Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Anti-H5N1 virus endocytosis antibody PTD-7B and application thereof

A PTD-7B, virus technology, applied in the direction of antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problem of being located on the inside of the virus envelope

Pending Publication Date: 2020-10-02
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
View PDF5 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Anti-H5N1 virus endocytosis antibody PTD-7B and application thereof
  • Anti-H5N1 virus endocytosis antibody PTD-7B and application thereof
  • Anti-H5N1 virus endocytosis antibody PTD-7B and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction, expression and purification of M1 protein recombinant expression plasmid pET-SUMO-M1 of H5N1 virus M1 protein

[0030] Design and synthesize primers P1 and P2 for M1:

[0031] P1: 5'-atgagtcttctaaccgaggtc-3'; its base sequence is shown in SEQ ID NO.1 in the sequence table.

[0032] P2: 5'-CCg gaattc ttaCttgaatcgctgcatctgcact-3'; its base sequence is shown in SEQID NO.2 of the sequence table.

[0033] 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...

Embodiment 2

[0036] Example 2 Screening of phage single-chain antibody library

[0037] 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

[0038] Example 3 Screening of anti-M1-scFv

[0039] 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 ;

[0040] 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;

[0041] Phage infection after...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an anti-H5N1 virus endocytosis antibody PTD-7B. The base sequence of the PTD-7B is shown as SEQ ID NO. 5 in a sequence table; the amino acid sequence of the fusion protein PTD-7B is shown as SEQ ID NO. 6 in a sequence table; the preparation method of the fusion protein PTD-7B comprises the following steps: 1) performing amplifying on a 7B gene of M1-ScFv by using a primer and using a screened phage antibody ScFv gene as a template; 2) connecting the amplified 7B gene into a PET28a-PTD-GFP vector, replacing a GFP gene fragment, and constructing a prokaryotic expression vector PET28a-PTD-7B; and 3) performing transforming on the prokaryotic expression vector into escherichia coli for expression and purification. The fusion protein PTD-7B is applied to preparation of anti-H5N1 human avian influenza virus drugs, and the result shows that the intracellular antibody has H5N1 virus neutralizing activity, and the titer is 350TCID50.

Description

technical field [0001] The invention belongs to the field of bioengineering and disease prevention and control, and specifically relates to the preparation and application of a fully human anti-highly pathogenic avian influenza H5N1 virus cell-infiltrating antibody PTD-7B. Background technique [0002] Avian influenza virus (AIV) is an RNA virus belonging to the group of orthomyxoviruses that spreads very rapidly. The known pathogenicity is very diverse, ranging from no clinical symptoms to almost 100% mortality after infection. All avian influenza viruses belong to type A and have multiple serotypes: hemagglutinin (HA) is classified into 16 subtypes, neuraminidase (NA) into 9 subtypes, which potentially allows 144 different combinations of influenza A viruses. [0003] Of these different subtypes of influenza A virus, the H5 and H7 subtypes are known to be pathogenic to birds, and the H1, H2 and H3 subtypes are known to cause influenza in humans. Avian influenza viruses ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N15/65A61K39/42A61P31/14
CPCC07K16/10C12N15/70C12N15/65A61P31/14C07K2319/10C07K2317/76A61K2039/505
Inventor 岳玉环张国利雍伟刘楚含高玉伟田园吴广谋王铁成李泽鸿刘雨玲卢士伟那漫孙赫邓欣
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com