Anti-CIB single-chain antibody, and screening method and application thereof

A single-chain antibody, light chain technology, applied in the field of bioengineering

Active Publication Date: 2021-09-10
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on sibuterol antibody, so the development of CIB antibody is of great significance to the construction and improvement of my country's drug residue detection system

Method used

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  • Anti-CIB single-chain antibody, and screening method and application thereof
  • Anti-CIB single-chain antibody, and screening method and application thereof
  • Anti-CIB single-chain antibody, and screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1CI

[0040] Preparation and identification of embodiment 1 CIB complete antigen

[0041] 1. Preparation of complete CIB antigen

[0042] (1) Diazotization of CIB

[0043] Accurately weigh CIB using a millionths analytical balance and dissolve in 0.1mol / L pre-cooled hydrochloric acid. Under the conditions of ice-water bath, low speed and slow stirring, slowly add 10mg / mL pre-cooled sodium nitrite solution (molar ratio 1:1.2); use starch-KI test paper to check whether the sodium nitrite solution is excessive, the standard is: test paper color Turned grayish purple. Protect from light, keep at low temperature (0-5°C), and stir for 1-2 hours.

[0044] (2) Coupling with carrier protein

[0045] BSA and OVA (molar ratio of CIB to carrier protein: 30:1) were accurately weighed and dissolved in 0.1 mol / L sodium borate solution with pH = 8.6, and stirred for 2 h under ice-bath conditions. The diazonium salt solution of CIB in step (1) is equally divided into two parts, and slowly adds ...

Embodiment 2

[0057] Embodiment 2 immune effect evaluation

[0058] Three mice were immunized with the prepared complete antigen CIB-BSA as the immunogen, and blood was collected by docking the tail on the 10th day after the fourth immunization, and the antibody titer in the mouse serum was determined by indirect ELISA. The basic steps are as follows:

[0059] (1) Coating: Coat the microtiter plate with the detection original CIB-OVA and the carrier proteins BSA and OVA respectively. The coating concentration is 2 μg / mL, 50 μL / well, and put in the refrigerator at 4°C overnight for coating. The next day, wash the plate, repeat 5 times, and let stand for 3 minutes each time.

[0060] (2) Blocking: add 200 μL of blocking solution (5 g of skimmed milk powder dissolved in 100 mL of PBST) to each well, block at 37° C. for 2 h, repeat washing the plate 5 times, and let stand for 3 min each time.

[0061] (3) Primary antibody incubation: blood was collected from each mouse, added to PBS, centrifug...

Embodiment 3CI

[0072] The construction of embodiment 3CIB phage single-chain antibody library

[0073] 1. Extraction of mouse spleen RNA

[0074] According to the identification of the immunization effect, the No. 2 mouse was immunized for the fifth time. By intraperitoneal injection, 50 μg of artificial complete antigen CIB-BSA was injected, which was a hyperimmune procedure without any adjuvant. After 5 days, the orbital blood was taken to separate the serum and it was a positive serum, and the patient was killed by neck dislocation and the spleen was removed. RNA was extracted according to the operating procedures of the Total RNA Extraction Kit of Treasure Biotech. Then immediately identified by 1.2% agarose gel electrophoresis.

[0075] Test results such as Figure 5 As shown, 28S and 16S RNA can be detected with clear bands, while 5S RNA is partially degraded. Subsequently, RT-PCR was used to reverse transcribe the identified RNA to obtain cDNA, and then PCR amplification of VH an...

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PUM

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Abstract

The invention discloses an anti-CIB single-chain antibody, and a screening method and application thereof. The amino acid sequence of the single-chain antibody is as shown in SEQ ID NO. 1; and the nucleotide sequence of a gene for coding the single-chain antibody is as shown in SEQ ID NO. 2. According to the invention, an antibody gene engineering technology and a phage display technology are utilized to construct a CIB murine immune phage single-chain antibody library; and the ScFv of CIB is screened out from the CIB murine immune phage single-chain antibody library, so that a theoretical basis and a material basis are provided for later establishment of a CIB immunological rapid detection method.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to an anti-CIB single-chain antibody and its screening method and application. Background technique [0002] At present, the methods for detecting sibuterol residues mainly focus on liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis (Capillary electrophoresis) , CE) and other methods. The above methods are instrument confirmation methods, and the detection precision and accuracy are very reliable. However, because the above methods have problems such as expensive instruments, cumbersome operations, and the need for professionals, it is difficult to meet the requirements of routine rapid detection. The immunochemical screening method based on the specific recognition reaction of antigen and antibody can meet the requirements of routine rapid detection. The core of this method is to obtain stable, specific a...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N15/13C12N15/70G01N33/53
CPCC07K16/44C12N15/70G01N33/5308C07K2317/622
Inventor 王选年郭东光李文明岳锋孙国鹏李鹏苄爽丽朱艳平潘鹏涛齐永华
Owner XINXIANG UNIV
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