Mini-NMD detection plasmid as well as construction method and application thereof

Abstract

The invention discloses a mini-NMD detection plasmid as well as a construction method and application thereof, a pNMD-3xFlag-Neo (EGFP) detection plasmid obtained by replacing an inserted gDNA fragment can realize detection of different gene mutations, and the mini-NMD detection plasmid is wide in application range and simple and convenient to operate; according to the method, a gDNA fragment needing to be inserted is smaller, vector construction is more efficient and rapid, and the problems that in-vitro NMD verification vector systems in the prior art are low in efficiency, poor in accuracy, tedious in detection process and the like are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a mini-NMD detection plasmid and a construction method and application thereof. Background Art

[0002] In eukaryotes, protein synthesis is a complex and precise process. Coding genes synthesize mRNA through the transcription process, and mRNA is used as a direct template to obtain the corresponding protein after translation by ribosomes. mRNA is composed of four types of ribonucleic acids (A, G, C, U), and their arrangement order determines the order of amino acids in the protein. During the translation process, the anticodon on tRNA can recognize the codon on mRNA (composed of three random ribonucleic acids) and perform base complementary pairing with it to confirm the specific amino acid sequence, thereby synthesizing protein. There are a total of 64 codons in eukaryotes corresponding to 20 amino acids, among which the stop codon (UAG, UAA, UGA) does not synthesize amino acids and is the termination signal of the translation process.

[0003] In gene mutations, there is a certain probability that new termination codons will be generated, causing the translation process to terminate prematurely. This type of gene mutation is called nonsense mutation. Nonsense mutations cause premature termination codons (PTC) to appear prematurely, resulting in truncated proteins. In eukaryotes, in order to prevent the production of abnormal truncated proteins and ensure the normal physiological function of cells, nonsense mutations will activate nonsense mutation-mediated mRNA degradation. Nonsense-mediated mRNA decay (NMD) is a mechanism for mRNA surveillance and gene homeostasis regulation, and may be the most typical eukaryotic RNA degradation pathway. Its main function is to reduce errors in gene expression by eliminating mRNA transcripts containing premature termination codons. mRNA with PTC is not only caused by nucleotide mutations in the coding region, but also by mutations that affect splicing, resulting in insertions or deletions in the coding region, frameshifts, and thus premature termination codons PTC. In some cases, the translation of these abnormal mRNAs may cause the resulting proteins to acquire harmful functions or become dominant negative. NMD can prevent the production of abnormal truncated proteins and ensure the normal physiological function of cells. It plays a very important role in the etiology of human genetic diseases and cancer.

[0004] Identifying the pathogenic genes of genetic diseases is a prerequisite for achieving precision medicine. With the improvement of high-throughput sequencing technology and the reduction of sequencing costs, more and more gene mutations have been discovered. Among these newly discovered mutations, a large number of mutations are listed as variants of uncertain significance (VUS) because of their unknown functions. Clarifying the correlation between these unknown significance variants and genetic diseases is an important issue to be solved in genetic diagnosis. Among them, whether the generation of nonsense mutations is related to the occurrence of the disease, whether it causes NMD, or whether it will produce non-functional or truncated proteins with normal functions, these are still difficult to detect. Therefore, how to quickly and efficiently verify NMD in vitro has always been a hot topic and difficulty in research. The vector system reported in the prior art for verifying NMD in vitro has problems such as low efficiency, poor accuracy, and cumbersome detection process. Summary of the invention

[0005] The present invention aims to provide a mini-NMD detection plasmid and a construction method and application thereof, which are used to solve the problems of low efficiency, poor accuracy, and complicated detection process in the vector system of in vitro NMD verification in the prior art.

[0006] In view of this, the scheme of the present invention is:

[0007] The first aspect of the present invention is to propose a mini-NMD detection plasmid, which contains a CMV enhancer, a pCMV promoter, a gene sequence, a protein tag, and a green fluorescent protein EGFP encoding gene; the gene sequence is a gDNA sequence near any PTC mutation to be detected.

[0008] Furthermore, the gDNA sequence is an upstream or downstream sequence of the PTC mutation, comprising three adjacent exons and their flanking intron sequences.

