Preparation method of phagocytophilia recombinant protein antigen and kit containing recombinant protein antigen

A recombinant protein and phagocytic cell technology, applied in the biological field, can solve the problems of long time-consuming and low sensitivity of detection methods, and achieve the effects of reducing detection cost, high expression and stability, and strong specificity.

Active Publication Date: 2022-04-12
河套学院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] This application provides a preparation method of anaplasma phagocytophilum recombinant protein antigen and a kit containing the recombinant protein antigen to solve the above-mentioned problems of low sensitivity and time-consuming detection methods for human granulocytic anaplasmosis , this application is used for the rapid detection of specific antibodies in the serum of patients infected with Anaplasma phagocytophilum, and provides a reliable basis for the rapid and simple detection of HGA

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  • Preparation method of phagocytophilia recombinant protein antigen and kit containing recombinant protein antigen
  • Preparation method of phagocytophilia recombinant protein antigen and kit containing recombinant protein antigen
  • Preparation method of phagocytophilia recombinant protein antigen and kit containing recombinant protein antigen

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preparation example Construction

[0039] First aspect, such as figure 1 As shown, the present application provides a method for preparing recombinant protein antigen of Anaplasma phagophilum, comprising:

[0040] S101. Reverse transcription and PCR amplification of the gene fragment containing p44 gene: use the RNA extraction kit to extract the RNA of the cultured THP-1 cells infected by Anaplasma phagophila, and use the extracted RNA as a template , reverse transcription to obtain the cDNA containing the P44 gene, and then perform PCR amplification on the obtained cDNA, the forward primer of the first round of amplification is:

[0041] P3726F 5'-GCTAAGGAGTTAGCTTATGA-3';

[0042] The reverse primer is:

[0043] P4183R 5'-CAATAGT(C / T)TTAGCTAGTAACC-3';

[0044] The forward primer for the second round of amplification is:

[0045] P3761F 5'-CTGCTCT(T / G)GCCAA(A / G)ACCTC-3';

[0046] The reverse primer is:

[0047] P4257R 5'-AGAAGATCATAACAAGCATTG-3'.

[0048] Optionally, the reverse transcription conditions ...

Embodiment 1

[0086] This example provides a method for synthesizing the recombinant protein antigen rP44-47E of Anaplasma phagophilum:

[0087] 1) Utilize the RNA extraction kit (Invitrogen) to extract the RNA of the THP-1 cultured cells infected by Anaplasma phagocytophilum, and use bioinformatics technology to design reverse transcription primers according to the sequence of the target p44-47E gene, And using the extracted mRNA as a template, use a reverse transcription kit (Invitrogen) to reverse transcribe the cDNA of the gene p44, and perform two rounds of PCR amplification on the obtained cDNA. The forward primer for the first round of amplification is: P3726F5 '-GCTAAGGAGTTAGCTTATGA-3'; the reverse primer is: P4183R 5'-CAATAGT(C / T)TTAGCTAGTAACC-3'; the second round of amplification forward primer is: P3761F5'-CTGCTCT(T / G)GCCAA(A / G) ACCTC-3'; the reverse primer is: P4257R5'-AGAAGATCATAACAAGCATTG-3'; the conditions of the two rounds of amplification are the same: denaturation at 94°C...

Embodiment 2

[0093] This embodiment provides a kit containing recombinant protein antigen rP44-47E of Anaplasma phagocytophilum. The detection card in the kit is made based on the double-antigen sandwich colloidal gold immunochromatography method, and its preparation method is as follows:

[0094] 1) Add 1mL of 10g / L chloroauric acid into 99mL of deionized double-distilled water, the concentration of the obtained chloroauric acid is 0.1g / L, place it in a flask with a condenser and heat it to boiling, and quickly add 10g / L of chloroauric acid under magnetic heating and stirring. L of trisodium citrate aqueous solution 1.6mL, continue to heat until the solution is wine color. After cooling, make it back to the original volume, put it in a brown bottle, and store it in a refrigerator at 2-8°C for later use.

[0095] 2) The recombinant protein antigen rP44-47E to be marked obtained in Example 1 was first blocked with 5% skim milk, purified by affinity chromatography, and then the recombinant ...

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Abstract

The invention provides a preparation method of an anaplasma phagocytophilum histone antigen. The preparation method comprises the following steps: carrying out reverse transcription and PCR (Polymerase Chain Reaction) amplification on a gene segment containing a p44 gene, recovering and cloning an amplification product, extracting and reconfirming a target gene, and synthesizing a recombinant protein antigen rP44-47E. According to the preparation method of the anaphagophilic recombinant protein antigen, the recombinant protein antigen rP44-47E can be singly, efficiently and massively prepared; the prepared recombinant protein antigen rP44-47E is high in expression quantity and stability, the defects that p44 whole genome antigen is low in expression quantity and not easy to prepare are overcome, and the prepared recombinant protein antigen rP44-47E has the advantages of being high in specificity, capable of effectively reducing the detection cost and easy to standardize when serving as antigen protein.

Description

technical field [0001] The application relates to the field of biotechnology, in particular to a method for preparing a recombinant protein antigen of Anaplasma phagocytic cells and a kit containing the recombinant protein antigen. Background technique [0002] Anaplasma phagocytophilum (Anaplasma phagocytophilum) is an obligate intracellular parasite of Rickettsiae, which can cause human granulocytic anaplasmosis (HGA). The disease was first discovered in 1994 in a patient with fever of unknown origin in the United States. The existence of HGA was confirmed for the first time in Anhui Province in my country in 2008. Anaplasma phagocytophilum infects neutrophils directionally in the body, and has the characteristics of forming parasitic empty cells (inclusion bodies) similar to morula in the cytoplasm to replicate and proliferate. On the surface of Anaplasma phagocytophilum, there is a major outer membrane protein group of P44 associated with antigenic variation, which is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/29C12N15/31C12N15/70C12N1/21G01N33/68G01N33/569G01N33/558G01N33/543C12R1/19
CPCY02A50/30
Inventor 高娃刘丹樊红霞李晓娜李方超
Owner 河套学院
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