阅读说明：本技术 环状RNA hsa_circ_0008602双荧光素酶报告基因载体制备方法 (Preparation method of circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector ) 是由 张梦娇 袁奕 唐林香 李昂 何胜祥 于 2020-04-10 设计创作，主要内容包括：一种环状RNA hsa_circ_0008602双荧光素酶报告基因载体制备方法,步骤(1),通过cirbase数据库获取hsa_circ_0008602的全长序列,全基因合成构建入pUC57-Amp载体；步骤(2),设计一对同源重组引物,将目的序列从pUC57-Amp载体上扩增出来；同时,对psiCHECK2骨架载体进行XhoI和NotI酶切线性化,通过琼脂糖凝胶对扩增产物和酶切产物进行纯化回收,再将扩增产物和酶切产物进行同源重组反应,得到重组产物；步骤(3),将重组产物转入感受态细胞中,挑取单克隆进行电泳验证和测序鉴定,得到载体psiCHECK2-8602。(A preparation method of a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector comprises the steps of (1) obtaining a full-length sequence of hsa _ circ _0008602 through a circase database, and constructing a pUC57-Amp vector through whole gene synthesis; step (2), designing a pair of homologous recombination primers, and amplifying a target sequence from a pUC57-Amp vector; meanwhile, XhoI and NotI enzyme digestion linearization is carried out on the psiCHECK2 skeleton vector, an amplification product and an enzyme digestion product are purified and recovered through agarose gel, and then homologous recombination reaction is carried out on the amplification product and the enzyme digestion product to obtain a recombinant product; and (3) transferring the recombinant product into a competent cell, and selecting a single clone for electrophoretic verification and sequencing identification to obtain a vector psiCHECK 2-8602.)

1. A preparation method of a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector is characterized in that: connecting the circular RNA sequence to a luciferase reporter gene vector by using a psiCHECK2 skeleton vector through a homologous recombination method; the method comprises the following steps:

step (1), acquiring a full-length sequence of hsa _ circ _0008602 through a circase database, and constructing a pUC57-Amp vector through whole gene synthesis;

step (2), designing a pair of homologous recombination primers, and amplifying a target sequence from a pUC57-Amp vector; meanwhile, XhoI and NotI enzyme digestion linearization is carried out on the psiCHECK2 skeleton vector, an amplification product and an enzyme digestion product are purified and recovered through agarose gel, and then homologous recombination reaction is carried out on the amplification product and the enzyme digestion product to obtain a recombinant product;

and (3) transferring the recombinant product into a competent cell, and selecting a single clone for electrophoretic verification and sequencing identification to obtain a vector psiCHECK2-8602, namely a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector.

2. The method for preparing the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector according to claim 1, wherein the method comprises the steps of: the method comprises the following steps:

step (1), inquiring a gene sequence of circular RNA hsa _ circ _0008602 according to circ Base, obtaining a full-length hsa _ circ _0008602 sequence in a full-gene synthesis mode, and constructing a pUC57-Amp vector;

step (2-1), inserting the circular RNA hsa _ circ _0008602 sequence and information of a luciferase vector psiCHECK2 into a luciferase reporter gene vector according to a structural diagram, and designing a homologous recombination primer;

step (2-2), amplifying a hsa _ circ _0008602 target fragment from a pUC57-Amp vector by using a homologous recombination primer, carrying out enzyme digestion linearization on a psiCHECK2 skeleton vector by using XhoI and NotI, purifying and recovering an amplification product and an enzyme digestion product by using agarose gel, and carrying out homologous recombination reaction on the two products;

and (3) transferring the recombinant product into a competent cell, and selecting a single clone for electrophoretic verification and sequencing identification to obtain a vector psiCHECK2-8602, namely a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector.

3. The method for preparing the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector according to claim 1 or 2, wherein the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector comprises: the homologous recombination primers are as follows:

8602-CZF：ATTCTAGGCGATCGCAAGCTTGTTTAAACC；

8602-CZR：TTTATTGCGGCCAGCGAATTCTTTATTGCG。

4. the method for preparing the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector according to claim 1 or 2, wherein the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector comprises: in the PCR reaction system, the PCR reaction system is,

the volume of p L V-CMV-EGFP-Neo DNA is 2 mu L;

forward primer (10. mu.M) in a volume of 1. mu. L;

reverse primer (10. mu.M) in a volume of 1. mu. L;

the volume of Taq DNA Polymerase was 25. mu. L;

ddH₂the volume of O was 21. mu. L.

