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PRRSV broad-spectrum neutralizing monoclonal antibody 5D9 and application thereof

A monoclonal antibody, broad-spectrum technology, applied in applications, antibodies, antiviral agents, etc., can solve the problems of unreported monoclonal antibody application, lack of neutralizing activity, etc., to prevent virus invasion and protect from infection. Effect

Active Publication Date: 2020-05-01
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Monoclonal antibodies with high neutralizing activity have also appeared in the prior art. For example, the MAb LM26 prepared by Liu Mengying et al. and activity, the highest neutralization titer is 1:50; B. Pirzadeh et al. ( Journal of General Virology (1997), 78, 1867–1873) the monoclonal antibody against GP5 had neutralizing activity against American type VR-2332, and the neutralizing potency was 1:32-1:128, but it had neutralizing activity against European type (Lelystad prototype) does not have neutralizing activity
However, monoclonal antibodies with broad-spectrum neutralization and their applications have not been reported in existing studies

Method used

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  • PRRSV broad-spectrum neutralizing monoclonal antibody 5D9 and application thereof
  • PRRSV broad-spectrum neutralizing monoclonal antibody 5D9 and application thereof
  • PRRSV broad-spectrum neutralizing monoclonal antibody 5D9 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Preparation and Identification of Monoclonal Antibody 5D9

[0026] 1.1 Establishment of hybridoma cell lines

[0027] PRRSV virus SD16 (provided by the Immunobiology Laboratory of Northwest Agriculture and Forestry College of Veterinary Medicine) was used as an immunogen, mixed and emulsified with Freund's complete adjuvant (Sigma company) 1:1, and immunized 6-week-old female Balb / c mice (provided by provided by Xi'an Jiaotong University), subcutaneously injected into the abdomen at a dose of (3×10^7 PFU / bird), and booster immunization every 14 days. 7 days after the third immunization, the tail vein blood was collected and the antibody titer against the immunogen in the mouse serum was detected by IFA. The mouse with the best titer was injected into the tail vein for shock immunization, and the immunogen was mixed with normal saline 1:1. , the dose is (3×10^7 PFU / bird).

[0028] 1.2 Cell Fusion

[0029] (1) Spleen cells of the immunized mice were aseptica...

Embodiment 2

[0053] Example 2: Determination of neutralizing activity of monoclonal antibody 5D9

[0054] 2.1 Determination of neutralizing activity

[0055] The virus neutralization experiment was carried out using the monoclonal antibody 5D9 to detect its neutralizing activity. Spread PAM cells into 24-well plates, insert 0.01 MOI of different types of PRRSV SD16 virus, each virus was incubated with 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml of antibodies, and incubated at 37°C for 1 hour before changing After 36 hours, Western blot and qPCR tests were performed to confirm that it had neutralizing activity. For specific results, see figure 1 .

[0056] based on figure 1 The results show that the alveolar macrophages (PAMs), the natural target cells of PRRSV in the host, were cultured in vitro, and the purified monoclonal antibody 5D9 was used in accordance with the concentrations of 0.05, 0.1, 0.2, 0.4 μM / mL (micromoles per milliliter) and 0.01M PRRSV-SD16 virus was cultured at 37...

Embodiment 3

[0062] Example 3: Preparation of monoclonal antibody 5D9 ascites and detection of neutralizing activity

[0063] 3.1 Preparation of ascites

[0064] The mice were sensitized by injecting paraffin oil one week in advance, and the screened hybridoma cells in the logarithmic growth phase were intraperitoneally injected into the mice one week later, 5×10^5 cells per mouse. Ascitic fluid was collected 7 days after the injection of hybridoma cells. The ascites fluid was taken out and centrifuged at 5000g×10min at 4°C. The supernatant ascites was collected in EP tubes and stored at -20°C.

[0065] 3.2 Detection of ascites neutralizing activity of monoclonal antibody 5D9

[0066] The ascites fluid containing the monoclonal antibody was used to conduct a virus neutralization experiment by 4-fold dilution method to detect the neutralization activity. Spread PAM cells into 24-well plates, insert 0.01MOI of PRRSV-SD16 virus (PRRSV-II type) or PRRSV-GZ11 (PRRSV-I type virus), respectivel...

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Abstract

According to the invention, PRRSV virus liquid-SD16 is used as an immunogen to immunize a Balb / c mouse. Through cell fusion and virus infection Marc145 cell screening and cloning, a positive hybridomacell line capable of efficiently secreting monoclonal antibodies is obtained, and the mouse monoclonal antibody 5D9 is obtained. An ELISA technology is used to determine that the subtype of the monoclonal antibody 5D9 is an IgM type monoclonal antibody. The PRRSV-I type virus and the PRRSV-II type virus infect Marc145 cells; the monoclonal antibody is used for detection by utilizing an IFA technology; and the monoclonal antibody 5D9 is proved to have broad-spectrum reactivity to the PRRSV-I type virus and the PRRSV-II type virus. The monoclonal antibody 5D9 is used for carrying out a virus neutralization experiment; a Western blot technology and a qPCR technology are used for proving that the monoclonal antibody has neutralization activity on PRRSV-I and PRRSV-II viruses, can prevent virus invasion and can protect an organism from being infected.

Description

technology field; [0001] The invention belongs to the biological field, and in particular relates to a PRRSV monoclonal antibody with broad-spectrum neutralizing activity, capable of broad-spectrum neutralizing type I and type II viruses, and can be used for the development of therapeutic drugs for porcine reproductive and respiratory syndrome. Background technique: [0002] Porcine reproductive and respiratory syndrome (PRRS) is a viral disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV), characterized by abortion in sows and respiratory disturbance in piglets. infectious disease and can cause severe immunosuppression. The virus has been widely spread in pig populations all over the world, causing huge economic losses to the pig industry in the world. PRRSV is an enveloped, single-stranded positive-sense RNA virus belonging to the family Arteriviridae and the genus Arterivirus. There are curren...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/577G01N33/569A61K39/42A61P31/14
CPCC07K16/10G01N33/577G01N33/56983A61P31/14C07K2317/56A61K2039/505
Inventor 南雨辰周恩民武春燕
Owner NORTHWEST A & F UNIV
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