Antibody gene for resisting pyrethriods pesticide scFv (Single Chain Fragment Variable) and application of antibody gene

A pyrethroid, scfv technology, applied in the fields of genetic engineering and immunological detection, can solve the problems of cumbersome preparation and screening process, easily unstable cell genetics, and high technical requirements for operation, and achieves easy development of downstream production, genetic information and other problems. Actionable and genetically stable effects

Active Publication Date: 2013-05-08
天津市明大百诚科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is an urgent need for a new rapid detection method; although the common enzyme-linked immunoassay has high sensitivity and large sample capacity, it has a large demand for experimental animals and a long feeding cycle, and different individual animals have different sensitivity to agricultural and veterinary drugs, resulting in There are problems such as large differences in antibodies; as the second-generation antibody, monoclonal antibodies are not conducive to wide application due to the cumbersome preparation and screening process, high requirements for operational technology, and easily unstable cell genetics after cloning.

Method used

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  • Antibody gene for resisting pyrethriods pesticide scFv (Single Chain Fragment Variable) and application of antibody gene
  • Antibody gene for resisting pyrethriods pesticide scFv (Single Chain Fragment Variable) and application of antibody gene
  • Antibody gene for resisting pyrethriods pesticide scFv (Single Chain Fragment Variable) and application of antibody gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The extraction of total RNA of embodiment 1PBA monoclonal cell and the synthesis of cDNA

[0036] 1. Extraction of total RNA from PBA monoclonal cell line 2G2E7

[0037] Take the fresh monoclonal cell line 2G2E7 screened by our laboratory, and use Qigen RNeasy Mini Kit to extract total RNA. The cell line 2G2E7 can produce monoclonal antibody against PBA.

[0038] ⑴Cultivate cells to 3-4×10 6 , centrifuged at 300×g for 5 min, and removed the supernatant;

[0039] (2) Preparation of lysate: Buffer RLT:β-mercaptoethanol=1ml:10μl;

[0040] (3) Add 350 μl Buffer RLT lysis buffer to the centrifuged cells, flick the tube wall, and blow with a pipette gun to make the buffer fully contact with the cells;

[0041] (4) Add the same volume of 70% ethanol solution, pipette gently to mix, and be careful not to centrifuge;

[0042] (5) Take the silica gel column in the Qigen RNeasy Mini Kit, put it into a 2ml collection tube, transfer all the samples to the column, centrifuge at 1...

Embodiment 2

[0059] Example 2 Construction of PBA-scFv single-chain antibody library

[0060] 1. PCR amplification of VH and VL

[0061] Primers used: (VL back primer and scFv back primer are primers used in equal amounts)

[0062]

[0063] Where K is a kappa primer, L is a lambda primer, the lambda chain can also be amplified with the kappa pre-primer, so there is no separate lambda pre-primer.

[0064]

[0065] PCR system (50μl):

[0066] PCR program:

[0067]

[0068] After PCR amplification, the product was detected by agarose gel electrophoresis, and the results were as follows: figure 1 shown.

[0069] 2. Use the Qiagen gel purification kit to recover the target fragment

[0070] (1) Prepare a clean centrifuge tube and weigh it;

[0071] (2) Cut off the target fragment on the agarose gel with a clean blade and put it into a centrifuge tube;

[0072] (3) Weigh, calculate the weight of the cut gel, and add 3 times the volume of Buffer QG to 1 times the volume of the gel...

Embodiment 3

[0125] The panning of embodiment 3PBA antibody library

[0126] 1. Preparation before panning

[0127] (1) Take 1ml of the primary antibody library, add it to 250ml of 2×YT-G medium, and incubate at 37°C with shaking at 250rpm for 1h;

[0128] (2) When the bacteria grow to an appropriate concentration, add 100 μg / ml AMP, and at the same time add freshly prepared phage M13K07 with moi=20:1 (preserved in the laboratory), shake gently in a water bath at 37°C for 30 minutes, then shake at 250 rpm at 37°C, and incubate for 30 minutes;

[0129] (3) Take out an appropriate amount of bacterial solution and dilute it by 2 times, and spread it on 2×YT-AG and 2×YT-KG plates (2×YT plates containing ampicillin and glucose) respectively, to confirm the success of phage infection;

[0130] (4) Centrifuge at 1000×g for 15 minutes, and take the supernatant carefully;

[0131] (5) Precipitate with 500ml 2×YT-AK suspended bacteria, culture overnight at 37°C and 250rpm;

[0132] (6) Centrifuge a...

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Abstract

The invention relates to an antibody gene for resisting pyrethriods pesticide scFv (Single Chain Fragment Variable), and particularly relates to construction of a pyrethriods pesticide antibody library, screening and sequence determination of a specific single-chain antibody gene and preparation of a specific phage display chain antibody. The antibody gene comprises nucleotide sequences of a coding heavy chain variable region (116 amino acids), a flexible chaining region (15 amino acids) and a light chain variable region (107 amino acids), shown as a SEQ ID No. 1 and a SEQ ID No.2 in a sequence table; and the antibody which is displayed on the surface of phage is prepared in a host cell by using phagemid. The antibody can be normally expressed and folded on the surface of the phage and can be used for well identifying pyrethriods antigens. Compared with a conventional antibody, the antibody has the advantages of high stability, convenient preparation and important potential application value.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and the field of immunological detection, in particular to an antibody gene and application of an anti-pyrethroid pesticide scFv. Background technique [0002] Pyrethroids (Pyrethroids) pesticides are an important class of biomimetic insecticides, which have been considered as safe pesticides in the past. Because of its advantages of being easily degraded by the oxidase system in the body and no accumulation, it is currently widely used and used in large quantities, accounting for 20% of the total production of insecticides and 25% of the total area of ​​insecticides used. However, recent studies have shown that most pyrethroid pesticides are environmental hormone substances, which have immune system toxicity, genotoxicity and cardiovascular toxicity, and seriously affect the normal physiological activities of mammals. Even at low doses, long-term exposure is easy Cause chronic diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C07K16/44G01N33/53
Inventor 杜欣军常继晨王硕
Owner 天津市明大百诚科技有限公司
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