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Anti-duck circovirus single-chain antibody and preparation method and application thereof

A technology of duck circovirus and single-chain antibody, which is applied in the field of bioengineering to achieve good virus recognition specificity and good blocking effect

Active Publication Date: 2020-08-07
青岛嘉智生物技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no research using phage antibody library technology to develop single-chain antibodies against duck circovirus, and to detect and diagnose duck circovirus

Method used

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  • Anti-duck circovirus single-chain antibody and preparation method and application thereof
  • Anti-duck circovirus single-chain antibody and preparation method and application thereof
  • Anti-duck circovirus single-chain antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of phage single-chain antibody library against duck circovirus

[0028] 1. Mash the liver and spleen of Cherry Valley duck infected with duck circovirus, add appropriate amount of PBS, and prepare purified virus liquid by sucrose density gradient ultracentrifugation. The obtained purified duck circovirus solution was inactivated with formaldehyde and emulsified with Freund's adjuvant 1:1 to immunize BALB / c mice three times with an interval of 14 days. When the duck circovirus was detected by the ELISA antibody and the titer reached more than 1:100000, the mice were killed, 50 mg of spleen was taken, and the total RNA was extracted by the Trizol kit.

[0029] 2. Removal of genomic DNA and synthesis of cDNA by reverse transcription

[0030] It should be noted that in this experiment, the kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. was preferred for the removal of genomic DNA and the synthesis of cDNA by reverse transcription.

[0031...

Embodiment 2

[0055] Example 2 Analysis of single-chain antibody diversity

[0056] Randomly pick 20 clones, extract the plasmids after amplification by shaken bacteria, double enzyme digestion with Sfi I and Not I, and preliminarily identify positive clones. Using S1 (5-CAACGTGAAAAAATTATTATT-3) and S6 (5-GTAAATGAATTTTCTGTATGAGG-3) as primers for sequencing analysis, the results showed that the gene sequences of the 20 clones were all consistent with the sequence of the mouse immunoglobulin variable region, which was consistent with the mouse light and heavy chains. Variable region gene structure, the arrangement is VH-Linker-VL. Among them, the VH part is about 357-367bp, the VL is about 320-330bp, and the linker base sequences between the heavy chain and the light chain are all correct. Sequence alignment shows that the homology is more than 80%.

Embodiment 3

[0057] Example 3 Enrichment of anti-duck circovirus single-chain antibody library

[0058] 1. Dilute the purified duck circovirus liquid by 1:10 (volume ratio), coat the immune tube with carbonate coating buffer, 2ml / tube, overnight at 4°C.

[0059] 2. After coating, wash the immunotube 3 times with PBS, seal the immunotube with blocking solution (2% skim milk PBS, MPBS), and seal at 37°C for 2 hours.

[0060] 3. Pour off the blocking solution, and wash the immunotube 3 times with PBS.

[0061] 4. Mix the supernatant of the above-mentioned primary phage single-chain antibody library with MPBS and phage supernatant according to the volume ratio of MPBS: supernatant = 2:3, and perform de-interference treatment at room temperature for 20 minutes.

[0062] 5. Add the mixed liquid processed in step 4 into the sealed immunotube, 2ml / tube, incubate with gentle shaking for 30min, and then incubate for 1.5 hours.

[0063] 6. Discard the phage supernatant in the immunotube, wash 3 tim...

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Abstract

The invention discloses an anti-duck circovirus single-chain antibody and a preparation method and application thereof, and relates to the technical field of bioengineering. The amino acid sequence ofthe anti-duck circovirus single-chain antibody is Seq ID NO.1 or Seq ID NO.3. The invention provides a coding gene of the anti-duck circovirus single-chain antibody. The nucleotide sequence of the coding gene is Seq ID NO.2 or Seq ID NO.4. The anti-duck circovirus single-chain antibody ScFv-DuCV is screened by adopting a phage antibody library technology, antibody structure modification is further carried out on the anti-duck circovirus single-chain antibody ScFv-DuCV so that the anti-duck circovirus single-chain antibody ScFv-mDuCV is obtained, and the anti-duck circovirus single-chain antibody ScFv-DuCV and the anti-duck circovirus single-chain antibody ScFv-mDuCV can be used for duck circovirus detection and diagnosis. The anti-duck circovirus single-chain antibody ScFv-mDuCV and the duck circovirus have good virus recognition specificity, meanwhile, the anti-duck circovirus single-chain antibody ScFv-mDuCV has a good blocking effect on the duck circovirus, and a foundation is laidfor research of novel drugs for treating and inhibiting the duck circovirus.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an anti-duck circovirus single-chain antibody and its preparation method and application. Background technique [0002] In order to detect duck circovirus disease and prevent and control its harm to duck industry, antibody technology has played an important role in duck industry disease. However, the commonly used monoclonal antibody technology has disadvantages such as easy loss of antibody genes, long production cycle, and difficult modification of antibody genes. People are gradually turning their attention to the preparation of antibodies by genetic engineering methods. [0003] The principle of the phage antibody library is to link the antibody variable region gene with the capsid protein gene of the filamentous phage, and express it on the surface of the phage, perform multiple rounds of affinity adsorption on the antibody library by the antigen, and screen out the...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12N15/70C12N1/21G01N33/569C12R1/19
CPCC07K16/081C07K2317/622C12N15/70G01N33/56983G01N2333/01
Inventor 李志中秦立廷魏笑笑戴荣莲
Owner 青岛嘉智生物技术有限公司
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