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Apolygus lucorum ultraspiracle protein specific polyclonal antibody as well as preparation method and application thereof

A polyclonal antibody, chlorotic bug technology, applied in the direction of serum immunoglobulin, anti-animal/human immunoglobulin, material test products, etc., can solve the difference in inhibitory effect, different protein expression, the effect of pesticide use drop and other problems, to achieve the effect of high sensitivity

Inactive Publication Date: 2014-08-20
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein expression of the USP gene of Lygus green bug is different at different ages and in different tissues, which leads to a large difference in the inhibitory effect of the target gene superventilin after the application of insect growth regulators, which can cause the use of pesticides. decline

Method used

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  • Apolygus lucorum ultraspiracle protein specific polyclonal antibody as well as preparation method and application thereof
  • Apolygus lucorum ultraspiracle protein specific polyclonal antibody as well as preparation method and application thereof
  • Apolygus lucorum ultraspiracle protein specific polyclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Obtaining Polyclonal Antibody of Aleus AluSP

[0026] First carry out antigen treatment: take the total amount of purified recombinant ALUSP protein about 5mg, the initial dose is 0.3mg / head, the second, third, and fourth immunization dose is 0.15mg / head; the primary antigen is protein antigen and equal volume Freund's complete adjuvant is mixed and emulsified; the second, third, and fourth immune antigens are protein antigens and an equal volume of Freund's incomplete adjuvant is mixed and emulsified. Then carry out animal immunization: use subcutaneous multi-point injection to immunize healthy female New Zealand white rabbits, a total of 4 times; the first immunization antigen is Freund's complete adjuvant and protein antigen; the second immunization after an interval of 21 days, the antigen is Freund's no Complete adjuvant and protein antigen; the third immunization after 35 days interval, the antigen is Freund's incomplete adjuvant and protein antigen, 1ml bl...

Embodiment 2

[0033] Example 2 ELLSA detection of ALUSP polyclonal antibody of Lygus lucorum

[0034] Coat the antigen with pH9.6, 0.05mol / l carbonate, and incubate overnight at 4°C; after removing the antigen, wash with PBST washing solution containing 0.05% Tween-20 three times for 3 minutes each; then add 5% skimmed milk powder to each well 100μl blocking solution, blocked at 37°C for 60 minutes; take out and wash with PBST washing solution containing 0.05% Tween-20 three times, 3 minutes / time; firstly dilute the rabbit serum according to 1:1000, then dilute by multiples, and incubate at 37°C for 1 Hours; take out and wash with PBST washing solution containing 0.05% Tween-20 three times, 3 minutes / time; add horseradish enzyme-labeled goat anti-rabbit IgG (H+L), dilute 1:8000, and incubate at 37°C for 45 min; take out and wash with PBST The solution contains 0.05% Tween-200 and washes five times, 3 minutes / time; finally, 100 μl / well of substrate solution (TMB) is added for 15 minutes, and 10...

Embodiment 3

[0036] Example 3: Western blot detection of ALUSP polyclonal antibody of Lygus lucorum

[0037] Take the ALUSP recombinant protein and load it in a gradient. After loading the sample, run the polyacrylamide gel at 90V to finish the stacking gel, and then increase the voltage to 200V until the end of electrophoresis. After the electrophoresis is over, remove the gel and transfer the membrane, and transfer the membrane at a constant voltage of 100V for 1.5 hours. After the electroporation, the membrane was removed and washed with PBS 4 times for 5 minutes each time. Then place it in 37°C, 5% skimmed milk powder blocking solution for 1 hour. Dilute the primary antibody (1:1000) with blocking solution, and react the membrane in the primary antibody dilution solution at 37°C for 1 hour. Wash the membrane 4 times, 5 minutes each time, dilute the secondary antibody (1:5000) with a blocking solution containing 5% milk. The membrane was reacted in the secondary antibody at 37°C for 1 ...

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Abstract

The invention discloses a specific polyclonal antibody of an apolygus lucorum ultraspiracle protein (ALUSP) as well as a preparation method and application thereof. An ALUSP recombinant protein after prokaryotic expression and purification is applied to an immune rabbit to prepare a polyclonal antibody; after determining titer of the polyclonal antibody by utilizing an ELISA (enzyme-linked immuno sorbent assay) method, the inventor adopts a Western blot method to determine that the polyclonal antibody can specifically recognize the ALUSP recombinant protein and can produce specific immunity reaction. On such as a basis, the inventor further establishes an efficient, high-sensitivity and accurate Western blot method, so that apolygus lucorum ultraspiracle protein can be specifically detected out from an apolygus lucorum polypide. The specific polyclonal antibody can be applied to providing a material basis and a technical support for tissue distribution of the ultraspiracle protein in the apolygus lucorum, rapid detection of the protein and molecular biology study.

Description

Technical field [0001] The present invention relates to the field of antibody engineering agricultural science and technology, in particular to a polyclonal antibody specific to A. lucorum super valve protein and a preparation method and application thereof. Background technique [0002] Apolygus lucorum (Hemiptera:Miridae) belongs to the family Hemiptera, and is an important pest on cotton, vegetables, fruit trees, pasture and other crops. In recent years, due to the large-scale planting of transgenic Bt cotton and the adjustment of the agricultural industry structure, the population of A. lucorum has increased sharply, coupled with long-term dependence on chemical pesticide control, resulting in the increasing resistance of the lucorum. The current agricultural production The prevention and control of A. lucorum faces severe challenges, and it is urgent to develop new pollution-free prevention and control methods to reduce the use of chemical pesticides. So far, a variety of n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/06G01N33/68
Inventor 谭永安肖留斌柏立新孙洋赵静
Owner JIANGSU ACAD OF AGRI SCI
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