Recombinant antigen protein rP44-60 for detecting granulocytoplasmosis and kit containing antigen

A technology of recombinant antigen protein and anaplasma, applied in the field of immunology, can solve the problems of strengthening the detection method of granulocytic anaplasmosis, lack of high sensitivity and high specificity, difficulty in antibody detection, etc., and achieves broad market prospects and application value. The effect of shortening the color time and reducing the missed diagnosis rate

Active Publication Date: 2022-03-11
河套学院
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] The invention provides a recombinant antigenic protein rP44-60 for detection of granulocytic anaplasmosis, which is used to solve the problems of difficult antibody detection, missed detection defects, and lack of high-sensitivity and high-specificity serum detection reagents in HGA serum detection
The recombinant antigen protein provided by the invention has high sensitivity and strong specificity, can quickly detect the specific antibody in the serum infected with Anaplasma phagophilum, improves the problem that the antibody is difficult to capture due to antigen variation in HGA, and makes up for the lack of detection of the existing antigen. defects, effectively reduce the rate of missed diagnosis, and help to strengthen the optimization of detection methods for granulocytic anaplasmosis, which has very important practical significance for the fields of medicine, public health and animal husbandry

Method used

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  • Recombinant antigen protein rP44-60 for detecting granulocytoplasmosis and kit containing antigen
  • Recombinant antigen protein rP44-60 for detecting granulocytoplasmosis and kit containing antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0059] A method for preparing recombinant antigen protein rP44-60 for detection of granulocytic anaplasmosis, comprising the following steps:

[0060] 1) Using bioinformatics technology, based on the existing p44 polygenome sequence as the basic template, the original proteome was synthesized, and then the original proteome was sent to Gene Script for sequencing to confirm that the above original proteome contained the rP44-60 target gene sequence.

[0061] 2) Using the RNA extraction kit (QIAGEN), the RNA extracted from the cultured cells of the susceptible THP-1 was used as a template, and reverse-transcribed into cDNA according to the instructions of the reverse transcription kit. Primers were designed according to the target gene sequence, followed by RT-PCR amplification, and the amplified product was recovered by agarose gel electrophoresis (TaKaRa's DNA recovery and purification kit) to obtain the recovered product. The above reverse transcription conditions are: 37°C, ...

Embodiment 2

[0070] This embodiment provides a kit based on a double-antigen sandwich colloidal gold immunochromatography method, which includes the recombinant antigen protein rP44-60 for the detection of granulocytic anaplasmosis.

[0071] Specifically, the preparation method of the colloidal gold immunochromatography test strip for the kit is as follows:

[0072] 1) Using the colloidal gold probe as a tracer marker, the colloidal gold probe solution is prepared, and then the recombinant antigen protein rP44-60 colloidal gold complex and the mouse IgG colloidal gold complex are prepared respectively.

[0073] 2) Mix the above-mentioned recombinant antigen protein rP44-60 colloidal gold complex and mouse IgG colloidal gold complex, resuspend with 0.1mmol / L Tris solution containing 5% sucrose, 5% BSA, and then adjust the resuspension to OD530 After reaching 2, soak the bonding pad and dry it at 37°C for 4 hours to obtain a gold standard pad.

[0074] 3) Use the recombinant antigenic prote...

experiment example 1

[0087] Antigenicity test

[0088] Experimental method: The recombinant antigen protein rP44-60 prepared in Example 1 was used as an antigen, and the serum of animals infected with Anaplasma phagocytophilum and positive patients was used to detect its antigenicity by WB technology, and its feasibility as an antigen was evaluated. Among the animals, there were 3 positive sera of mice, 3 positive serums of sheep, and 3 positive patient sera. The secondary antibody used Goat Anti-Human IgG Alkaline phosphatase (Invitrogen). First, the recombinant antigen protein rP44-60 was loaded on the SDA-PAGE gel, the protein was transferred to the nitrocellulose membrane, the membrane was cut into strips, blocked for 30min, incubated with the primary antibody positive serum (1:2000), washed with PBS for 3 times, incubated with secondary antibody (1:5000), washed 4 times with PBS, and developed. The result is as figure 1 shown.

[0089] figure 1 It is the result of WB technology detection...

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Abstract

The invention provides a recombinant antigen protein rP44-60 for detecting granulocytoplasmosis and a kit containing the antigen, and belongs to the technical field of immunology, and the recombinant antigen protein comprises an amino acid sequence as shown in SEQ ID NO.1 in a sequence table. The kit comprises a recombinant antigen protein rP44-60, a sample pad, a combination pad, an absorption pad, a nitrocellulose membrane, a colloidal gold probe, mouse IgG and goat anti-mouse IgG. The recombinant antigen protein disclosed by the invention is high in sensitivity and strong in specificity, and can be used for rapidly detecting an antibody in anaplasma phagocytophilum infected serum, so that the problem that the antibody is difficult to capture due to antigen variation of HGA is solved, the defect of existing antigen missing detection is made up, and the missed diagnosis rate is effectively reduced; according to the preparation method, cross reactivity caused by irrelevant sequences is reduced, and the expression quantity and stability of recombinant antigen protein are high; the kit has the characteristics of rapidness, sensitivity, high specificity, no limitation of experimental conditions and the like, and has low requirements on detection conditions.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular to a recombinant antigen protein rP44-60 for detecting granulocytic anaplasmosis and a kit containing the antigen. Background technique [0002] Human granulocytic anaplasmosis (HGA) is caused by Anaplasma phagocytophilum (Anaplasma phagocytophilum) infecting neutrophils in human peripheral blood, mainly characterized by fever accompanied by leukopenia, thrombocytopenia, and multiple organ dysfunction Clinically manifested tick-borne disease. The disease is caused by a parasitic bacterium that lives in cells and is mainly transmitted through tick bites. The incubation period of the disease is 1-2 weeks, mostly acute onset, persistent high fever, and the fever can reach above 40°C. The clinical manifestations of the disease are mainly general malaise, fatigue, headache, muscle pain, nausea, vomiting, anorexia, diarrhea, etc., which may be accompanied by damage to multiple organs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/29C12N15/31C12N15/70G01N33/569G01N33/58G01N33/558G01N33/543
CPCC07K14/29C12N15/70G01N33/56911G01N33/558G01N33/587G01N33/54346C12N2800/101G01N2333/29G01N2469/20Y02A50/30
Inventor 高娃刘丹樊红霞李晓娜李方超乌兰图雅
Owner 河套学院
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