阅读说明：本技术 一种监测pre-mRNA剪接效率的双报告基因探针及制备方法 (Double-reporter gene probe for monitoring pre-mRNA splicing efficiency and preparation method thereof ) 是由 王福 郭镔 郑海锋 解锦荣 陈思 王希楠 施潇蕊 毛文杰 于 2019-11-28 设计创作，主要内容包括：本发明公开了一种监测pre-mRNA剪接效率的双报告基因探针及制备方法,所述的探针包括载体pcDNA3.1、基因片段A、B、C,所述的基因片段A、B、C按照A-B-C顺序与线性化的pcDNA3.1载体重组,所述的片段A为Fluc,所述的片段B由exon 1、Intron、exon 2组成,所述的片段C为Rluc。所述的探针的核苷酸序列如SEQ ID NO.5所示。通过对本发明的报告基因分子影像探两种荧光素酶产生的信号比值Fluc/Rluc进行分析,实时、无创地分析在体pre-mRNA的剪接效率,为调控pre-mRNA剪接的药物研究提供了有效的筛选工具。(The invention discloses a double-reporter gene probe for monitoring the splicing efficiency of pre-mRNA and a preparation method thereof, wherein the probe comprises a vector pcDNA3.1 and a gene segment A, B, C, the gene segment A, B, C is recombined with a linearized pcDNA3.1 vector according to an A-B-C sequence, the segment A is Fluc, the segment B consists of exon 1, Intron and exon 2, and the segment C is Rluc. The nucleotide sequence of the probe is shown as SEQ ID NO. 5. By analyzing the signal ratio Fluc/Rluc generated by the two luciferases by the reporter gene molecule image probe, the splicing efficiency of in-vivo pre-mRNA is analyzed in real time and noninvasively, and an effective screening tool is provided for the research of medicines for regulating and controlling the splicing of the pre-mRNA.)

1. A double-reporter gene probe for monitoring the splicing efficiency of pre-mRNA is characterized by comprising a vector pcDNA3.1 and a gene segment A, B, C, wherein the gene segment A, B, C is recombined with the linearized pcDNA3.1 vector according to the sequence of A-B-C, the segment A is Fluc, the segment B consists of exon 1, Intron and exon 2, and the segment C is Rluc.

2. The dual-reporter gene probe for monitoring pre-mRNA splicing efficiency according to claim 1, wherein the nucleotide sequence of the fragment A is shown as SEQ ID No.1, the nucleotide sequence of the fragment B is shown as SEQ ID No.2, and the nucleotide sequence of the fragment C is shown as SEQ ID No. 3.

3. The dual reporter gene probe for monitoring pre-mRNA splicing efficiency according to claim 1, wherein the nucleotide sequence of the probe is shown as SEQ ID No. 5.

4. The method for preparing a dual-luciferase reporter gene imaging probe according to claim 1, comprising the steps of:

1) the vector pcDNA3.1 is digested by KpnI and XhoI, so that the vector is linearized;

2) a, B, C fragments are amplified by PCR;

3) the gene fragment A, B, C is recombined with the linearized pcDNA3.1 vector according to the sequence A-B-C;

4) and transforming the recombinant vector, extracting plasmid, and carrying out enzyme digestion identification to obtain the Dual-Luc reporter gene probe.

5. The method according to claim 5, wherein the fragment A is Fluc, the fragment B is exon 1, Intron, exon 2, and the fragment C is Rluc.

6. The method according to claim 5, wherein the nucleotide sequence of the gene fragment amplified in step (2) is represented by SEQ ID NO. 4.

Technical Field

The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a double-reporter gene image probe for monitoring pre-mRNA and a preparation method thereof.

Background

The chemical nature of the Pre-mRNA splicing process involves a two-step transesterification reaction. The chemical reaction is difficult to occur in cells by itself, and needs the participation of spliceosomes to complete. Spliceosomes are multicomponent complexes formed when Pre-mRNA splicing is performed, consisting mainly of small nuclear RNAs and proteins. It is formed at various stages of the splicing process with the addition of SNRNA. That is to say a splicing intermediate formed on the complete pre-mRNA. The spliceosome itself requires the participation of some small nuclear RNA. These small nuclear RNAs do not translate into any protein, but play an important role in regulating genetic activities. Since many human diseases can be due to mis-splicing of genes or mis-regulation of spliceosomes. It is known that 35% of human genetic disorders are caused by alternative splicing of individual genes due to gene mutations. Yet another cause of some diseases is that mutations in the spliceosome proteins affect the splicing of many transcripts. Still other cancers are also associated with the misregulation of splicing factors.

