Apolygus lucorum soluble trehalase (AlTre-1) monoclonal antibody as well as preparation method and application of thereof

A monoclonal antibody, chlorotic bug technology, applied in anti-enzyme immunoglobulins, measuring devices, instruments, etc., can solve problems such as differences in inhibitory effects and decline in pesticide use effects, and achieve high-sensitivity effects.

Inactive Publication Date: 2015-08-12
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein expression level of Lygus green bug water-soluble trehalase (AlTre-1) gene in different ages and different tissues is not the same, resulting in the inhibitory

Method used

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  • Apolygus lucorum soluble trehalase (AlTre-1) monoclonal antibody as well as preparation method and application of thereof
  • Apolygus lucorum soluble trehalase (AlTre-1) monoclonal antibody as well as preparation method and application of thereof
  • Apolygus lucorum soluble trehalase (AlTre-1) monoclonal antibody as well as preparation method and application of thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Obtaining of the monoclonal antibody of Lygus chlorophylla AlTre-1

[0028]Antigen treatment is carried out first: the total amount of purified recombinant AlTre-1 protein is about 2 mg, and the doses of the primary, secondary, third and fourth immunizations are all 100 μg per mouse; the primary immunization antigen is protein antigen and an equal volume of Freund’s complete Mix and emulsify the agent; the second, third and fourth immune antigens are protein antigens mixed with an equal volume of Freund's incomplete adjuvant and emulsified. Then animal immunization: healthy 6-8 week-old female BALB / c mice were immunized by subcutaneous multi-point injection for a total of 4 times; the first immunization antigen was Freund's complete adjuvant and protein antigen; the second time after an interval of 21 days immunity. The third immunization antigen was used for the third immunization after an interval of 35 days; the fourth immunization antigen was used for the...

Embodiment 2

[0041] Example 2: Western blot detection of Lygus chlorophylla AlTre-1 monoclonal antibody

[0042] Take the AlTre-1 recombinant protein and load it according to the gradient. After loading the sample, run the polyacrylamide gel at 75V to finish the stacking gel, and then increase the voltage to 110V until the end of electrophoresis. After the electrophoresis, the gel was removed and transferred to the membrane at a constant voltage of 100V for 1.5 hours. After electroporation, remove the membrane and wash it with PBS 4 times, 5 minutes each time. Then place it at 37°C in 5% non-fat dry milk blocking solution for 1 hour. The primary antibody was diluted with blocking solution, and the membrane was reacted in the primary antibody diluent at 37°C for 1 hour. Wash the membrane 4 times, 5 minutes each time, dilute the secondary antibody with blocking solution containing 5% milk. The membrane was reacted in the secondary antibody at 37°C for 1 hour. Washed membrane ECL develop...

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Abstract

The invention discloses an Apolygus lucorum soluble trehalase (AlTre-1) monoclonal antibody as well as a preparation method and application of thereof. The method comprises the following steps: firstly, applying AlTre-1 recombinant protein subjected to prokaryotic expression and purification to an immunized mice so as to prepare antiserum, and determining the titer of the antiserum by an indirect ELISA method; and preparing myeloma cells and spleen cells and then performing cell fusion, screening positive porocyte by an ELISA method, then performing subcloning, and picking out positive monoclonal holes for enlarging cultivation and strain fixing, thereby finally obtaining the AlTre-1 monoclonal antibody. Besides, a Western blot method determines that the monoclonal antibody can specifically identify the AlTre-1 recombinant protein and can generate specific immune reaction. By virtue of the application of the AlTre-1 monoclonal antibody, a material base and a technical support can be provided for the tissue distribution of soluble trehalase in Apolygus lucorum and the quick detection and molecular biology study of the protein.

Description

technical field [0001] The invention relates to the field of antibody engineering agricultural science and technology, in particular to a water-soluble trehalase monoclonal antibody of Lygus viridans and its preparation method and application. Background technique [0002] The green mirid bug Apolygus lucorum (Hemiptera: Miridae) belongs to the Miridae family of Hemiptera, and is an important pest on various crops such as cotton, vegetables, fruit trees, pasture and so on. In recent years, due to the large-scale planting of transgenic Bt cotton and the adjustment of the agricultural industrial structure, the population of Lygus spp. has increased sharply, and the long-term dependence on chemical pesticide control has led to the increasing resistance of Lygus spp. The prevention and control of the green Lygus is facing 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 o...

Claims

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Application Information

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IPC IPC(8): C07K16/40G01N33/577G01N33/68
Inventor 谭永安肖留斌柏立新赵静孙洋
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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