Polyclonal antibody against outer membrane protein of Candidatus liberobacter asiaticum, and preparation method and application thereof

A technology of citrus Huanglongbing bacteria and polyclonal antibody, which is applied in the field of genetic engineering to achieve the effects of shortening the production cycle, increasing yield and purity, and improving efficiency

Inactive Publication Date: 2011-10-12
ZHEJIANG CITRUS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has not been reported to obtain a large amount of this protein through in vitro expression, and use it as an immunogen to prepare antiserum and use it to detect Huanglongbing citrus

Method used

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  • Polyclonal antibody against outer membrane protein of Candidatus liberobacter asiaticum, and preparation method and application thereof
  • Polyclonal antibody against outer membrane protein of Candidatus liberobacter asiaticum, and preparation method and application thereof
  • Polyclonal antibody against outer membrane protein of Candidatus liberobacter asiaticum, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 Amplification of outer membrane protein gene and construction of expression vector

[0033] 1. Design and synthesis of specific primers

[0034] Using bioinformatics software to analyze the outer membrane protein gene sequence of Huanglongbing citri, design specific primers for specific amplification of the N-terminal of the gene: OMPN5: 5′-AGCGGATCCCAATTGAAGATGATAGTTCGCT-3′ and OMPN3: 5′-AGCAAGCTTTTACACATAATCGGATACATCAT-3′, using Primers OMPC5 at the C-terminus of the amplified gene: 5′-AGCGGATCCTATTTTTAGGGAGTCCTATATC-3′ and OMPC3: 5′-AGCAAGCTTTTACATGCGATTACCTATACG-3′, the designed primers were sent to the company for synthesis.

[0035] 2. Extraction of total DNA from citrus leaves

[0036] Collect citrus leaves infected by citrus Huanglongbing bacteria in the field, take the midrib of the leaves and weigh about 0.1-0.2g, grind them into powder with liquid nitrogen in a sterile mortar, add appropriate amount of PVP and mercaptoethanol, and extract with t...

Embodiment 2

[0045] Example 2 Induced expression and separation and purification of outer membrane protein

[0046] 1. Induced expression of outer membrane proteins

[0047] Using CaCl 2 Escherichia coli BL21 competent cells were prepared by the above method, and the above-mentioned recombinant plasmids pIGH3-OMP-N and pIGH3-OMP-C were transformed into competent cells respectively, and the transformed products were coated on LB medium plates, and cultured at 37°C until colonies grew out. . Pick the monoclonal colony on the plate, shake culture in LB liquid medium, add 0.5mM IPTG to induce the expression of the target protein in the logarithmic growth phase (OD600 is 0.5-0.6), and perform SDS-PAGE electrophoresis on the expressed product, Detection of protein expression. The result shows that there are specific bands with a size of about 58KD and 55KD in the induced bacterial protein (see attached Figure 5 ), consistent with the expected target protein size.

[0048] 2. Separation and...

Embodiment 3

[0050] Example 3 Preparation and Purification of Outer Membrane Protein Polyclonal Antibody

[0051] 1. Antiserum preparation and potency determination

[0052]Take 500 μg of OMP-N protein and OMP-C protein respectively, supplement it with PBS to 500 μL, add an equal amount of Freund’s complete adjuvant, emulsify for 3 times, inject into white rabbits subcutaneously (take negative serum before immunization), and inject 250 μg of protein 2 weeks later. / 500μL protein plus the same amount of Freund's incomplete adjuvant to boost the immunization, and then 250ug / 500ul protein plus the same amount of Freund's incomplete adjuvant to boost the immunization after 5 weeks and 8 weeks respectively, sacrifice the white rabbits in the 10th week, and extract the anti- serum. Coat OMP-N protein and OMP-C protein on the microtiter plate respectively, dilute negative serum at 1:1000 and 1:4000, and dilute positive antiserum at 1:1000, 1:4000, 1:16000, 1:32000 , 1:64000, 1:128000 dilution, ...

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Abstract

The invention relates to a polyclonal antibody against an outer membrane protein of Candidatus liberobacter asiaticum, and a preparation method and application thereof. The polyclonal antibody is characterized by being prepared through injecting target proteins acquired by prokaryotic expression and used as an immunogen into a rabbit, and can effectively detect Candidatus liberobacter asiaticum existing in citrus by separately and specifically combining with the N terminal and C terminal of the outer membrane protein of Candidatus liberobacter asiaticum. According to the invention, plenty of high-purity target proteins are acquired by carrying out prokaryotic expression on the outer membrane protein genes of Candidatus liberobacter asiaticum, and the target proteins are used as the immunogen and injected into the rabbit, thus preparing and purifying the polyclonal antibody against the outer membrane protein. The titer assay and enzyme-linked immune experiments show that the prepared antibody has high titer and high specificity, and can be used for detecting field samples infected by Candidatus liberobacter asiaticum, thus laying a good foundation for the further development of colloidal gold immunochromatography assay test strips used for the field rapid detection of Candidatus liberobacter asiaticum.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a polyclonal antibody for the outer membrane protein of citrus huanglongbing bacteria, a preparation method thereof, and an application of the antibody in detecting citrus huanglongbing bacteria. Background technique [0002] Citrus huanglongbing is a devastating disease that is difficult to control, seriously harmful and threatening in citrus production. It is called "AIDS" on citrus and is widely distributed in almost all citrus producing areas in my country. The disease was first discovered in the south of Wenzhou in Zhejiang Province in the 1980s. With the warming of the climate and the development of citrus production, the epidemic spread northward. Except for Quzhou and Hangzhou, which have not been reported yet, the disease is serious in major citrus producing areas such as Taizhou and Wenzhou. As a devastating disease, citrus infected plants often ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/70G01N33/569C12R1/19
Inventor 鹿连明陈国庆杜丹超胡秀荣张利平
Owner ZHEJIANG CITRUS RES INST
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