5. The method for preparing the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector according to claim 1 or 2, wherein the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector comprises: in the PCR reaction system, the PCR reaction conditions are as follows:

the temperature is 95 ℃, the time is 2min, and the cycle number is 1;

the temperature is 95 ℃, the time is 30s, and the cycle number is 35 x;

the temperature is 58 ℃, the time is 30s, and the cycle number is 35 x;

the temperature is 72 ℃, the time is 30s, and the cycle number is 35 x;

the temperature was 65 ℃ for 5min, cycle number 1.

6. The method for preparing the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector according to claim 1 or 2, wherein the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector comprises: in a system of enzyme digestion linearization, the enzyme digestion linearization,

vector volume is 10u L;

volume of XhoI was 1u L;

NotI has a volume of 1u L;

volume of 10 XNEB Buffer was 5u L;

ddH₂the volume of O was 33u L.

7. The method for preparing the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector according to claim 1 or 2, wherein the circular RNA comprises: the sequence information for circular RNA hsa _ circ _0008602 is:

CCTCAGACGATACAGCAGACTCTCAAGAGGACACTGCAGTATTATGAACATCAAGTTATTGGTTACAGGGATGCAGAAAAGAATTTCCACAATATCTCTAACAGATGCTCCTATGCAGACCACTCCAACAAAGAAGAAATTGAAGATGTCTCAGGAATTCTTCAGTGTACTGCTAATATACTCG。

Technical Field

The invention relates to the field of genetic engineering, in particular to a preparation method of a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector.

Background

Circular RNA (circRNA) is a special non-coding RNA molecule (a small amount of RNA molecules can also code protein), is not influenced by RNA exonuclease, has stable expression, is not easy to degrade, and is evolutionarily conserved. CircRNA is an endogenous non-coding RNA that has no 5 'end cap or 3' end poly (A) tail, and is used as a covalent closed loop formed by the mechanical post-splicing of a precursor mRNA by a spliceosome. The cyclization mode is divided into intron cyclization, exon cyclization, intergenic cyclization and the like, and currently, the mainstream cyclization mechanism can be classified into three types: shear-dependent pegging cyclization; the cis-acting element promotes circRNA formation; RNA Binding Proteins (RBPs) regulate circRNA formation.

Functionally, recent studies have shown that circRNA can be involved in a wide variety of processes in cell life activities, such as cell differentiation, proliferation, apoptosis, chromosome modification and inactivation, through regulation of gene expression and function. Compared with the broad spectrum of the expression of the coding gene and miRNA, the expression of the circRNA is often characterized by cell and tissue specificity, space-time difference of differentiation and development and the like. In recent years, with the development of technologies such as sequencing transcription profiling of the new generation, the role of circRNA in cancer is gradually being explored and emphasized by researchers, and more data show that the abnormal expression and/or mutation of circRNA exists in various tumors and is closely related to the occurrence and development of the tumors.

The research shows that the circRNA is rich in miRNA binding sites, plays a role in competitively binding miRNA in cells and is a high-efficiency ceRNA, the research reports that the circ L ARP4 is mainly positioned in cytoplasm, the biological function of gastric cancer cells is inhibited by a sponge adsorbent serving as miR-424, the ectopic expression of miR-424 promotes the proliferation and invasion of gastric cancer cells by targeting L ATS1 gene, and the circ L ARP4 is possibly a new tumor inhibition factor and a potential biomarker in gastric cancer, the circRNA-miRNA network can regulate the tumor inhibition factor by competitive endogenous RNA (ceRNA), and a new gene expression regulation model of interaction of coding RNA and non-coding RNA and a complex network system related to the ceRNA are established.

