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Nucleic acid aptamer capable of being specifically bound to tubercle bacillus antigen and application thereof

A nucleic acid aptamer and specific binding technology, applied in sugar derivatives, instruments, analytical materials, etc., can solve the problems of difficulty and randomness in obtaining high-affinity aptamers, and achieve low cost and simple preparation , to overcome the effect of low sensitivity

Active Publication Date: 2012-10-10
武汉顺可达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the selection of ssDNA aptamers is highly random, and it is difficult to obtain effective high-affinity aptamers

Method used

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  • Nucleic acid aptamer capable of being specifically bound to tubercle bacillus antigen and application thereof
  • Nucleic acid aptamer capable of being specifically bound to tubercle bacillus antigen and application thereof
  • Nucleic acid aptamer capable of being specifically bound to tubercle bacillus antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Screening of aptamers

[0032] 1. Purification and identification of CFP10-ESAT6 fusion protein

[0033] Construction of prokaryotic expression plasmid pET28a-CFP10-ESAT6 (or pET28a-CE, Li Juan, Wu Xueqiong, Zhang Junxian, etc. High expression of Mycobacterium tuberculosis CFP10-ESAT6 fusion protein in Escherichia coli[J]. Chinese Journal of Antituberculosis, 2004,26(4):204-208.), adding a linker sequence between CFP-10 and ESAT-6 fragments, the amino acid sequence corresponding to this sequence is GGGGSGGGGSGGGGS, the existence of this sequence can improve the CFP10-ESAT6 fusion protein ( The flexibility between the CFP-10 and ESAT-6 sequences in the CE fusion protein or CE protein for short, thereby fully maintaining their respective natural conformations.

[0034] Transform the correct plasmid into Escherichia coli BL21, from kanamycin resistance (Kan r ) plate to pick a single clone, inoculated in 5ml sterile LB medium with kanamycin added at 1:1000 (try...

Embodiment 2

[0057] Example 2 The sensitivity of antibody-coated aptamer detection sandwich method to detect CE protein

[0058] After the 96-well ELISA plate (Costar), the rabbit anti-CE protein antiserum (or rabbit anti-tuberculosis H37Rv polyantiserum) was diluted at 1:200~1:500 to coat the ELISA plate, and the CE protein and BSA were used Carbonate buffer at pH 9.6 (Na 2 CO 3 1.5g,NaHCO 3 2.9g,Na 2 N 3 0.2g, add water 1000ml, adjust the pH to 9.6) to prepare 10μg / ml, 1μg / ml, 100ng / ml, 10ng / ml, 1ng / ml, 0.1ng / ml, 0.01ng / ml concentration gradient, add 50μl to each well, After incubation overnight at 4°C, wash with PBST 3 times, 2 minutes each time; then add 100 μl blocking solution (0.2% gelatin) to each well and incubate at 37°C for 1 hour, then wash 3 times with PBST, 2 minutes each time; Add 50 μl of 100 nM biotin-labeled CE24 aptamer to the well, incubate at 37°C or room temperature (25°C) for 1 hour, wash with PBST, and wash 3 times for 2 minutes each time. Then add 50 μl strep...

Embodiment 3

[0060] Example 3 Aptamer Direct Method Detection of CE Protein in Intestinal Tuberculosis, Pulmonary Tuberculosis, and Intestinal Crohn's Patient Serum

[0061] Add 100 μl of various sera (including intestinal tuberculosis, pulmonary tuberculosis, intestinal Crohn’s disease and healthy volunteers’ sera) to each well of the 96-well ELISA plate, incubate overnight at 4°C, wash with PBST 3 times, 2 minutes each time . Add 100 μl of 2% BSA to each well, block for 1 hour at 37°C, wash with PBST three times, 2 minutes each time; then add 100 μl of 300 nM biotin-labeled CE24 aptamer to each well, and incubate at 37°C or room temperature (25°C) For 1 hour, wash with PBST 3 times, 2 minutes each time. Then add 50 μl of streptavidin-labeled horseradish peroxidase (HRP) (diluted 1:2000) to act for 30 minutes at 37°C, wash with PBST three times for 2 minutes each time; finally wash with 50 μl of tetramethyl Benzidine (TMB) was used as the chromogenic substrate and incubated at 37°C for ...

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Abstract

The invention discloses a nucleic acid aptamer capable of being specifically bound to a tubercle bacillus antigen and application thereof. The aptamer is micromolecule single chain DNA (deoxyribonucleic acid) (ssDNA) as to mycobacterium tuberculosis secretion antigen CFP10-ESAT6 (culture filtrate protein 10-early secreted antigenic target-6kDa) fusion protein, and a nucleotide sequence is shown in SEQ ID NO (sequence identifier number).1. The micromolecule single chain DNA has the function similar to a monoclonal antibody, and can directly and specifically be bound to the CFP10-EAST6 protein. By adopting an aptamer direct method or a sandwich method (or enzyme-linked oligonucleotide sandwich absorption method) of the aptamer and a polyclonal antibody, the CFP10 and ESAT6 antigens in blood serum (or blood plasma) of patients with phthisis and extrapulmonary tuberculosis can be fast and effectively detected within 2 hours. The technique of building the aptamer can sensitively detect early secretory antigen CFP10-ESAT6 protein or antibody of tuberculosis specificity in a blood sample or body fluid of the patient with tuberculosis, and sensitivity of CFP10-ESAT6 antigen detection in the blood serum can be up to below 0.01ng / ml.

Description

technical field [0001] The invention belongs to the fields of molecular microbiology and infection immunity, and relates to a 30nt long single strand DNA (single strand DNA, ssDNA) aptamer and its application. The aptamer can bind tuberculosis branch specifically and with high affinity Bacillus-specific secreted proteins CFP10 and ESAT6 can be used for serological diagnosis of early tuberculosis infection. Background technique [0002] Tuberculosis is a disease caused by Mycobacterium tuberculosis (M.tuberculosis) infection of the body, involving multiple organs throughout the body. At present, about 1 / 3 of the people in the world have been infected with Mycobacterium tuberculosis, and about 2 million people die of tuberculosis infection every year (Dye C SS, Dolin PP PV, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence , and mortality by country.W.H.O.Global Surveillance and Monitoring Project.J Am Med Assoc.1999.282:677-68...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10C07H21/04G01N33/569
Inventor 章晓联唐晓磊周亚雄
Owner 武汉顺可达生物科技有限公司
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