Nucleic acid aptamer combined with nucleocapsid protein of novel coronavirus SARS-CoV-2 and application of nucleic acid aptamer

A nucleic acid aptamer and nucleocapsid protein technology, which is applied in the direction of viruses/phages, viruses, viral peptides, etc., can solve the problems of renal failure, rapid transmission, and imperfect detection methods of new coronaviruses, and achieve small molecular weight, Chemically stable, easy to store and label

Active Publication Date: 2020-10-09
安徽省昂普拓迈生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The new coronavirus can be transmitted from person to person. Compared with SARS coronavirus, SARS-CoV-2 has a lower fatality rate, but spreads faster, because people infected with SARS-CoV-2 will develop acute and severe respiratory diseases, accompanied by fever, cough, shortness of breath and Difficulty breathing, kidney failure and death in severe cases, currently there is no effective treatment
The detection method of the new coronavirus is not perfect at present, it needs to rely on specific instruments, special laboratories and professional technicians, and the detection process is complicated, it cannot meet the needs of timely detection, and there is a certain rate of missed detection

Method used

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  • Nucleic acid aptamer combined with nucleocapsid protein of novel coronavirus SARS-CoV-2 and application of nucleic acid aptamer
  • Nucleic acid aptamer combined with nucleocapsid protein of novel coronavirus SARS-CoV-2 and application of nucleic acid aptamer
  • Nucleic acid aptamer combined with nucleocapsid protein of novel coronavirus SARS-CoV-2 and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Screening of nucleic acid aptamers that specifically bind to the new coronavirus SARS-CoV-2 nucleocapsid protein

[0043] 1. Synthesize the random single-stranded DNA library and primers shown in the following sequence:

[0044] Random single-stranded DNA library (denoted as lib13 library):

[0045] 5’~3’

[0046] TTCAGCACTCCACGCA-TAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTATGCGTGCTACCGTGAA

[0047] Wherein, "N" represents a sequence formed by linking arbitrary nucleotide bases, and the library is synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.;

[0048] The primer information is shown in Table 1, synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0049] Table 1 Primers and their sequences

[0050]

[0051] Remarks: ①In the primer name, S1 stands for forward primer, A2 stands for reverse primer, S1-FAM stands for fluorescently labeled forward primer, A2-ployA is a reverse primer with polyA tail attached, polyA is composed of 19 A (Adenylate) composed of...

Embodiment 2

[0097] Example 2 Surface Plasmon Resonance (SPR) detection of the affinity between the nucleic acid aptamer shown in SEQ ID No. 1-2 and the N protein

[0098] Sample solution: Take the nucleic acid aptamer shown in SEQ ID No. 1-2 and dilute it with DPBS buffer to a concentration of 500 nM.

[0099] Other operations are the same as those in step 2.5 of Example 1 figure 2 The detection method of S1, the coupling amount of N protein is 1500Ru.

[0100] Test results such as image 3 As shown, image 3 It is the result of the binding force of the nucleic acid aptamer shown in SEQ ID No. 1 and 2 to the N protein detected by SPR, where SARS-CoV-2-N1 is the nucleic acid aptamer shown in SEQ ID No.1 SARS-CoV-2-N2 is a nucleic acid aptamer shown in SEQ ID No.2. Each curve is the curve after channel 2 minus channel 1.

[0101] by image 3 It can be seen that the nucleic acid aptamers shown in SEQ ID No. 1 and 2 have strong binding to the SARS-CoV-2 nucleocapsid protein detected by the SPR inst...

Embodiment 3

[0105] Example 3 Surface plasmon resonance (SPR) detects the binding sites of nucleic acid aptamers shown in SEQ ID No. 1 and 2 and N protein.

[0106] Solution preparation:

[0107] Sample 1: Take the nucleic acid aptamer shown in SEQ ID No. 1, and dilute it with DPBS buffer to a concentration of 500 nM.

[0108] Sample 2: Take the nucleic acid aptamer shown in SEQ ID No. 2, and dilute it with DPBS buffer to a concentration of 500 nM.

[0109] Mixed solution: an equal volume mixed solution of 0.4M EDC aqueous solution and 0.1M NHS aqueous solution.

[0110] operating:

[0111] S1. Couple N protein to the second channel on the surface of the CM5 chip: first clean the chip with 50mM NaOH aqueous solution, inject 20μl at a flow rate of 10μl / min, and then inject 50μl of the activated chip with a mixed solution at a flow rate of 5μl / min; The protein was diluted with a pH=5.5, 10mM sodium acetate aqueous solution to a final concentration of 50μg / mL and then injected. The injection volume was...

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Abstract

The invention discloses a nucleic acid aptamer specifically combined with nucleocapsid protein of novel coronavirus SARS-CoV-2. The sequence of the nucleic acid aptamer comprises at least one of the following nucleotide sequences: A, any one DNA sequence as shown in SEQ ID No. 1-2; B, a DNA sequence which has more than 60% of homology with any DNA sequence shown as SEQ ID No. 1-2; C, a DNA sequence which is hybridized with any one DNA sequence shown as SEQ ID No. 1-2 under strict conditions; D, an RNA sequence transcribed by any one DNA sequence shown as SEQ ID No. 1-2; wherein the nucleotidesequences can be specifically combined with the nucleocapsid protein of the novel coronavirus SARS-CoV-2. The invention further discloses conjugates and derivatives of the aptamer and an application of the conjugates and the derivatives.

Description

Technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a nucleic acid aptamer that specifically binds to the nucleocapsid protein of the novel coronavirus SARS-CoV-2 and its application. Background technique [0002] Coronavirus 2 (severe acute respiratory syn-dromecoronavirus 2, SARS-CoV-2) is a new type of coronavirus, which has the spike protein S (S protein) and nucleocapsid protein (N protein) Several major structural proteins such as, envelope protein (E protein) and membrane protein (M protein). Studies have shown that S protein is the main protective antigen of the virus, and N protein and M protein can also induce immune effects, especially N protein It has strong immunogenicity. Around January 10, 2020, the full sequence of the novel coronavirus genome was submitted to the NCBI GenBank database by the Shanghai Public Health Clinical Center of Fudan University. Among them, 28274-29533 bits are "N" genes, which can pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N7/02C07K14/165C07K1/14G01N33/569A61K47/54A61K45/00A61P31/14A61P11/00C12R1/93
CPCC12N15/115C12N7/00C07K14/005G01N33/56983A61K47/549A61K45/00A61P31/14A61P11/00C12N2310/16C12N2770/20051G01N2333/165C12N2310/315Y02A50/30
Inventor 何磊张峥徐涛朱建国张师杨
Owner 安徽省昂普拓迈生物科技有限责任公司
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