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Swine anti-PDCoV-N protein scFv and expression vector, construction method and application thereof

A construction method and expression vector technology, applied in the field of molecular biology, can solve the problems of inability to enter cells smoothly, limited research on virus antibodies, poor specificity, etc.

Active Publication Date: 2021-10-22
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the late discovery and prevalence of PDCoV, the research on virus antibodies is relatively limited. Most of the antibodies against PDCoV developed at present are polyclonal serum and hybridoma scFv. Polyclonal antibodies are easy to prepare, but the specificity is poor. For example, patent CN201611180957 .4 Disclosed the preparation method and application of anti-pseudorabies virus porcine monoclonal antibody, the method adopts the hybridoma cells of pseudorabies virus prepared first, and uses the hybridoma cells as templates for cloning; the traditional scFv has good specificity, but it is Mouse-derived, so immune rejection may occur during treatment, and due to the relatively large molecular weight of scFv, it may also fail to enter cells smoothly

Method used

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  • Swine anti-PDCoV-N protein scFv and expression vector, construction method and application thereof
  • Swine anti-PDCoV-N protein scFv and expression vector, construction method and application thereof
  • Swine anti-PDCoV-N protein scFv and expression vector, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of PDCoV porcine phage single-chain antibody library

[0043] 1.2 Method

[0044] 1.2.1 Extraction of total RNA from pig spleen

[0045] The total RNA of pig spleen was extracted by TRIzol method, and the specific operation steps were as follows:

[0046] (1) Take the spleen tissue stored at -80°C, cut a small part, put it in a sterilized and pre-cooled mortar, add 1mL TRIzol to grind thoroughly, transfer the ground liquid to a 1.5mL centrifuge tube, and let it stand for 5min.

[0047] (2) Add 200 μL of chloroform, vortex to mix, let stand for 3 minutes, and centrifuge at 12000 r / min at 4°C for 10 minutes.

[0048] (3) Extract the upper aqueous phase into a new 1.5mL centrifuge tube, add 500μL isopropanol, shake vigorously and mix well, let stand for 20min, and centrifuge at 12000r / min at 4°C for 10min.

[0049] (4) Discard the supernatant, add 1mL of 70% ethanol, resuspend the pellet, and centrifuge at 12000r / min at 4°C for 10min. This step ...

Embodiment 2

[0112] Example 2: Enrichment screening of phage single-chain antibody library

[0113] 2.2 Method

[0114] 2.2.1 Expression and purification of PDCoV-N protein

[0115] 2.2.1.1 Prokaryotic expression of PDCoV-N protein

[0116] (1) Take 1 μL of the pET28a-PDCoV-N plasmid stored in the laboratory and add it to 100 mL of BL21 (DE3) competent cells, mix gently, then ice-bath for 30 minutes, heat shock at 42°C for 90 sec, and ice-bath for 5 minutes, add 900 μL LB to culture based on 37 Cultivate with shaking at ℃ for 1 h, take 200 μL of culture solution to spread on LBA plate, and incubate at 37 ℃ for 16 h.

[0117] (2) Pick a single colony from the plate grown overnight, inoculate it in 5 mL LB-A medium, inoculate it in 200 mL fresh LB-A medium after shaking culture at 37°C for 5 hours, and continue shaking culture at 37°C until OD 600 About 0.6.

[0118] (3) Add the inducer IPTG 1mL, that is, the final concentration is 0.8mM, and induce with shaking at 37°C for 6h.

[0119]...

Embodiment 3

[0175] Example 3: Expression and functional identification of specific single-chain antibodies

[0176] 3.2 Method

[0177] 3.2.1 Construction of scFv eukaryotic recombinant expression vector

[0178] The three specific strains obtained from the screening were expanded and cultivated, and the plasmids were extracted according to the steps in 1.2.6.1 in Test 1. The recombinant pSEX-scFv plasmid and the eukaryotic expression vector pFUSE plasmid were used at the same time sfiI Restriction endonucleases were used for enzyme digestion, and the enzyme digestion system (50 μL) was: DNA 1 μg, sfiI 1 μL, Cutsmart Buffer 5 μL, add deionized water to a total volume of 50 μL, and the reaction condition is 50°C for 3h. After the reaction, 5 μL was taken for identification with 1% agarose gel, and the rest were purified and recovered according to the steps 1.2.4 in Test 1 after gel electrophoresis.

[0179] The recovered target gene was ligated with the pFUSE vector. The reaction syst...

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Abstract

The invention belongs to the field of molecular biology, and relates to a porcine anti-PDCoV-N protein scFv, in particular to a porcine anti-PDCoV-N protein scFv as well as an expression vector, a construction method and application of the porcine anti-PDCoV-N protein scFv. The scFv of the porcine anti-PDCoV-N protein has an amino acid sequence as shown in SEQ ID No.2; the scFv comprises a heavy chain variable region VH and a light chain variable region VL, the nucleotide sequence of the heavy chain variable region VH is as shown in SEQ ID NO.3, and the nucleotide sequence of the light chain variable region VL is as shown in SEQ ID NO.4. The specific scFv-25 screened and expressed by the invention not only can be used for developing PDCoV diagnosis and treatment reagents, but also can provide help for further research of an N protein mechanism.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a pig-derived scFv against PDCoV-N protein, in particular to a pig-derived scFv against PDCoV-N protein and its expression vector, construction method and application. Background technique [0002] Members of the coronavirus genus are very easy to mutate during transmission to increase their virulence. Therefore, there have been many outbreaks of coronaviruses in recent years and there are certain difficulties in prevention and control. PDCoV belongs to the deltacoronavirus genus and mainly infects pigs, but can also infect chickens or mice across species. Since the outbreak of PDCoV in pig herds in the United States in 2014, it has spread rapidly to all parts of the world, causing huge losses to the pig industry. Since PDCoV is often co-infected with other pig enteric pathogens, and there is currently no vaccine or specific drug against PDCoV, its pathogenic mechanism and the spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C40B50/06C40B30/04G01N33/569A61P31/14
CPCC07K16/10C07K16/005C12N15/70C40B50/06C40B30/04G01N33/56983A61P31/14C07K2317/622C07K2317/565G01N2333/165G01N2469/10
Inventor 魏战勇王林青宋月张艺璇刘中原刘石
Owner HENAN AGRICULTURAL UNIVERSITY
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