[0009] Furthermore, corresponding restriction sites are provided at both ends of the gDNA sequence for use in the detection of the gene to be tested.

[0010] The second aspect of the present invention is to propose a method for constructing the mini-NMD detection plasmid described in the first aspect, wherein the gDNA sequence near the PTC mutation is obtained by PCR amplification and inserted into pCMV-3xFlag-Neo (EGFP) for construction.

[0011] Furthermore, the restriction sites used for connecting the gDNA fragment near the PTC mutation with the vector plasmid are EcoRⅠ and HindⅢ.

[0012] The third aspect of the present invention is to propose the use of the mini-NMD detection plasmid described in the first aspect in the preparation of a detection reagent, wherein the detection reagent is used to verify nonsense-mediated mRNA degradation in eukaryotic organisms.

[0013] Furthermore, the process of verifying nonsense-mediated mRNA degradation in eukaryotic organisms comprises the following steps:

[0014] 1) Using the sample as a template, PCR amplification was performed to obtain a gDNA sequence fragment near the PTC mutation site; using the normal human genome as a template, a wild-type gDNA sequence fragment was constructed; the gDNA sequence in pNMD-3xFlag-Neo(EGFP) was replaced with the above fragment to construct an NMD recombinant expression plasmid expressing the wild-type and mutant genes;

[0015] 2) The obtained wild-type and mutant recombinant plasmids were transformed into Escherichia coli DH5α competent bacteria respectively, and the recombinant plasmids were extracted after cultivation;

[0016] 3) transfecting eukaryotic cells with the recombinant NMD expression vectors of the wild-type and mutant genes constructed in step 2), and collecting the cells;

[0017] 4) Detect the mRNA expression level in the cells; detect the expression level of the corresponding protein in the cells by immunoblotting; after inhibiting NMD by using UPF1 knockdown plasmid, detect the recovery of the mRNA and corresponding protein expression levels in the cells to determine whether mRNA degradation occurs.

[0018] Furthermore, the PTC mutation site is the PRRT2 gene c.649dup mutation, and the PCR amplification primers comprise the nucleotide sequences shown in SEQ ID NOs: 1-4.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The mini-NMD detection plasmid of the present invention can detect different gene mutations simply by replacing the inserted gDNA fragment, has a wide range of applications, and is easy to operate.

[0021] The pNMD-3xFlag-Neo (EGFP) detection plasmid constructed by the method of the present invention requires a smaller gDNA fragment to be inserted, and the vector construction is more efficient and rapid.

[0022] The present invention provides a complete and feasible method for verifying that nonsense mutations in eukaryotic genes lead to mRNA degradation (NMD) based on pNMD-3xFlag-Neo (EGFP) detection plasmid, which can quickly, efficiently and accurately verify in vitro that nonsense mutations in genes lead to mRNA degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a map of the pNMD-3xFlag-Neo (EGFP) detection plasmid constructed in the embodiment of the present invention.

[0024] Figure 2 This is a plasmid map of NMD detection for the PRRT2 c.649dup mutation constructed in an embodiment of the present invention.

[0025] Figure 3 This is a diagram showing the detection results of NMD caused by the PRRT2 c.649dup mutation in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In one embodiment, a mini-NMD detection plasmid is provided, namely, a detection plasmid pNMD-3xFlag-Neo (EGFP). The vector comprises a CMV enhancer, a pCMV promoter, a genomic DNA (gDNA) fragment, a 3×Flag tag, and a green fluorescent protein EGFP encoding gene. The mini-NMD detection plasmid can detect different gene mutations simply by replacing the inserted gDNA fragment, has a wide range of uses, and is easy to operate.

[0028] In another embodiment, the method for constructing the pNMD-3xFlag-Neo (EGFP) detection plasmid is to obtain the gDNA sequence near the PTC mutation by PCR amplification, insert it into pCMV-3xFlag-Neo (EGFP), transform the pNMD-3xFlag-Neo (EGFP) recombinant plasmid into Escherichia coli DH5α competent cells by heat shock method, and obtain it by ampicillin resistance screening.

