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Shuttling intracellular antibody TAT-2C for H3N2-type canine influenza virus

A TAT-2C, canine influenza virus technology, applied in the direction of antibodies, antiviral agents, recombinant DNA technology, etc., can solve problems such as weak immune cross-reaction

Active Publication Date: 2018-11-02
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, influenza virus is highly variable, and the immune cross-reaction between different variant branches is weak

Method used

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  • Shuttling intracellular antibody TAT-2C for H3N2-type canine influenza virus
  • Shuttling intracellular antibody TAT-2C for H3N2-type canine influenza virus
  • Shuttling intracellular antibody TAT-2C for H3N2-type canine influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Construction and expression of M1 recombinant expression plasmid pET-SUMO-M1 and purification of M1 protein

[0025] Design and synthesize primers M1P1 and M1P2, use canine H3N2 cDNA as a template to PCR amplify the M1 protein gene, and clone it into the PET-SUMO vector to construct the plasmid pET-SUMO-M1, and then transfer it into T-shot competent cells containing kan Resistant agar plates were used for preliminary screening. A single colony was selected and cultured in LB liquid medium; the plasmid was extracted with a plasmid recovery kit, identified by PCR, and the product was analyzed by 1% agarose gel electrophoresis, and a band of about 750 bp was obtained, which was consistent with the inserted target gene. The sequence was determined to prove that the target fragment was correctly inserted into the vector, and the recombinant plasmid pET-SUMO-M1 was successfully constructed (see figure 1 ).

[0026] The recombinant plasmid pET-SUMO-M1 was transfor...

Embodiment 2

[0028] Example 2: Amplification of phage single-chain antibody library

[0029] 1. Construction of canine phage antibody library construction

[0030] Separation of canine lymphocytes: Separation of 6 peripheral blood lymphocytes of Beagle dogs, extraction of total RNA, reverse transcription to synthesize cDNA; amplification of VH gene, adding NcoI and XhoI restriction sites to the upstream and downstream respectively; amplification of VL gene, upper , and downstream respectively add SalI and NotI enzyme cutting sites; VH enzyme digestion and ligation into the PIT2 vector; VL enzyme digestion and ligation into the PIT2 vector.

[0031] 2. The constructed vector was electrotransformed into Escherichia coli TG1, coated on a TYE plate (containing a final concentration of 100 μg / mL Amp + 1% glucose), cultured overnight at 37°C, and a single clone was randomly picked from the plate for bacterial liquid PCR identification, and calculated Recombination rate, and estimated storage ca...

Embodiment 3

[0032] Example 3: Screening of anti-M1-scFv

[0033] The purified M1 protein was antigen-coated on a 96-well microtiter plate and kept overnight at 4°C. Discard the supernatant the next day, block with 2% Milk-PBS at 37°C for 2 hours, and add the prepared phage antibody library (titer: 1.0×10 13 pfu), shake vigorously at room temperature for 60 minutes, and let stand for 60 minutes. Discard the liquid, wash 10 times with PBS containing 0.1% Twenn-20, pat dry the remaining liquid in each well after washing, add 50 µL eluent (5 mg / mL trypsin-PBS) to each well, Vigorously shake at room temperature for 10 min to elute the phage, collect and store at 4°C.

[0034] Infect E.coli TG1 with the eluted phage, spread on TYE plates (containing 100μg / mL Amp and 1% glucose) and culture overnight at 37°C. The phage library was amplified using the helper phage KM13, and the phage were recovered by PEG / NaCl. Repeat the above process 3 times, a total of 4 rounds of screening.

[0035] Phag...

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Abstract

The invention discloses a shuttling intracellular antibody for H3N2-type canine influenza virus. The H3N2 virus is A-type influenza virus, the main neutralizing antibody is from hemagglutinin (HA), therefore, the HA becomes an original main research target; however the influenza virus has high variability, the immune cross reaction among different variable branches is weaker, M1 antibody can be combined with M1 protein to inhibit the activity and disturb multiplication, transcription and release of the influenza virus so as to play a role in resisting the virus; therefore, the M1 protein is selected for preparing the corresponding antibody to obtain stable titer, coupling the antibody with TAT protein PTD (Protein Transduction Domain) capable of transducing biomacromolecules into cells andexpressing fusion protein TAT PTD-M1 ScFv, and then a shuttling antibody for resisting the canine influenza virus is prepared to provide a new path for treating the canine influenza virus.

Description

technical field [0001] The invention belongs to the fields of bioengineering and disease prevention and control, and in particular relates to the preparation of a shuttle intracellular antibody TAT-2C directed at the conserved antigen of H3N2 canine influenza virus, and the antiviral effect of the antibody on H3N2 canine influenza virus. Background technique [0002] For a long time, it has been generally believed that dogs are not susceptible to various subtypes of influenza viruses in a natural state, so dogs have been excluded from the susceptibility range of influenza viruses. However, in 2004, virologists isolated dogs from Florida greyhounds. This understanding was changed only after Canine influenza virus (CIV) was confirmed and CIV infection in various dog breeds was confirmed. Since then, studies have shown that the flu virus that has caused panic among the American racing dog industry and pet breeders is actually a mutant strain of the equine flu virus H3N8. Canin...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00A61K39/42A61K47/42A61P31/16G01N33/569
CPCA61P31/16C07K16/1018C07K2319/10C07K2317/76C07K2317/622
Inventor 田园岳玉环张国利吴广谋祝令伟余晓颖孙红刘雨玲马洪圆
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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