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Coding gene, vector, anti-idiotypic nano antibody and preparation method and application thereof

A nanobody and anti-idiotype technology, applied in the field of anti-idiotype nanobody and its preparation, can solve the problems of uneven quality of antibodies, complex preparation, long production cycle, etc.

Active Publication Date: 2020-07-31
NORTHWEST A & F UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of anti-idiotypic antibodies by polyclonal antibody technology has the disadvantages of uneven antibody quality between batches and cumbersome purification work.
Monoclonal antibody technology has disadvantages such as high production cost, long production cycle, complicated preparation and high storage cost

Method used

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  • Coding gene, vector, anti-idiotypic nano antibody and preparation method and application thereof
  • Coding gene, vector, anti-idiotypic nano antibody and preparation method and application thereof
  • Coding gene, vector, anti-idiotypic nano antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] This example provides a gene encoding an anti-idiotypic Nanobody, the nucleotide sequence of which is shown in SEQ ID NO:3 in the sequence listing. Specifically, the screening method of the coding gene comprises the following steps:

[0025] S1. 2 mg of commercially available monoclonal antibody 1E7 was emulsified with an equal volume of Freund's adjuvant to immunize Bactrian camels, and blood was collected before immunization to detect serum antibody titer.

[0026] S2. Collect 250 mL of peripheral blood from Bactrian camels after immunization using blood collection bags containing anticoagulants and separate lymphocytes.

[0027] S3. Extracting the total RNA of the lymphocytes and reverse-transcribing to synthesize cDNA, and amplifying the VHH gene by nested PCR.

[0028] S4. Construct the VHH-pCANTAB 5E recombinant phage expression vector and electrotransform into TG1 competent cells, successfully constructing a library with a capacity of 7×10 9 phage library.

[...

Embodiment 2

[0031] This embodiment provides an anti-idiotypic nanobody, the amino acid sequence of which is shown in SEQ ID NO: 2 in the sequence listing. The preparation method of the anti-idiotype nanobody comprises the following steps:

[0032] S1. Digest the coding gene provided in the above example 1, and then connect it to the expression vector of Pichia pastoris for recombination to obtain a vector containing the above coding gene, which is denoted as pPICZαA-VHH vector; wherein, the expression vector of Pichia pastoris The existing commercially available pPICZαA vector can be used.

[0033] S2. Linearize the above-mentioned pPICZαA-VHH vector by restriction enzyme Sac I, and then electrotransfer it into X-33 Pichia competent cells for activation treatment; then, activate the activated X-33 Pichia The competent cells of red yeast were smeared on the YPD / agar plate containing 100 μg / mL bleomycin and cultured to obtain monoclonal colonies.

[0034] S3. Carry out PCR identification ...

Embodiment 3

[0036] This embodiment provides an anti-idiotypic nanobody, the amino acid sequence of which is shown in SEQ ID NO: 2 in the sequence listing. The preparation method of the anti-idiotype nanobody comprises the following steps:

[0037] S1. Digest the coding gene provided in the above example 1, and then connect it to the expression vector of Pichia pastoris for recombination to obtain a vector containing the above coding gene, which is denoted as pPICZαA-VHH vector; wherein, the expression vector of Pichia pastoris The existing commercially available pPICZαA vector can be used.

[0038] S2. Linearize the above-mentioned pPICZαA-VHH vector by restriction enzyme Sac I, and then electrotransfer it into X-33 Pichia competent cells for activation treatment; then, activate the activated X-33 Pichia The competent cells of red yeast were spread on LB plates containing 80 μg / mL bleomycin and cultured to obtain monoclonal colonies.

[0039] S3. Carry out PCR identification on the abov...

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Abstract

The invention is applicable to the technical field of biology, and provides a coding gene, a vector, an anti-idiotypic nano antibody and a preparation method and application thereof. The amino acid sequence of the anti-idiotypic nano antibody is shown as SEQ ID NO: 2 in a sequence table. The anti-idiotypic nano-antibody is prepared by the following steps: successfully screening the coding gene ofa required anti-idiotypic nano-antibody through a phage display technology, then connecting the coding gene to a pichia pastoris expression vector, and electrically transforming the vector to pichia pastoris competent cells for culturing. The anti-idiotypic nanometer antibody can specifically block the combination of monoclonal antibody 1E7 and PCV2 Cap protein, has the effect of simulating PCV2 Cap key antigenic epitopes, and can be used for developing anti-idiotypic nanometer antibody vaccine, diagnosis reagent, biological reagent and the like for preventing and treating PCV2.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a coding gene, a carrier, an anti-idiotypic nanobody and a preparation method and application thereof. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is divided into non-pathogenic PCV1 (Porcine circovirus type 1, PCV1) and pathogenic PCV2 (Porcine circovirus type 2, PCV2) two types. PCV2 infection is closely related to the occurrence of Postweaning multisystemic wasting syndrome (PMWS), and its ORF2 encodes the capsid protein (Capsid protein, Cap) of the virus, which is the main immunogenic protein that induces host immunity, including virus major antigenic epitopes. In the prior art, the "mirror image" anti-idiotypic antibody (Ab2β) mimics the antigen in structure or function, and can be used to prepare anti-idiotypic antibody vaccines, identify and identify PCV2 cell receptors, and be used in the establishment of PCV2 diagnostic methods. ...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12N15/81C12P21/02A61K39/42A61P31/20G01N33/569
CPCA61K2039/505A61K2039/552A61P31/20C07K16/081C07K2317/14C07K2317/22C07K2317/569C07K2317/76C12N15/815G01N33/56983G01N2333/01G01N2469/10
Inventor 周恩民张桂夕张璐蔡雪辉涂亚斌王刚
Owner NORTHWEST A & F UNIV
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