Until now, research aiming at cervical cancer has found that the expression change of some circRNAs is related to the occurrence and development of cervical cancer. However, in general, our current research on circRNAs in cervical cancer is still not comprehensive and deep enough, and in the early exploration, we have significantly down-regulated the circular RNA hsa _ circ _0008602 in cervical cancer tissues through high-throughput sequencing and bioinformatics analysis and confirmed that the molecule is closely related to cervical cancer progression. hsa _ circ _0008602 is located at chr 11: 106849344 and 106856857 derived from the 6 th and 7 th exons of the parental gene GUCY1A 2. At present, no research report on the molecule in the tumor process exists. In order to further explore the functions and corresponding mechanisms of hsa _ circ _0008602 in tumors represented by cervical cancer, a luciferase reporter gene vector is constructed, miRNAs combined with the luciferase reporter gene vector are further screened, whether the circular RNA hsa _ circ _0008602 can act through miRNA sponge (miRNA sponge) is explored, and a cerRNA regulatory network driven by hsa _ circ _0008602 is verified.

In the current report, the dual-luciferase reporter gene system is more rapid and sensitive compared with other reporter gene detection systems, can be carried out in a 96-well plate, is convenient for large-scale detection, and the detection amount of luciferase can be as low as 10^-20mol, has extremely high sensitivity, and is safe and non-radioactive. At present, the detection method for recognizing target genes by using a dual-luciferase reporter gene system has been widely applied, for example, in receptor activity, intracellular signal transduction, mRNA processing, protein-protein folding machine protein-protein interaction and the like.

However, the prior art also has the following disadvantages:

(1) when miRNAs are adsorbed by circRNA, the total amount of intracellular miRNA would not theoretically change, so that it is impossible to determine whether miRNA is adsorbed by qRT-PCR detection. The function of the circRNA span can be indirectly reflected only by predicting miRNA which is likely to be adsorbed and detecting the downstream gene expression difference of miRNA after the circRNA is over-expressed. However, this method cannot be said to be effective as a result of the direct action of circRNA-miRNA. The direct evidence lies in the construction and activity detection of the Sensor reporter and the accurate judgment of the relationship between the miRNAs and the circRNA.

(2) At present, many circular RNA luciferase reporter gene-based vectors are constructed by constructing a small predicted RNA sequence behind L uciferase in a vector and then constructing luciferase plasmids, namely, the construction and verification of the individual circular RNA luciferase reporter gene vector need to be carried out on each candidate miRNA molecule.

(3) Currently, no study on the expression and mechanism of circular RNAhsa _ circ _0008602 is reported, and no luciferase reporter gene detection system based on hsa _ circ _0008602 is available, so that the study is not beneficial to further discussing the action and mechanism of the molecule in the tumor process.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a preparation method of a circular RNAhsa _ circ _0008602 dual-luciferase reporter gene vector.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for preparing a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector utilizes a psiCHECK2 skeleton vector to connect a circular RNA sequence to the luciferase reporter gene vector by a homologous recombination method; the method comprises the following steps:

step (1), acquiring a full-length sequence of hsa _ circ _0008602 through a circase database, and constructing a pUC57-Amp vector through whole gene synthesis;

step (2), designing a pair of homologous recombination primers, and amplifying a target sequence from a pUC57-Amp vector; meanwhile, XhoI and NotI enzyme digestion linearization is carried out on the psiCHECK2 skeleton vector, an amplification product and an enzyme digestion product are purified and recovered through agarose gel, and then homologous recombination reaction is carried out on the amplification product and the enzyme digestion product to obtain a recombinant product;

and (3) transferring the recombinant product into a competent cell, and selecting a single clone for electrophoretic verification and sequencing identification to obtain a vector psiCHECK2-8602, namely a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector.

Compared with the prior art, the invention has the advantages that:

advantages (1) the invention constructs the full-length sequence of the circular RNAhsa _ circ _0008602 on a psiCHECK2 framework vector, thereby facilitating the subsequent screening of a plurality of target molecules. Compared with a vector constructed aiming at a single target site, the invention not only saves time, but also saves cost.

Advantages (2) the invention can detect luciferase activity by co-transfecting miRNAs or negative control to be detected and a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector into cells, and judges the interaction of miRNA-hsa _ circ _0008602 according to luciferase activity difference.