Researchers initially establish the generation processes of assembly, activation and depolymerization of spliceosomes, and complex RNA splicing regulation networks of interaction and mutual regulation between proteins and nucleic acids by means of research measures such as immunoprecipitation, gene knockout, cross-linking mass spectrometry, establishment of in vitro splicing reaction systems and the like. However, due to the high dynamics and complexity of spliceosomes, there has been a lack of reliable systems to detect splicing efficiency. Therefore, the invention develops a dual-luciferase reporter gene imaging probe, monitors the pre-mRNA splicing efficiency in real time, and provides a robust, rapid and convenient detection tool for researching the pre-mRNA splicing.

The invention utilizes a molecular imaging means to develop a developing system based on dual-luciferase reporter gene to quantitatively analyze the splicing efficiency of pre-mRNA. Provides a detection tool for real-time, rapid and quantitative research on splicing efficiency. The stable expression of the gene system in the living body is reported by the carrier, the real-time, dynamic and visual molecular splicing information of the living body is provided in a non-invasive and repeated way, the quantitative research is carried out simultaneously, the two fluorescent signals are analyzed, the splicing process and the splicing efficiency of the spliceosome in the body can be evaluated in real time, and the gene expression condition in the body can be detected. Imaging splicing-related genes plays a key role in optimizing gene therapy protocols and evaluating efficacy.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a double-reporter gene probe for monitoring the pre-mRNA splicing efficiency and a preparation method thereof, and the generation and development of the splicing process and the splicing efficiency are monitored according to the fluorescence intensity generated by two luciferases.

The invention is realized by the following technical scheme:

a double-reporter gene probe for monitoring the splicing efficiency of pre-mRNA comprises a vector pcDNA3.1 and a gene segment A, B, C, wherein the gene segment A, B, C is recombined with the linearized pcDNA3.1 vector according to the sequence A-B-C, the segment A is Fluc, the segment B consists of exon 1, Intron and exon 2, and the segment C is Rluc.

The nucleotide sequence of the fragment A is shown as SEQ ID NO.1, the nucleotide sequence of the fragment B is shown as SEQ ID NO.2, and the nucleotide sequence of the fragment C is shown as SEQ ID NO. 3.

The nucleotide sequence of the probe is shown as SEQ ID NO. 5.

In another aspect of the present invention, there is provided a dual reporter gene probe for monitoring pre-mRNA splicing efficiency and a method for preparing the same, comprising the steps of:

1) the vector pcDNA3.1 is digested by KpnI and XhoI, so that the vector is linearized;

2) a, B, C fragments are amplified by PCR;

3) the gene fragment A, B, C is recombined with the linearized pcDNA3.1 vector according to the sequence A-B-C;

4) and transforming the recombinant vector, extracting plasmid, and carrying out enzyme digestion identification to obtain the Dual-Luc reporter gene probe.

The fragment A is Fluc, the fragment B is composed of exon 1, Intron and exon 2, and the fragment C is Rluc.

The nucleotide sequence of the gene fragment obtained by amplification in the step (2) is shown as SEQ ID NO. 4.

The invention has the beneficial effects that:

the invention constructs a double-reporter gene probe for monitoring the pre-mRNA splicing efficiency, and has the following advantages because the double-reporter gene probe can be well expressed in organisms:

(1) provides a real-time and non-invasive monitoring tool for simulating the dynamic change of the splicing process of the living body.

(2) Overcomes the defect that the in vitro content analysis possibly has deviation with the actual in vivo effect in the traditional method.

(3) The reporter gene system has the advantages of strong targeting property, good safety, high flux, relatively low price, convenient operation and the like, provides an effective screening tool for the research of the medicament acting on the spliceosome, and has very important value in the research of new medicaments and the observation of the physiological and pathological processes in organisms.

Drawings

FIG. 1 is a schematic diagram of the Dual-Luc reporter probe of the present invention;

FIG. 2 is a structural diagram of the molecular probe of the present invention;

FIG. 3 is the activity test of reporter gene under the action of Pladienolide B with different concentrations;

FIG. 4 is the reporter gene activity assay at different time points after the action of Pladienolide B;

FIG. 5 is a diagram of in vivo activity assay of the Dual-Luc reporter gene system.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.