Dual-luciferase recombinant vector, transformant, transfection body and application

Abstract

The embodiment of the invention discloses a new application of a plasmid, and the plasmid has a nucleotide sequence as shown in SEQ ID NO: 1 and can be used for ensuring the height of a fluorescence ratio in activity detection of a 3 'UTR sequence. The embodiment of the invention further provides a dual-luciferase recombinant vector, a transformant, a transfection body and application. The dual-luciferase recombinant vector, the transformant, the transfection body and the application can be used for detecting the activity of a 3 'UTR sequence.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a dual-luciferase recombinant vector, a transformant, a transfectant and uses thereof. Background Art

[0002] 3'UTR is the 3' untranslated region in the transcript, referred to as 3'UTR. It is well known that post-transcriptional regulation of gene expression is regulated by 3'UTR. 3'UTR is a sequence located after the stop codon of the gene and extends to before the transcription termination signal.

[0003] 3'UTR plays a vital role in regulating mRNA stability, mRNA localization and translation. The stability and translation level of mRNA directly affect the expression abundance of proteins in organisms, while the localization of mRNA has an important impact on the function of proteins. More and more studies have shown that SNPs in the 3'UTR region have an important impact on traits.

[0004] The dual-luciferase reporter gene system is an important tool for detecting the activity of SNPs (single nucleotide polymorphism) in 3'UTR sequences. The luciferase reporter gene system refers to a method for indirectly screening potential regulatory factors that affect the expression of target genes and quantifying their regulatory activity by quantifying the bioluminescence emitted by luciferase-catalyzed oxidation of luciferin to oxyluciferin. The dual-luciferase reporter gene system contains the firefly luciferase gene and the Renilla luciferase gene, which serve as reporter genes and internal reference genes, respectively. The fluorescence ratio refers to the ratio of the activity of firefly luciferase and the activity of Renilla luciferase, and is usually used to evaluate the regulatory ability of the inserted target sequence on the expression of the reporter gene. A high fluorescence ratio indicates that the inserted sequence has a higher activity, while a low fluorescence ratio may indicate a lower activity. If a vector can only produce a significant fluorescence ratio difference with the control group when a high-activity sequence is inserted, the detection sensitivity of the vector is low and the suitable sequence activity range is small. Therefore, for some low-activity sequences, the vector may not be able to effectively detect their activity.

[0005] A commonly used dual-luciferase reporter plasmid for verifying the functional activity of 3'UTR is the pmirGLO plasmid, but the dual-luciferase recombinant vector (pmirGLO vector) constructed by inserting the sequence to be tested based on the pmirGLO plasmid is more focused on exploring the activity of miRNA bound to the 3'UTR region. There is an obvious problem in applying the pmirGLO vector to identify the functional activity of the SNP sequence in the 3'UTR region, that is, the fluorescence ratio is low when detected by the pmirGLO vector.

[0006] In addition to the pmirGLO plasmid, some research groups also use plasmids such as pgl4.23 to construct dual luciferase reporter vectors. However, these plasmids are mainly used in the study of enhancers, 5'UTR, etc. Using them to detect 3'UTR will also result in the defect of low fluorescence ratio in many cases.

[0007] Therefore, how to ensure a high fluorescence ratio in the activity detection of 3'UTR sequences is a problem to be solved. Summary of the invention

[0008] An object of embodiments of the present application is to solve at least the above-mentioned problems and to provide at least the advantages to be described below.

[0009] Another purpose of the embodiments of the present application is to provide a new use of a plasmid. When the plasmid is used to detect the activity of a 3'UTR sequence, a higher fluorescence ratio can be generated, which is beneficial for activity detection.

[0010] Another object of the embodiments of the present application is to provide a dual luciferase recombinant vector, which can generate a higher fluorescence ratio when used to detect the activity of a 3'UTR sequence, thereby facilitating activity detection.

[0011] Another object of the present embodiment is to provide a transformant that can be used to propagate the dual-luciferase recombinant vector.

[0012] Another object of the present embodiment is to provide a transfectant that can be used to detect the activity of a 3'UTR sequence.

[0013] To achieve the above objectives, the embodiments of the present application provide the following technical solutions.

[0014] In a first aspect, an embodiment of the present application provides a use of a plasmid as a vector for detecting the activity of a 3'UTR sequence, wherein the plasmid has a nucleotide sequence as shown in SEQ ID NO:1.

[0015] In a second aspect, an embodiment of the present application provides a use of a plasmid in preparing a dual-luciferase recombinant vector, wherein the plasmid has a nucleotide sequence as shown in SEQ ID NO:1.