[0029] In a preferred embodiment, the specific method of resistance screening is as follows: pick a single colony and inoculate it into LB liquid culture medium containing ampicillin, culture it overnight at 37°C and 200rpm, and use a commercial plasmid mini-extraction kit to extract the recombinant pNMD-3xFlag-Neo (EGFP) plasmid.

[0030] In another embodiment, the pNMD-3xFlag-Neo (EGFP) plasmid is used in in vitro verification of nonsense-mediated mRNA degradation in eukaryotic organisms, and the application method comprises the following steps:

[0031] (1) The gDNA sequence in pNMD-3xFlag-Neo(EGFP) was replaced with the wild-type and mutant gDNA fragments obtained by PCR amplification to construct NMD recombinant expression plasmids expressing wild-type and mutant genes;

[0032] (2) transfecting eukaryotic cells with the recombinant expression vectors of the wild-type and mutant genes constructed in step (1), and collecting the cells 24 hours after transfection;

[0033] (3) Reverse transcription real-time quantitative PCR (qRT-PCR) was used to detect the expression level of intracellular mRNA; Western Blot was used to detect the expression level of the corresponding protein of intracellular mRNA; UPF1-sh1 knockdown plasmid was used to inhibit NMD and then detect the level of replenishment of intracellular mRNA and corresponding protein expression levels.

[0034] In a preferred embodiment, the in vitro verification of nonsense-mediated mRNA degradation in eukaryotes can be used for in vitro verification of human cells, and gDNA sequence fragments near the PTC mutation site are obtained by PCR amplification based on the subject sample as a template; wild-type gDNA sequence fragments are constructed using the normal human genome as a template; the gDNA sequence in pNMD-3xFlag-Neo (EGFP) is replaced by the above fragments to construct an NMD recombinant expression plasmid expressing wild-type and mutant genes, and in vitro verification is performed according to the above steps.

[0035] Example Detection of NMD degradation caused by c.649dup mutation in human PRRT2 gene

[0036] 1. Construction of pNMD-3xFlag-Neo(EGFP)-PRRT2 wt and pNMD-3xFlag-Neo(EGFP)-PRRT2 mut vectors

[0037] The c.649dup mutation of the PRRT2 gene is located in the first exon of the PRRT2 gene. Using the normal human genome as a template, the primers PRRT2-F-HindⅢ and PRRT2-R-EcoRI were used to amplify the wild-type gDNA fragment; the primers PRRT2-F-HindⅢ, PRRT2-R-EcoRI, PRRT2-c.649dup-F, and PRRT2-c.649dup-R were used to amplify the PRRT2-c.649dup point mutation gDNA fragment; the gDNA fragment contained the complete sequence of exons 2, 3, and 4 of PRRT2 and the truncated sequence of introns 2 and 3, with a total length of 1585 bp; the sequences of the primers are shown in the following table:

[0038]

[0039] After the above PCR fragments were purified and recovered, the amplified fragments were double-digested with EcoRI / HindⅢ, and the pNMD-3xFlag-Neo(EGFP) plasmid was double-digested with EcoRI / EcoRV; the fragments and the vector were connected using T4 DNA ligase, and the ligation products were transformed into Escherichia coli DH5α competent cells using the heat shock method. The positive clones screened by ampicillin resistance were double-verified by colony PCR detection and sequencing detection to obtain pNMD-3xFlag-Neo(EGFP)-PRRT2 wt and pNMD-3xFlag-Neo(EGFP)-PRRT2 mut expression vectors; the map of the pNMD-3xFlag-Neo(EGFP)-PRRT2 wt expression plasmid is shown in Figure 1 As shown; the map of pNMD-3xFlag-Neo(EGFP)-PRRT2 mut expression plasmid is shown Figure 2 shown.

[0040] 2. Detection of NMD degradation caused by c.649dup mutation of PRRT2 gene

[0041] The obtained pNMD-3xFlag-Neo(EGFP)-PRRT2 wt and pNMD-3xFlag-Neo(EGFP)-PRRT2mut plasmids were transfected into 293T cell lines, and UPF1 siRNA was used to co-transfect 293T cell lines with pNMD-3xFlag-Neo(EGFP)-PRRT2 wt and pNMD-3xFlag-Neo(EGFP)-PRRT2 mut plasmids, respectively; cells were collected 24 hours after transfection, and the results of Western blotting were shown in Figure 2. Figure 3 .

