Monoclonal antibody specifically combined with porcine rotavirus and application thereof

A monoclonal antibody and porcine rotavirus technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulin, etc., can solve the problems of expensive instruments, high background, and time-consuming separation and identification, and achieve rapid detection and high sensitivity Effect

Active Publication Date: 2021-06-01
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods for detecting porcine rotavirus include virus isolation and identification, serum examination and PCR method, but the isolation and identification, serum examination take too long, and the PCR method is sensitive but requires expensive instruments and professional operators
Al

Method used

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  • Monoclonal antibody specifically combined with porcine rotavirus and application thereof
  • Monoclonal antibody specifically combined with porcine rotavirus and application thereof
  • Monoclonal antibody specifically combined with porcine rotavirus and application thereof

Examples

Experimental program
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Example Embodiment

[0060]Example 1 Preparation, purification and identification of monoclonal antibodies of pig-shaped virus

[0061]1.1 Preparation of monoclonal antibodies

[0062]Cultivation of porcine-shaped virus OSU strain (purchased from ATCC), centrifuge with the harvested virus liquid for 5000 rpm for 30 minutes, and centrifuged at 32,000 rpm and centrifugation for 2 hours, and the precipitate was resuspended; formulated 55%, 65 %, 75% density sucrose solution, adding a centrifuge tube to prepare 55% -75% sucrose concentration gradient, adding a precipitated resuspension, 4 ° C, centrifugation for 2 hours, 30% -70% concentration gradient The virus liquid is purified, and the protein concentration is 0.98 mg / ml after purification; it is emulsified with the Frego adjuvant, and the mice were immunized by 30 μg / per 2 weeks. Indirect immunofluorescence (IFA) (PROV vaccination cells) of 30% of lesions were fixed with 80% acetone at 4 ° C for 30 min, and washed 3 times with pH 7.4 phosphate buffer sol...

Example Embodiment

[0104]Example 2 Preparation and application of the kit

[0105]2.1 pairing study of monoclonal antibodies

[0106]The monoclonal antibody 8c3d6, 6d4a8 was respectively used as a gold bond antibody, and the immobilized compound was prepared. Dilute the Prov OSU strain, HN03 strain to 105.0TCID50 / ml, the clinical negative feces identified by the PROV OSU strain, HN03 strain and RT-PCR, and the results are shown in Table 5: Terminal use of gold label antibody 6D4A8, fixed antibody 8C3D6 pairing detection of PROV OSU strain, HN03 strain dilution is positive, clinical The negative feces is weak positive, which is false positive. Therefore, the gold label antibody 6D4A8 is selected, and the immobilized antibody 8C3D6 is prepared for the kit.

[0107]Table 5 Parappiness results of monoclonal antibodies

[0108] Fixed antibody Gold bond antibody OSU strain HN03 strain Clinical negative feces 6D4A8 8c3d6 +++ 8c3d6 6D4A8 ++-

[0109]Note: "+" means positive, "-" indicates negative.

[0110]2.2 Preparation...

Example Embodiment

[0154]Example 3 Effect of monoclonal antibodies 8C3D6 prevention and / or treatment of viral infections

[0155]3.1 Evaluation of Monoclonal Antibody 8C3D6 to prevent viral infection

[0156]Filter PEDV, TGEV, PROV antigen, antibody bungeic 2-3 years old, randomly divided into three groups, first group and second groups, orally, the monoclonal antibody prepared in Example 1, 3C3D6 2ml, third Group Oral PBS buffer 2ml, 24 hours after oral administration and the third group of oral PROV epidemic strain HN03 small intestine poison 2ml (including 1000 minimum pathogeneous dose), the second set Oral OSU strain small intestine poison (including 1000 The minimum pathogeneity is the daily clinical symptom of piglets, 14 days, continuous observation, through clinical incidence and onset, mortality and use PCR method to detect the number of virals in anal flue to evaluate the number of monoclonal antibodies 8C3D6 The effect of porcine-shaped viral infection is shown in Table 13.

[0157]Table 13 Test ...

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Abstract

The invention provides a variable region sequence of a mouse monoclonal antibody specifically combined with porcine rotavirus, an antibody or an antibody fragment prepared from the variable region sequence, an application of the antibody, and a kit and a pharmaceutical composition prepared from the antibody. The antibody disclosed by the invention can be used for indirect immunofluorescence, immunohistochemistry and the like; the kit disclosed by the invention can be used for effectively detecting the porcine rotavirus, and is high in sensitivity and rapid in detection; and the pharmaceutical composition disclosed by the invention can be used for effectively preventing and treating porcine rotavirus infection.

Description

technical field [0001] The invention relates to a pair of porcine rotavirus monoclonal antibodies, a pharmaceutical composition prepared by using the monoclonal antibodies, a kit and applications thereof, and the invention belongs to the field of biomedicine. Background technique [0002] Porcine rotavirus disease is an acute intestinal infectious disease of pigs caused by porcine rotavirus (PRoV). The main symptoms are anorexia, vomiting and diarrhea. The incidence rate of suckling piglets is high, the incidence rate is generally 50% to 80%, and the mortality rate is generally within 10%. Porcine rotavirus is a virus that can infect both humans and animals. It has a high infection rate in pigs. After infection, the immunity of pigs, especially piglets, will drop sharply, which will induce mixed infections of various pathogens and lead to an increase in the mortality of piglets. or growth retardation. Porcine rotaviruses are divided into 4 subgroups: A, B, C, and E. The ge...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20A61K39/42A61P31/14G01N33/577G01N33/569G01N33/558
CPCC07K16/10A61P31/14G01N33/577G01N33/56983G01N33/558C07K2317/56C07K2317/92C07K2317/76C07K2317/33A61K2039/505G01N2469/10G01N2333/14
Inventor 田克恭张云静
Owner LUOYANG PULIKE WANTAI BIOTECH
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