Canine parvovirus single domain antibody and its preparation method and application
A technology of canine parvovirus and single-domain antibody, which is applied in the field of virus antibody and canine parvovirus single-domain antibody, can solve the problems of difficult to stimulate T cell immune response, complex molecular structure of whole antibody, low VHH immunogenicity, etc., to achieve Shorten the treatment cycle, high affinity, small molecular weight effect
Inactive Publication Date: 2016-01-20
SHANXI AGRI UNIV
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Problems solved by technology
At present, antibodies are limited to cell surface targets in the treatment of diseases. Full antibodies are difficult to express in cells due to their complex molecular structure, which limits their application in cells. VHH has low immunogenicity and is difficult to stimulate T cell immune response. , can be used as a therapeutic antibody for long-term administration
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[0022] The present invention will be further described in detail through the following examples. It should be understood that the following embodiments are only used to explain the present invention more clearly, but cannot be used to limit the protection scope of the present invention. Within the scope of protection defined by the claims of the present invention, any changes, modifications or equivalent changes made to them without substantial changes shall fall within the scope of protection of the present invention.
[0023] 1. Immunization of alpaca with inactivated canine parvovirus vaccine
[0024] Put the CPV virus solution into a Erlenmeyer flask, add analytical pure formaldehyde to dilute to a final concentration of 0.25wt%, shake it up while adding, add a stopper and wrap it with sterile gauze, and act at 37°C for 24 to 48 hours; remove the stopper and wrap it with gauze The mouth of the Erlenmeyer flask is shaken at intervals until the smell of formaldehyde is gone.
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Abstract
The invention discloses a canine parvovirus single-domain antibody, and a preparation method and an application thereof. The canine parvovirus single-domain antibody has a nucleotide sequence as shown in SEQIDNO.1. The preparation method comprises the steps: immunizing an alpaca by a canine parvovirus inactivated vaccine, and taking peripheral blood to obtain alpaca anti-canine parvovirus serum; extracting RNA in the peripheral blood, and carrying out reverse transcription to generate cDNA; utilizing specific primers for amplification, and purifying a VHH fragment; connecting the VHH fragment with a phagemid vector, transforming TG1, and constructing a canine parvovirus single-domain antibody library; and enriching the antibody library by utilizing a canine parvovirus structural protein VP2, introducing into an expression bacterial strain E.coil HB2151, and utilizing IPTG to induce the canine parvovirus single-domain antibody VHH having the biological activity to be expressed. The prepared canine parvovirus single-domain antibody has the detection activity of 15 [mu]g / ml and the detection sensitivity of 250 ng / ml, and can be used as drugs applied for treating dogs with canine parvovirus disease.
Description
Technical field [0001] The present invention belongs to the technical field of clinical veterinary medicine and antibody engineering, and relates to a virus antibody, in particular to a canine parvovirus single domain antibody that mainly relies on alpaca heavy chain antibody to achieve its biological expression, and a preparation method of the antibody And application. Background technique [0002] Canine parvovirus disease is caused by canine parvovirus (Canineparvovirus, CPV). It is characterized by high morbidity, high mortality, and strong infectivity. It can occur all year round. Deaths from hemorrhagic enteritis and myocarditis canine parvovirus disease The rates are 10%-50% and 60%-100%, which can cause serious economic losses. Vaccines for preventing canine parvovirus disease mainly include inactivated vaccines, attenuated vaccines and polyvalent vaccines. The optimized immunization program can achieve better immune effects. However, a survey and research on the immune...
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IPC IPC(8): C07K16/08C12N15/63A61K39/42A61P31/20
Inventor 段智变孙耀贵程志学员世宇詹俊杰杨妍丽李小茜
Owner SHANXI AGRI UNIV