[0042] Figure 3The results showed that the expression level of PRRT2 protein was high in cells transfected with pNMD-3xFlag-Neo(EGFP)-PRRT2 wt, indicating that the wt PRRT2 recombinant fragment could be transcribed and expressed normally; the expression level of PRRT2 truncated protein was very low in cells transfected with pNMD-3xFlag-Neo(EGFP)-PRRT2mut, indicating that the PRRT2 c.649dup mutation caused the cells to produce PRRT2 truncated protein and the truncated protein was degraded; in cells co-transfected with UPF1 siRNA and pNMD-3xFlag-Neo(EGFP)-PRRT2mut, a certain complementation was obtained, and the expression level was close to that of cells co-transfected with UPF1 siRNA and pNMD-3xFlag-Neo(EGFP)-PRRT2 wt, indicating that inhibiting UPF1, a key component of the NMD pathway, could significantly reverse the post-transcriptional degradation of the mut recombinant fragment. The above results indicate that the PTC generated by the c.649dup mutation will lead to mRNA degradation through the NMD pathway. The mini-NMD detection plasmid constructed by the present invention can quickly, efficiently and accurately verify in vitro that nonsense mutation of a gene leads to mRNA degradation (NMD).

[0043] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A mini-NMD detection plasmid, characterized in that: The plasmid contains a CMV enhancer, a pCMV promoter, a gene sequence, a protein tag, and a green fluorescent protein EGFP coding gene; the gene sequence is a gDNA sequence near any PTC mutation to be detected.

2. The mini-NMD detection plasmid according to claim 1, characterized in that The gDNA sequence is an upstream or downstream sequence of the PTC mutation, comprising three adjacent exons and their flanking intron sequences.

3. The mini-NMD detection plasmid according to claim 1, characterized in that Enzyme cleavage sites are arranged at both ends of the gDNA sequence.

4. The method for constructing the mini-NMD detection plasmid according to claim 1, characterized in that: The gDNA sequence near the PTC mutation was obtained by PCR amplification and inserted into the vector plasmid pCMV-3xFlag-Neo (EGFP) to construct it.

5. The construction method according to claim 4, characterized in that: The restriction sites used for connecting the gDNA fragment near the PTC mutation with the vector plasmid are EcoRⅠ and HindⅢ.

6. Use of the mini-NMD detection plasmid according to claim 1 in preparing a detection reagent, characterized in that: The somatic detection reagent is used to verify the nonsense-mediated mRNA degradation of eukaryotic organisms.

7. The use according to claim 6, characterized in that The process of verifying eukaryotic nonsense-mediated mRNA degradation comprises the following steps: 1) Using the sample as a template, PCR amplification was performed to obtain a gDNA sequence fragment near the PTC mutation site; using the normal human genome as a template, a wild-type gDNA sequence fragment was constructed; the gDNA sequence in pNMD-3xFlag-Neo(EGFP) was replaced with the above fragment to construct an NMD recombinant expression plasmid expressing the wild-type and mutant genes; 2) The obtained wild-type and mutant recombinant plasmids were transformed into Escherichia coli DH5α competent bacteria respectively, and the recombinant plasmids were extracted after cultivation; 3) transfecting eukaryotic cells with the recombinant NMD expression vectors of the wild-type and mutant genes constructed in step 2), and collecting the cells; 4) Detect the mRNA expression level in the cells; detect the expression level of the corresponding protein in the cells by immunoblotting; after inhibiting NMD by using UPF1 knockdown plasmid, detect the recovery of the mRNA and corresponding protein expression levels in the cells to determine whether mRNA degradation occurs.

8. The use according to claim 7, characterized in that The PTC mutation site is the PRRT2 gene c.649dup mutation, and the PCR amplification primers contain the nucleotide sequences shown in SEQ ID NOs: 1-4.

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