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An anti-H5N1 viropexis antibody PTD (Protein transduction domain)-3F and application

A PTD-3F, virus technology, applied in the direction of antiviral agents, antibodies, antibody mimics/stents, etc., can solve the problem of being located inside the virus envelope

Active Publication Date: 2020-10-16
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

M1 protein antibody can produce antiviral activity against various subtypes of avian influenza virus, but it is located inside the viral envelope, and the antibody needs to enter the infected cell to exert its effect

Method used

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  • An anti-H5N1 viropexis antibody PTD (Protein transduction domain)-3F and application
  • An anti-H5N1 viropexis antibody PTD (Protein transduction domain)-3F and application
  • An anti-H5N1 viropexis antibody PTD (Protein transduction domain)-3F and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027]Example 1 Construction, expression and purification of M1 protein recombinant expression plasmid pET-SUMO-M1 of H5N1 virus M1 protein

[0028] Design and synthesize primers P1 and P2 for M1 protein:

[0029] P1: 5'-atgagtcttctaaccgaggtc-3'; as shown in SEQ ID NO.1 of the sequence listing.

[0030] P2: 5'-CCg gaattc ttaCttgaatcgctgcatctgcact-3'; as shown in SEQ ID NO.2 of the sequence table.

[0031] The M1 protein gene was amplified by PCR using H5N1 cDNA as a template, and cloned into the PET-SUMO vector to construct the plasmid pET-SUMO-M1, which was then transferred into T-shot competent cells and carried out on an agar plate containing kanamycin resistance. initial screening. A single colony was selected and cultured in LB liquid medium; the plasmid was extracted with a plasmid recovery kit and identified by PCR. 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,...

Embodiment 2

[0034] Example 2 Screening of phage single-chain antibody library

[0035] Inject all the frozen Tomlinson I and J libraries into 200 mL 2×TY medium (containing 100 µg / mL Amp and 1% glucose), and culture with shaking at 37°C until the OD600 value is about 0.4, from Take out 50 mL bacterial liquid from the culture medium, add 2×10 11 The helper phage KM13 was placed in a water bath at 37°C for 30 minutes, centrifuged at 3000×g for 10 minutes at 4°C, and the pellet was resuspended with 50 mL of 2×TY medium (containing 100 µg / mL Amp, 50 µg / mL Kan and 0.1% glucose). Suspended and cultured overnight at 30°C with shaking. Centrifuge the overnight product at 4°C, 3500×g for 30 min, collect 40 mL of the supernatant, add 10 mL of ice-cold PEG / NaCl solution (final concentration is 20% PEG-6000, 2.5 mol / L NaCl), mix well and store on ice Place for more than 1 h, centrifuge at 4°C, 3500×g for 30 min, discard the PEG / NaCl solution, resuspend the pellet in 2 mL of PBS, centrifuge at 11600...

Embodiment 3

[0036] Example 3 Screening of anti-M1-scFv

[0037] Coat the purified M1 protein on a 96-well microtiter plate overnight at 4°C. Discard the supernatant the next day, block with 2% Milk-PBS at 37°C for 2 hours, add the prepared secondary phage antibody library, incubate with vigorous shaking at room temperature for 60 minutes, discard the liquid after standing for 60 minutes, and replace with PBS containing 0.1% Twenn-20 Wash 10 times, after washing, gently pat dry the remaining liquid in each well, add 50 µL eluent (5 mg / mL trypsin-PBS) to each well, shake vigorously at room temperature for 10 min, elute phage, collect and store at 4 °C ;

[0038] Infect E.coli TG1 with the eluted phage, spread on TYE plates (containing 100 μg / mL ampicillin and 1% glucose) and culture overnight at 37°C. Use the helper phage KM13 to amplify the phage library, and recover the phage by PEG / NaCl; repeat the above process 3 times, a total of 4 rounds of screening;

[0039] Phage infection after...

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PUM

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Abstract

The invention discloses an anti-H5N1 viropexis antibody PTD (Protein transduction domain)-3F. The base sequence of the anti-H5N1 viropexis antibody PTD-3F is disclosed in SEQ ID NO. 5. The amino acidsequence of the fusion protein PTD-3F is disclosed in SEQ ID NO. 6. A preparation method for the fusion protein PTD-3F comprises the following steps of: 1) utilizing a primer, and taking a screened bacteriophage antibody ScFv gene as a template to carry out 3F gene amplification; 2) connecting an amplified 3F gene into a PET28a-PTD-GFP carrier to construct a prokaryotic expression vector PET28a-PTD-3F; and 3) converting the prokaryotic expression vector into escherichia coli to be expressed and purified. The invention discloses application of the PTD-3F for preparing a medicine capable of resisting H5N1 type human avian influenza viruses. A result displays that an intracellular antibody can neutralize H5N1 virus activity, and titer is 400 TCID50 (median tissue culture infective dose).

Description

technical field [0001] The invention belongs to the field of bioengineering and disease prevention and control, and specifically relates to a fully human anti-highly pathogenic avian influenza H5N1 virus cell-infiltrating antibody PTD-3F and its application. Background technique [0002] Human infection with highly pathogenic avian influenza (referred to as human avian influenza) is a systemic or respiratory infectious disease caused by type A influenza virus H5N1, with a fatality rate of over 60%. Under natural circumstances, the host range of influenza virus infection has certain specificity, based on which viruses can be divided into different groups, such as human influenza, avian influenza, swine influenza, etc., but the boundary of the host range of influenza virus infection is not very strict , the virus can spread between different species. Since the first report of human infection with the H5N1 subtype avian influenza virus in Hong Kong in 1997, the highly pathogen...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/70A61K39/42A61K47/64A61P31/16
CPCC07K16/1018C12N15/70A61K39/42A61K47/64A61P31/16C07K2319/00A61K2039/505
Inventor 张国利田园岳玉环李泽鸿高玉伟邓欣吴广谋刘楚含刘雨玲王铁成雍伟卢士伟王冬冬那漫
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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