular to an anti-hepatitis B virus surface antigen monoclonal antibody, a hybridoma cell line producing the antibody and the application of the antibody. Background technique

[0002] Hepatitis B virus (Hepatitis B Virus, HBV) is a hepatotropic virus and a DNA retrovirus of the hepatovirus family. It was discovered by Dana in 1965 and is called Dan-style particles. It mainly exists in liver cells and damages liver cells. The pathogen can infect the body through blood, mother to child and sexual transmission, causing liver cell inflammation, necrosis, fibrosis, damage to the liver, and triggering viral hepatitis B disease, referred to as hepatitis B. There are two types of hepatitis B: acute and chronic. Acute hepatitis B can be cured in 90% of adults, while chronic hepatitis B has different manifestations, including chronic hepatitis B carriers, chronic active hepatitis ...

Examples

Embodiment 1

[0030] Embodiment 1 immunogen preparation and animal immunization

[0031] (1) The p5GK3 plasmid was constructed by molecular cloning technology, and the uracil synthase (orotidine-5-phosphate decarboxylase) gene Ura3 of Hansenula was knocked out by electrotransformation to obtain the corresponding uracil deficiency After the Hansenula strains were selected, the constructed uracil-auxotrophic Hansenula strains were screened through the auxotrophic medium. The HBsAg expression plasmid pDKXMPHT was constructed by molecular cloning technology, transformed into uracil auxotrophic Hansenula, and the Hansenula strains capable of expressing recombinant HBsAg were screened.

[0032] (2) Take the recombinant Hansenula working seed batch bacterial strain expressing the surface antigen of hepatitis B virus constructed by DNA recombination technology, carry out three-stage fermentation through Erlenmeyer flask, seed tank and production tank, and harvest the bacteria.

[0033] (3) Break u...

Embodiment 2

[0039] Example 2 Cell Fusion and Strain Construction

[0040] (1) Resuscitate and culture the SP2 / 0 cell line before cell fusion, expand the culture 3 days before fusion, remove RPMI1640 cell culture medium (Gibco) 1 day before fusion, and add culture medium again to prepare SP2 / 0 cells.

[0041] (2) The immunized mice were sacrificed, and the mouse splenocyte suspension was prepared according to conventional methods.

[0042] (3) Add an appropriate amount of incomplete IMDM culture medium (Gibco) according to the counting results of splenocytes and SP2 / 0 cells, shake and mix the SP2 / 0 cells, and pipette the splenocytes evenly. Then the splenocytes and SP2 / 0 cells were combined at a ratio of 1:2 in a 50ml centrifuge tube and mixed.

[0043] (4) Add incomplete IMDM culture medium to 50ml, centrifuge for 5 minutes, and pour out the supernatant. Lightly tap the bottom of the fusion tube to loosen and evenly precipitate the cells, and place the centrifuge tube in a 37°C water ba...

Embodiment 3

[0050] Example 3 Monoclonal Antibody Cell Line Ascites Preparation and Antibody Titer Detection

[0051] Resuscitate the frozen hybridoma cells obtained in Example 2 according to conventional methods, and cultivate them. When the cells cover more than 50% of the bottom of the 25ml cell culture bottle, BALB / c mice can be inoculated intraperitoneally according to conventional methods, and the ascites can be collected regularly. HBV-1.

[0052] The hepatitis B vaccine stock solution is diluted with 0.01M PBS1:60, 100 μl / well is coated with an enzyme-labeled plate, overnight at 4°C, and the antibody titer of the hepatitis B virus surface antibody ascites HBV-1 secreted by the hybridoma cells of the present invention is detected, and the antibody is detected OD value / negative serum test OD value (calculated as 0.05 if less than 0.05) is greater than or equal to 2.1, the antibody result is positive, and the dilution factor corresponding to the antibody at this time is the antibody t...

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