Advantage (3) the invention can further break random binding sites of MRE response elements of candidate miRNAs to obtain mutant MUTs on the basis of advantage (2), then transfer the mutant MUTs into cells together with a dual-luciferase reporter vector of the circular RNA hsa _ circ _0008602, detect luciferase activity, and make accurate judgment on the relationship between a pair of hsa _ circ _0008602 and the candidate miRNAs according to the luciferase activity difference.

Advantage (4) the invention can calculate the ratio of the firefly luciferase activity to the internal reference Renilla luciferase activity, perform differential analysis on each group, and judge the interaction of miRNA-hsa _ circ _0008602 according to the luciferase activity difference.

Advantage (5) currently, no study on the expression and mechanism of circular RNA hsa _ circ _0008602 is reported, no luciferase reporter gene detection system based on hsa _ circ _0008602 exists, and the study is not beneficial to further discussing the action and mechanism of the molecule in the tumor process, and the invention effectively fills up the blank of related research.

The invention has the advantages that by constructing the dual-luciferase reporter gene vector of the circular RNA hsa _ circ _0008602, candidate microRNAs regulated and controlled by the circular RNA hsa _ circ _0008602 can be screened and verified, and whether the circular RNA hsa _ circ _0008602 can be used as endogenous competitive RNA to compete miRNAs through MREs (miRNA response elements) is discussed, so that the expression of miRNA downstream target genes is indirectly regulated and controlled, and data support is provided for analyzing the physiological/pathological functions and mechanisms of the circular RNA hsa _ circ _ 0008602.

The invention has the advantages that (7) the dual-luciferase vector reporting system of the circular RNA hsa _ circ _0008602 is constructed, the hsa _ circ 0008602 sequence is synthesized through the whole gene, the whole sequence is constructed into the psiCHECK2 luciferase vector, and then candidate miRNAs and reporter gene plasmids are co-transfected into 293T cells or other related cell lines, the intensity of fluorescence is detected to determine the activity of luciferase, so that the miRNA-circRNA interaction is judged (if the miRNA can be combined with the hsa _ circ _0008602, the luciferase gene is down-regulated, and the expression amount of the luciferase is inversely proportional to the action intensity of the miRNA-hsa _ circ _ 0008602).

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the hsa _ circ _0008602 site information of the circular RNA of the present invention;

FIG. 2 is luciferase vector information of the present invention;

FIG. 3 is a schematic diagram of the structure of the circular RNA hsa _ circ _0008602 dual-luciferase reporter vector of the present invention;

FIG. 4 is a schematic diagram of a circular RNA hsa _ circ _0008602 sequence amplicon of the present invention;

FIG. 5 is a graph showing the result of the monoclonal validation of the present invention;

FIG. 6 is a diagram showing the sequencing alignment of psiCHECK2-8602 vector according to the present invention;

FIG. 7 shows the sequencing peaks of the circular RNA hsa _ circ _0008602 of the present invention.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings, in order that the present disclosure may be more fully understood and fully conveyed to those skilled in the art. While the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the invention is not limited to the embodiments set forth herein.

A method for preparing a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector is characterized in that a circular RNA sequence is connected to a luciferase reporter gene vector by a homologous recombination method by utilizing a psiCHECK2 skeleton vector; the method comprises the following steps:

step (1), acquiring a full-length sequence of hsa _ circ _0008602 through a circase database, and constructing a pUC57-Amp vector through whole gene synthesis;

step (2), designing a pair of homologous recombination primers, and amplifying a target sequence from a pUC57-Amp vector; meanwhile, XhoI and NotI enzyme digestion linearization is carried out on the psiCHECK2 skeleton vector, an amplification product and an enzyme digestion product are purified and recovered through agarose gel, and then homologous recombination reaction is carried out on the amplification product and the enzyme digestion product to obtain a recombinant product;

and (3) transferring the recombinant product into a competent cell, and selecting a single clone for electrophoretic verification and sequencing identification to obtain a vector psiCHECK2-8602, namely a circular RNA hsa _ circ _0008602 dual-luciferase reporter gene vector.