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Hybridoma cell strain capable of secreting anti-rift valley fever virus NSs protein monoclonal antibody and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in antiviral immunoglobulin, analytical materials, biological material analysis, etc., can solve the problem of inability to distinguish infection antibodies and immune antibodies, and achieve good specificity and repeatability Effect

Active Publication Date: 2020-04-21
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The laboratory detection of RVFV mainly includes ELISA, RT-PCR, virus isolation, etc. Among them, the ELISA method with N protein as the target antigen has been reported to be used for virus antibody detection, but this method cannot distinguish infection antibodies from immune antibodies

Method used

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  • Hybridoma cell strain capable of secreting anti-rift valley fever virus NSs protein monoclonal antibody and application thereof
  • Hybridoma cell strain capable of secreting anti-rift valley fever virus NSs protein monoclonal antibody and application thereof
  • Hybridoma cell strain capable of secreting anti-rift valley fever virus NSs protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of embodiment 1 recombinant protein

[0022] 1. Construction of recombinant plasmids and protein expression

[0023] According to the Rift Valley fever virus NSs gene sequence (HE687307) published by Genbank, the sequence shown in SEQ ID NO:1 was obtained. The sequence shown in SEQ ID NO:1 was synthesized and cloned into pET-28a(+) to obtain the recombinant plasmid pET-28a-NSs. The recombinant plasmid pET-28a-NSs was transformed into Escherichia coli competent cells, and the positive colonies were picked and named pET-28a-NSs (BL21) after PCR identification. Inoculate pET-28a-NSs (BL21) into LB liquid medium containing kanamycin for culture, when OD 600 When it reaches 0.6-0.8, add 0.5mmol / L IPTG, induce expression at 37°C for 5h, collect the cells and lyse them with ultrasonic waves, collect the supernatant and precipitate of the lysate, and identify the expression of the recombinant protein by SDS-PAGE gel electrophoresis. Transform pET-28a(+) into ...

Embodiment 2

[0028] Embodiment 2 Establishment of Monoclonal Antibody Hybridoma Cell Line

[0029] 1. Immunization of BALB / c mice

[0030] 100 μg of recombinant NSs protein and Freund's adjuvant were mixed and emulsified at a volume ratio of 1:1, and then subcutaneously injected into BALB / c mice (100 μg / mouse). Afterwards, two booster immunizations were performed, with an interval of two weeks between each time and the previous immunization. For each booster immunization, a mixture of 100 μg of recombinant NSs protein and incomplete Freund's adjuvant was mixed and emulsified at a ratio of 1:1. A total of 3 times of immunization. Blood was collected 2 weeks after the third immunization, and the serum titer of immunized mice was detected by indirect ELISA. The indirect ELISA method is as follows: the recombinant NSs protein was coated with a concentration of 2 μg / mL on a 96-well microtiter plate, 100 μL / well, overnight at 4°C. Wash with PBST 3 times and pat dry; add PBST containing 0.5% B...

Embodiment 3

[0040] Example 3 Establishment of RVFV NSs Blocking ELISA Antibody Detection Method

[0041] 1. Determination of the coating concentration of the antigen and the dilution of the enzyme-labeled monoclonal antibody

[0042] The recombinant NSs protein was diluted to 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL with antigen coating solution (0.05 mol / L, pH 9.6 carbonate buffer) by square array titration method, Add 100 μL to each well, incubate overnight at 4°C; wash three times with PBST (PBS containing 0.5% Tween-20); add 300 μL PBST containing 0.5% BSA to each well, block at 37°C for 2 hours; wash three times with PBST; Add 1:10 diluted RVFV negative and positive sera to the antigen concentration, incubate at 37°C for 1 hour; wash with PBST 3 times; add 100 μL respectively and use PBST according to the dilution of 1:500, 1:1000, 1:1500, 1:2000, 1 :2500, 1:3000 diluted HRP-2D6D10 (the concentration of HRP-2D6D10 before dilution was 1mg / mL), incubated at 37°C for 1h; washed 3 times wit...

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Abstract

The invention provides a hybridoma cell strain capable of secreting an anti-rift valley fever virus NSs protein monoclonal antibody and an application thereof, and belongs to the technical field of biology. The preservation number of the hybridoma cell strain 2D6D10 capable of secreting an anti-rift valley fever virus NSs protein monoclonal antibody is CCTCC NO: C2019269. The invention further provides an anti-rift valley fever virus NSs protein monoclonal antibody secreted by the hybridoma cell strain and an application of the anti-rift valley fever virus NSs protein monoclonal antibody in preparation of a rift valley fever virus NSs protein antibody detection kit. The hybridoma cell strain 2D6D10 disclosed by the invention can generate a monoclonal antibody aiming at RVFV non-structuralprotein NSs protein, and the monoclonal antibody has a blocking effect, so that the blocking ELISA kit disclosed by the invention can be used for differential diagnosis of virus infection and inactivated vaccines or deletion vaccine immune antibodies.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a hybridoma cell strain secreting monoclonal antibody against NSs protein of Rift Valley fever virus and application thereof. Background technique [0002] Rift valley fever (RVF) is a zoonotic disease caused by Rift valley fever virus (RVFV) in ruminants and humans. The disease has a serious impact on ruminants such as sheep, goats, and cattle. Symptoms of suspected influenza will appear after infection. In severe cases, it will cause abortion of pregnant female animals, and there is a high mortality rate in young animals. Humans are susceptible to RVFV and can be infected by contacting or handling infectious materials or biting mosquito vectors. After infection, it can manifest as fatigue, headache, fever, muscle and joint pain, jaundice, and even encephalitis, hemorrhagic fever, etc., and severe cases can lead to die. [0003] Rift Valley fever virus (RVFV) belongs to the genus Ph...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/68G01N33/577G01N33/569G01N33/535C12R1/91
CPCC07K16/10G01N33/68G01N33/577G01N33/56983G01N33/535G01N2333/175
Inventor 李文良张聪张纹纹陈亚玲杨蕾蕾毛立李基棕孙敏刘茂军
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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