CN-122012513-A - Nucleic acid aptamer specifically binding MMLV as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a nucleic acid aptamer specifically combined with MMLV, wherein the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO.1 or SEQ ID NO. 2; the nucleic acid aptamer capable of specifically binding with MMLV is obtained through screening based on a SELEX technology, has high affinity to MMLV protein, can be applied to MMLV protein enrichment reagents, MMLV protein separation reagents, MMLV protein detection reagents, test paper and biosensors, and is beneficial to recognition and activity research of MMLV protein.

Inventors

• WANG YINGZHI
• Ran Liuyi
• LEI YANG
• WANG XIAOLIANG
• LIANG GUANGYAN

Assignees

• 重庆医科大学附属大学城医院

Dates

Publication Date

20260512

Application Date

20260324

Claims (5)

1. 1. A nucleic acid aptamer specifically binding to MMLV is characterized in that the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. 2. Use of a nucleic acid aptamer that specifically binds MMLV according to claim 1 for the preparation of a reagent for enriching MMLV proteins.
3. 3. Use of a nucleic acid aptamer that specifically binds MMLV according to claim 1 in the preparation of an isolated MMLV protein reagent.
4. 4. The use of the aptamer specifically binding to MMLV according to claim 1 for preparing MMLV protein detection reagents, test paper, biosensors.
5. 5. The method for preparing a nucleic acid aptamer specifically binding to MMLV according to claim 1, comprising the steps of: S1, designing and constructing a random nucleic acid library and a primer; S2, annealing the random nucleic acid library, incubating with MMLV protein, and carrying out combination screening by adopting Ni-NTA magnetic beads to obtain an MMLV-library complex; s3, amplifying the MMLV-library complex obtained after screening to obtain an amplified product, and carrying out ssDNA separation on the amplified product by STREPTAVIDIN MAGPOLY BEADS to obtain ssDNA, namely screening a nucleic acid library for the first round; S4, repeating the steps S2-S3, screening more than 5 times, wherein the nucleic acid library obtained by screening more than one time is used as an initial library in each operation, and obtaining the target nucleic acid aptamer after screening is finished.

Description

Nucleic acid aptamer specifically binding MMLV as well as preparation method and application thereof Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to a nucleic acid aptamer specifically combined with MMLV, and a preparation method and application thereof. Background The aptamer is a single-stranded DNA or RNA oligonucleotide obtained by artificial screening, and can be combined with a target molecule with high affinity and high specificity through a specific three-dimensional space structure formed by the aptamer. Compared with antibody, the said antibody has many outstanding advantages, such as high affinity and high specificity, can distinguish proteins in different conformations, can be synthesized in vitro, is easy to modify, has wide target range, can screen corresponding aptamer no matter ion, small molecule or protein or cell, and has good stability. The SELEX technique (SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT) is an in vitro method for screening specific nucleic acid aptamers. Classical SELEX generally comprises several steps of incubation, isolation and amplification, and after screening for specific oligonucleotide ligands that specifically adsorb phage T4DNA polymerase and organic dye molecules using this technique from Tuerk et al, the SELEX technique has become an important research tool and tool after more than a decade of development. MMLV enzyme (Moloney Murine Leukemia Virus REVERSE TRANSCRIPTASE), which is called Moloney murine leukemia virus reverse transcriptase, is an extremely important tool enzyme in molecular biology, has 5 '. Fwdarw.3' DNA polymerase activity and RnaseH activity, and is mainly applied to aspects such as RT-PCR, RT-qPCR, CDNA library construction, RNA sequencing, retrovirus life cycle and the like. MMLV reverse transcriptase is a key molecular tool for converting RNA information into DNA, and mutants (such as M-MLV and SuperScript series) thereof greatly improve performance by removing RNase H activity and improving thermostability, so that the MMLV reverse transcriptase becomes an indispensable reagent in gene expression analysis, diagnosis and biotechnology research. Currently, nucleic acid aptamers have been used in many fields, and the development of nucleic acid aptamers against MMLV can help researchers to study the use of MMLV enzymes more deeply. Disclosure of Invention The invention aims to provide a nucleic acid aptamer specifically binding to MMLV, which provides assistance for research application of MMLV enzyme. In order to achieve the aim, the scheme of the invention is that the nucleic acid aptamer specifically binds to MMLV, and the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO.1 or SEQ ID NO. 2. Use of the above described aptamer specifically binding MMLV for the preparation of a reagent for enriching MMLV proteins. Use of the above described aptamer specifically binding MMLV for the preparation of a reagent for isolating MMLV proteins. The application of the nucleic acid aptamer specifically binding to MMLV in preparing MMLV protein detection reagents, test paper and biosensors. The preparation method of the nucleic acid aptamer specifically binding to MMLV comprises the following steps: S1, designing and constructing a random nucleic acid library and a primer; S2, annealing the random nucleic acid library, incubating with MMLV protein, and carrying out combination screening by adopting Ni-NTA magnetic beads to obtain an MMLV-library complex; s3, amplifying the MMLV-library complex obtained after screening to obtain an amplified product, and carrying out ssDNA separation on the amplified product by STREPTAVIDIN MAGPOLY BEADS to obtain ssDNA, namely screening a nucleic acid library for the first round; S4, repeating the steps S2-S3, screening more than 5 times, wherein the nucleic acid library obtained by screening more than one time is used as an initial library in each operation, and obtaining the target nucleic acid aptamer after screening is finished. Compared with the prior art, the invention has the following advantages: according to the scheme, after verification, the nucleic acid aptamer obtained by screening based on the SELEX technology can specifically bind MMLV protein and has high affinity for binding MMLV protein, so that the nucleic acid aptamer can be used in enrichment, separation and detection products of MMLV protein, and is beneficial to recognition and activity research of MMLV protein. Drawings FIG. 1 is a graph showing the results of gel electrophoresis of amplified products and nucleic acid aptamers prepared as a sequencing library during each round of screening in an embodiment of the present invention; FIG. 2 is a diagram showing the results of quality testing of a sequencing library according to an embodiment of the present invention; FIG. 3 is a diagram showing the binding between MMLV protein and the aptamer PG7572 and PG7