A kind of bacterial endotoxin nucleic acid aptamer and its application
A bacterial endotoxin and nucleic acid aptamer technology, applied in the field of nucleic acids, can solve the problems of unfavorable endotoxin specific removal, toxicity, and low specificity, and achieve the effects of small molecular weight, simple detection, and high sensitivity
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Embodiment 1
[0036] Example 1 In vitro screening of nucleic acid aptamers that specifically bind to bacterial endotoxin
[0037] In vitro screening of nucleic acid aptamers that specifically bind to bacterial endotoxins is performed in the following steps:
[0038] 1. In vitro synthesis of single-stranded DNA random oligonucleotide library, the two ends of the single-stranded DNA random oligonucleotide library are fixed sequences, and the middle is a random sequence of 35 bases, which is 5'-ATGAGAGCGTCGGTGTGGTA-35nt-TGTAGGAGGGTGCGGAAGTA -3' with a storage capacity of 10 10 Article above, the library entrusted Shanghai Sangon Bioengineering Co., Ltd. to synthesize. Dissolve the synthetic single-stranded DNA nucleic acid library in binding buffer 1 (20mmol / L HePes, 5mmol / L KCl, 120mmol / L NaCl, 1mmol / L MgCl 2 , 1 mmol / L CaCl 2 , pH7.3) to obtain a 0.1 μM nucleic acid solution, heat the solution: heat at 90°C for 5 minutes, place on ice for 15 minutes, and then place at room temperature for...
Embodiment 2
[0054] Example 2 Determination of the affinity of the resulting single-stranded DNA and bacterial endotoxin with the PMB-Elisa method
[0055] The biotin-labeled EAQ1 sequence, EAQ2 sequence and EAQ3 sequence obtained through screening in Example 1 were used for experiments.
[0056] Add 100 μL of polymyxin B (PMB) diluted to 10 μg / mL with coating solution (0.05M carbonate buffer, pH9.6) into a 96-well plate, overnight at 4°C; remove the coating solution and wash Plate 3 times, 5 min each time; add blocking solution (1% BSA) to block at 37°C for 1 h; add 100 μL, 10 μg / mL bacterial endotoxin after washing, and incubate at 37°C for 1 h; add binding buffer 1 (20 mmol / L HePes, 5mmol / L KCl, 120mmol / L NaCl, 1mmol / L MgCl 2 , 1 mmol / L CaCl 2 , pH 7.3) diluted to 0.1 μM single-stranded DNA (ie, EAQ1 sequence, EAQ2 sequence and EAQ3 sequence), incubated at 37°C for 1 h; patted the liquid in the well, and added 200 μL washing buffer (20 mmol / L HePes, 5 mmol / L KCl, 120mmol / L NaCl, 1mmo...
Embodiment 3
[0057] Example 3 The specificity of the obtained single-stranded DNA to bacterial endotoxin was determined by PMB-Elisa method.
[0058] Using the method in Example 2, bovine serum albumin and ovalbumin were used instead of bacterial endotoxin for affinity determination, and whether the nucleic acid aptamer is specific to bacterial endotoxin can be determined by comparing the OD value with bacterial endotoxin.
[0059]
[0060] The results showed that none of the three aptamers could combine with BSA and OVA, and the three aptamers screened had specific affinity for bacterial endotoxin.
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