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Heavy chain and light chain variable region for resisting to canine parvovirus antibody and gene engineering antibody

A technology of genetically engineered antibodies and canine parvovirus, applied in genetic engineering, antiviral immunoglobulin, plant gene improvement, etc., can solve problems such as reducing immunogenicity, reducing curative effect, immune rejection or allergic reaction, and achieve inhibition of agglutination , good active effect

Active Publication Date: 2019-04-16
CHANGCHUN SR BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, mouse-derived monoclonal antibodies have strong heterogeneity and immunogenicity to non-mouse-derived organisms, and they are likely to cause immune rejection or allergic reactions in the host when used in vivo, which reduces the curative effect of mouse-derived monoclonal antibodies, and even in vivo Serious consequences have greatly limited the clinical application of mouse monoclonal antibodies. Therefore, it is necessary to transform mouse monoclonal antibodies. The modified mouse monoclonal antibodies reduce their immunogenicity and can be more effectively used in Clinical and treatment of various diseases
[0005] Currently, the monoclonal antibodies against canine parvovirus on the market are mouse-derived monoclonal antibodies. Monoclonal antibodies and antiserum have strong immunogenicity, and there will be side effects during the treatment process, which will reduce the curative effect, which greatly limits its clinical application in dogs with canine parvovirus. application

Method used

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  • Heavy chain and light chain variable region for resisting to canine parvovirus antibody and gene engineering antibody
  • Heavy chain and light chain variable region for resisting to canine parvovirus antibody and gene engineering antibody
  • Heavy chain and light chain variable region for resisting to canine parvovirus antibody and gene engineering antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Preparation and screening of embodiment 1 mouse CPV monoclonal antibody

[0022] (1) Preparation of hybridoma cell lines

[0023] Animal immunization Select 5 female BALB / c mice aged 6-8 weeks, inject the concentrated and purified canine parvovirus third-generation virus and the same amount of complete Freund's adjuvant emulsion into the mice, and inject 200 μl subcutaneously into each mouse . Two weeks later, the second immunization was carried out, and the mice were injected with concentrated and purified canine parvovirus and an equal amount of Freund's incomplete adjuvant emulsion, and the injection volume and method were the same as the first immunization. Two weeks after the second immunization, the third immunization was carried out according to the second immunization. Two weeks after the third immunization, mice with high serum titers determined by indirect ELISA were selected for intraperitoneal injection.

[0024] Cell Fusion BALB / c mice about 8 weeks old w...

Embodiment 2

[0043] The preparation of embodiment 2 mouse origin CPV genetic engineering antibody

[0044] (1) Acquisition of variable region sequence of CPV monoclonal antibody

[0045] Utilize the operation method of RNA extraction kit to extract the total RNA of hybridoma cell line, and then use RACE 5' / 3' kit (Takara Company), cDNA was obtained by reverse transcription, and primers were designed according to the subtype of the monoclonal antibody, as shown in Table 3.

[0046] Table 3 Antibody Amplification Primers

[0047] serial number

Primer name

Primer sequence

SEQ ID 5

Heavy Chain External Primer (5'-3')

AGG ACA GGG GTT GAT TGT TGA

SEQ ID 6

Light chain external primer (5'-3')

CTC ATT CCT GTT GAA GCT CTT GAC

SEQ ID 7

Heavy Chain Internal Primer (5'-3')

CTC AAG TTT TTT GTC CAC CGT GGT GC

SEQ ID 8

Light Chain Internal Primer (5'-3')

CTC ATT CCT GTT GAA GCT CTT GAC AAT GGG

[0048] The DNA fragments o...

Embodiment 3

[0053] Example 3 Activity Research of Mouse CPV Genetic Engineering Antibody

[0054] (1) Hemagglutination inhibition method was used to measure the resistance effect of CPV genetic engineering antibody on the combination of canine parvovirus and red blood cells

[0055] Prepare the diluted CPV to be eight units of virus (that is, a CPV virus solution with three hemagglutination units). Add 15mM PBS of pH 6.5 to a 96-well V-type hemagglutination plate, 25μl / well, and add 25μl CPV to the first column For monoclonal antibody, do 3 repetitions for each antibody, do 2-fold gradient dilution, dilute to the last row and discard 25 μl of liquid, the last two rows are used as the porcine red blood cell control area, sample area (including positive control group, experimental group, Empty plasmid control group, blank control group) were added 25 μl eight-unit CPV virus solution to each well, and 25 μl PBS was added to the porcine red blood cell control area, and kept in a 37° C. incuba...

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Abstract

The invention discloses a heavy chain and light chain variable region for resisting to a canine parvovirus antibody and a gene engineering antibody, and belongs to the technical field of antibody engineering. An amino acid sequence and a nucleotide sequence of the heavy chain and light chain variable region for resisting to the canine parvovirus antibody provide supports for construction of the high-affinity and low-immunity canine parvovirus antibody. A variable region sequence of a monoclonal antibody for canine parvovirus and a murine constant region are further assembled, so that the geneengineering antibody for resisting to canine parvovirus is obtained, good activity for neutralizing CPV viruses is shown, and the gene engineering antibody has a restraining effect on erythrocyte agglutination of canine parvovirus, can be applied to research of a canine source of the monoclonal antibody for canine parvovirus and other fields, and has important meaning for pushing of development ofcanine source monoclonal antibody medicine.

Description

technical field [0001] The present invention relates to the technical field of antibody engineering, in particular to a heavy chain and light chain variable region of an anti-canine parvovirus antibody, including its amino acid sequence and nucleotide sequence, and a genetically engineered antibody against canine parvovirus constructed using it . Background technique [0002] Canine parvovirus (Canine parvo-virus, CPV) is a member of the genus Parvovirus in the family Parvoviridae. It mainly infects puppies and can cause severe vomiting and a significant decrease in white blood cells. The main clinical symptoms are hemorrhagic enteritis or non-suppurative myocarditis. The characteristics of puppies are sudden onset, rapid infection and high mortality, which seriously endangers the development of the dog industry and causes serious economic losses to the dog industry. [0003] Vaccines to prevent canine parvovirus mainly include: inactivated vaccines, attenuated vaccines, an...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13
CPCC07K16/081C07K2317/33C07K2317/35C07K2317/56C07K2317/92C07K2317/76
Inventor 石晶李雪殷玉和李希辰赵健刘伟付玉张馨月
Owner CHANGCHUN SR BIOLOGICAL TECH
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