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Preparation method and application of HAO rabbit polyclonal antibody

A technology of polyclonal antibody and antibody serum, applied in the direction of anti-enzyme immunoglobulin, immunoglobulin from serum, measuring devices, etc., can solve the problems of inability to detect and express HAO expression, undisclosed HAO antibody preparation methods, etc., to achieve The effect of improving detection efficiency and accuracy

Inactive Publication Date: 2019-12-20
INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0004] At present, fluorescent quantitative PCR method is mostly used to detect HAO, but this method characterizes the mRNA level of HAO gene, and cannot directly express the protein expression level of HAO, so that it is impossible to directly detect the expression level of HAO; while using ELISA method , the HAO antibody can be combined with the HAO antigen, and the protein can be directly quantitatively analyzed, which is simple and convenient, and has high accuracy; however, the preparation method of the HAO antibody has not been disclosed in the prior art

Method used

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  • Preparation method and application of HAO rabbit polyclonal antibody
  • Preparation method and application of HAO rabbit polyclonal antibody
  • Preparation method and application of HAO rabbit polyclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Preparation of HAO recombinant protein

[0032] Using the ATCC 19718 culture as a template, the nucleic acid sequence of Hydroxylamine oxidoreductase 25-570 amino acids was amplified by PCR with Pfu high-fidelity DNA polymerase (Tiangen ep101), the fragment length was 1638bp, identified by 1% agarose gel electrophoresis, see figure 1 ; Use the DNA product recovery kit (Axygen, AP-GX-250) to recover the target band by cutting the gel, and the recovered product is digested with restriction endonucleases Bam HI and XhoI, and the gel is recovered and the same enzyme digestion and gel recovery are carried out. The treated pET28a(+)-SUMO plasmid fragments were ligated overnight at 4°C, transformed into DH5α competent cells (Tiangen), picked clones, and after PCR identification and sequencing confirmed positive, plasmid DNA mini-extraction kit (Axygen, AP-MN-P-250) to extract the plasmid and carry out the next step of transformation. The resulting plasmid was transf...

Embodiment 2

[0033] Example 2 Identification of protein expression for immunization

[0034] 4000rpm centrifugation 10min of the expression bacterial strain that has expressed the protein of interest obtained in embodiment 1 is collected thalline and discards supernatant, phosphate buffered saline (Solei Bao) resuspended thallus, add the β-mercaptoethanol of 1 / 1000 volume ( Sigma-Aldrich), put the bacterial solution in ice cubes, crush the bacterial cells with an ultrasonic breaker, the power is 400W, the ultrasonic time is 3s and the interval is 3s for a total of 10min, and the bacterial supernatant 1 and inclusion body precipitation are obtained by centrifugation at 9000rpm for 10min. 2M urea solution was added to the precipitate, ultrasonicated under the same conditions and then centrifuged to obtain supernatant 2 and inclusion body precipitate. 8M urea solution was used to dissolve the inclusion body precipitate, and centrifuged at 12000rpm for 1min to obtain the supernatant as the inc...

Embodiment 3

[0035] Example 3 Antiserum Affinity Purification Using Protein Detection

[0036]The process of Example 1 and Example 2 was repeated to obtain an inclusion body protein solution, which was stained with Coomassie Brilliant Blue after SDS-PAGE electrophoresis. The results showed that the protein concentration was 2 mg / ml, and the purity was equivalent to that of the antigen protein, which could be used for antigen affinity purification. see results image 3 ;

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Abstract

The invention discloses a preparation method of an HAO rabbit polyclonal antibody. The preparation method comprises the process of performing prokaryotic expression to obtain HAO recombination protein, preparing the HAO recombination protein into suspension, performing injection on animals, after 2-4h, performing separation on animal serum to obtain an HAO antibody, and performing purification onthe HAO antibody by an antigen affinity purification method so as to obtain the polyclonal antibody. The polyclonal antibody prepared by the method is good in specificity and can directly detect HAO protein.

Description

technical field [0001] The present invention relates to the technical field of polyclonal antibody preparation, and more specifically relates to a preparation method of Hydroxylamineoxidoreductase rabbit polyclonal antibody Background technique [0002] Ammonia oxidizing bacteria are Gram-negative bacteria, which belong to the class β-Proteobacteria or class γ-Proteobacteria of the phylum Proteobacteria. It is considered to be the most sensitive and fragile metabolic pathway in the sewage biological treatment system, and it is the key link for the stable operation of the system. [0003] Nitrification plays an important role in the nitrogen cycle in nature and is a key reaction for biological nitrogen removal in sewage systems. The nitrosation reaction is the rate-limiting step of the whole process, which is completed by ammonia oxidizing bacteria (AOB). Oxidoreductase, HAO) catalyzes the process of generating nitrite; therefore, the expression level of HAO can be used as ...

Claims

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Application Information

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IPC IPC(8): C07K16/40C07K16/06G01N33/573
CPCC07K16/06C07K16/40G01N33/573G01N2333/90694
Inventor 赵辰邱志刚王景峰谌志强薛斌王尚李辰宇
Owner INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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