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A monoclonal antibody for distinguishing virulent strain of classical swine fever virus 2.1 subtype and its antibody

A monoclonal antibody, swine fever virus technology, applied in antiviral immunoglobulins, instruments, peptides, etc., can solve the problems of swine fever virulent purification and inability to distinguish positive swine fever virus antibodies in pigs and other problems

Active Publication Date: 2022-06-28
GUANGDONG HAID GROUP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a high degree of serological cross-reactivity between the C-strain vaccine strain and the Shimen strain and other subtypes of virulent strains. Existing technologies, such as the ELISA method for detecting antibody levels in pigs, cannot clinically distinguish positive swine fever in pigs Does the virus antibody come from infection with a virulent strain (in my country, the virulent strain is usually the Shimen strain of swine fever virus), or from the immune vaccine virus C strain
In addition, the C-strain vaccine is widely used in pig herds in my country, so that the antibody to CSF ​​is always at a high level, thus causing great trouble for the purification of the virulent CSF
However, designing primers according to the differences in the conserved sequences of the virus and using fluorescent quantitative PCR to directly identify the genotype of CSFV is only suitable for detecting that pigs are in the active phase of the virus or when they are shedding the virus. Infected pigs cannot be identified

Method used

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  • A monoclonal antibody for distinguishing virulent strain of classical swine fever virus 2.1 subtype and its antibody
  • A monoclonal antibody for distinguishing virulent strain of classical swine fever virus 2.1 subtype and its antibody
  • A monoclonal antibody for distinguishing virulent strain of classical swine fever virus 2.1 subtype and its antibody

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0133] Example 1 Preparation of monoclonal antibodies for identifying virulent strains of swine fever virus 2.1 subtype

[0134] 1. Expression of recombinant protein

[0135] according to figure 1 In the design scheme of middle A, the genes encoding swine fever virus E2 (the E2 protein of the swine fever virus 2.1 subtype virulent strain and the E2 protein of the C-strain vaccine strain) were inserted into Bac respectively through the restriction endonuclease sites BamHI and XhoI. - Plasmid pFastBac in the to-Bac insect baculovirus system (purchased from Invitrogen) TM 1, then follow "Bac-to- Baculovirus Expression System" using the instruction manual to package the recombinant virus step by step. After infecting insect cells H5, two recombinant proteins were expressed and purified. The specific method of protein purification is as follows:

[0136] (1) Dilute the well-grown H5 cells to 40 mL with a concentration of 3 × 10 6 pcs / mL;

[0137] (2) Insert recombinant bacul...

Embodiment 2

[0155] Example 2 Identification of the recognition site of monoclonal antibody on E2

[0156] Based on the amino acid sequences of E2ab-C / S-2.1, E2ab-C strain, and Shimen strain E2ab fragments, a series of polypeptides were designed. The corresponding positions and sequences of the polypeptides on the E2 protein are as follows: Figure 4 Medium A and Figure 4 As shown in B, the specific amino acid sequences are as follows: the sequence of 2.1-1 is shown in SEQ ID NO.6, the sequence of 2.1-2 is shown in SEQ ID NO.7, and the sequence of 2.1-3 is shown in SEQ ID NO.8 The sequence of 2.1-4 is shown in SEQ ID NO.9, the sequence of 2.1-5 is shown in SEQ ID NO.10, the sequence of 2.1-6 is shown in SEQ ID NO.11, and the sequence of 2.1-7 is shown in SEQ ID NO.11. As shown in SEQ ID NO.12, the sequence of C-1 is shown in SEQ ID NO.13, the sequence of C-2 is shown in SEQ ID NO.14, and the sequence of C-3 is shown in SEQ ID NO.15 , the sequence of C-4 is shown in SEQ ID NO.16, the seq...

Embodiment 3

[0158] Example 3 Precise identification of antigenic determinants of monoclonal antibodies on E2

[0159] In order to analyze the antigenic determinants recognized by MM5, the amino acid sequences of polypeptides 2.1-1 and 2.1-2 derived from the virulent strain of swine fever virus 2.1 subtype were compared with the amino acid sequence of polypeptide C-1 derived from the C-strain vaccine strain. yes, as Figure 5 Shown in A: the black and underlined parts are non-conserved amino acids, the amino acid difference between the polypeptide 2.1-1 derived from the virulent strain of swine fever virus 2.1 subtype and SM-1 derived from the polypeptide C-1 derived from the C strain vaccine strain There are 2 numbers, spanning a length of 5 amino acids; since MM5 only recognizes polypeptide 2.1-1, " P LGA E (SEQ ID NO.21)" is the specific recognition site of MM5. In order to further confirm the role of the two amino acid residues P and E in the antigenic determinant, three polypeptides...

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Abstract

The invention belongs to the field of biotechnology, and discloses a monoclonal antibody for distinguishing 2.1 subtype virulent strains of classical swine fever virus and antibodies thereof. The monoclonal antibody can specifically bind to the virulent strain of CSFV 2.1 subtype, but not to the vaccine strain of CSFV, and can be used to detect the E2 protein of the virulent strain of CSFV 2.1 subtype and detect the Type virulent strains and identification of CSFV 2.1 subtype virulent strains and C-strain vaccine strains can also be used to identify antibodies against CSFV 2.1 subtype virulent strains and C-strain vaccine strains.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody for identifying the virulent strain of swine fever virus 2.1 subtype and its antibody. Background technique [0002] Swine fever (Classical Swine Fever, CSF) is an acute, febrile and highly contagious infectious disease caused by swine fever virus (Classical Swine Fever Virus, CSFV). It is listed as a Class I infectious disease in my country, and the World Organization for Animal Health (OIE) stipulates it as one of the animal infectious diseases that must be declared. There is only one serotype of swine fever virus, and it can be divided into multiple subtypes according to the genes encoding the virus. The current popular subtypes in my country are mostly 2.1 subtypes and 2.2 subtypes, of which 2.1 subtypes can be further subdivided into four types: 2.1a, 2.1b, 2.1c, and 2.1d. The virulent strain of swine fever virus isolated in my country in the 2...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08C12N5/20C12N15/13G01N33/577G01N33/569
CPCC07K16/081G01N33/577G01N33/56983C07K2317/56C07K2317/565G01N2333/01G01N2469/10
Inventor 潘春根黄攀张俊赵玉林王宁
Owner GUANGDONG HAID GROUP