[0016] In a third aspect, the present application provides a dual luciferase recombinant vector, comprising:

[0017] A plasmid having the nucleotide sequence shown in SEQ ID NO: 1; and

[0018] A target recombinant sequence, wherein the target recombinant sequence includes a target gene sequence, wherein the target gene sequence includes a SNP selected from a 3'UTR sequence;

[0019] Wherein, the target recombinant sequence is inserted into the plasmid.

[0020] In a fourth aspect, the present invention provides a transformant, comprising:

[0021] The dual luciferase recombinant vector according to the third aspect; and

[0022] The host cell is selected from Escherichia coli competent cells.

[0023] In a fifth aspect, the present application provides a transfectant, comprising:

[0024] The dual luciferase recombinant vector according to the third aspect; and

[0025] The transfected cells are selected from C2C12 cells.

[0026] In a sixth aspect, an embodiment of the present application provides a use of the dual luciferase recombinant vector described in the third aspect in preparing transfected cells for detecting the activity of a 3'UTR sequence.

[0027] The beneficial effects that can be achieved by the embodiments of the present application include: :

[0028] In some embodiments, according to The embodiments of the present application provide The new use of the plasmid can ensure a high fluorescence ratio in the activity detection of the 3'UTR sequence, thereby overcoming the defects in the background technology.

[0029] In some embodiments, the present invention constructs a dual luciferase recombinant vector based on a plasmid having a sequence as shown in SEQ ID NO: 1, which can ensure a high fluorescence ratio in the activity detection of the 3'UTR sequence, thereby overcoming the defects in the background technology.

[0030] In some embodiments, compared with existing plasmids such as pmirGLO, pgl4.23, and pgl3-promoter, the present invention constructs a dual-luciferase recombinant vector based on a plasmid having a sequence as shown in SEQ ID NO: 1, which can improve the fluorescence ratio of the dual-luciferase recombinant vector when used to detect the activity of the 3'UTR sequence.

[0031] In some embodiments, the present invention constructs a dual luciferase recombinant vector based on a plasmid having a sequence as shown in SEQ ID NO: 1. Since the fluorescence ratio during detection is improved, the detection activity range is wider.

[0032] In some embodiments, from the perspective of detecting the SNP effect of the 3'UTR sequence, when the dual luciferase recombinant vector constructed according to the plasmid having the sequence shown in SEQ ID NO: 1 in the embodiment of the present application is used to detect the activity of the 3'UTR sequence, the detection effect is more stable and the difference between the SNP sites is more significant, that is, the dual luciferase recombinant vector can more sensitively capture the expression difference of the SNP in the 3'UTR sequence.

[0033] Other advantages, objectives and features of the present application will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic diagram of a fluorescence ratio of a first target sequence and its corresponding first reference sequence using different recombinant vectors in the experimental example of this application;

[0035] Figure 2 A schematic diagram of a fluorescence ratio of a second target sequence and its corresponding second reference sequence using different recombinant vectors in the experimental example of this application;

[0036] Figure 3 A schematic diagram of a fluorescence ratio of detecting a third target sequence and its corresponding third reference sequence using different recombinant vectors in the experimental example of this application. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0038] The terms "first", "second", and "third" in the embodiments of the present application are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second", and "third" may explicitly or implicitly include at least one of the features.

[0039] It should also be noted that, in the present application, “plurality” means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0040] Furthermore, the terms "comprises," "includes," "has," and the like, and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to the process, method, product, or apparatus.

[0041] In addition to the above, it is still necessary to emphasize that the reference to "embodiment" in this article means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0042] For the same sequence to be detected, when different vectors are used for detection, the sequence activity is certain; however, the fluorescence ratios detected by different vectors may be different, which may lead to the defect that some sequences may be difficult to be detected by certain vectors. Assume that for a sequence A to be detected with a sequence activity of 10, different vectors 1 and 2 are used for detection, and the fluorescence ratios detected are 3 and 30 respectively; then, if vector 1 and vector 2 are used to detect another sequence B to be detected with very low sequence activity (for example, activity of 1), the sequence activity detected by vector 2 with a high fluorescence ratio may be 3, but the activity of sequence B to be detected will basically not be detected by vector 1 with a low fluorescence ratio. Therefore, it can be understood that, compared with vector 1, vector 2 can detect the activity of some sequences with lower activity, that is, it can be considered that the range of activity it can detect is wider.

[0043] <New Uses of Plasmids>

[0044] A plasmid is a DNA molecule.

[0045] To overcome the problems raised in the background technology, in the first aspect, the embodiments of the present application provide a new use of a plasmid as a vector for detecting the activity of a 3'UTR sequence; wherein the plasmid is an SCP1 plasmid having a nucleotide sequence as shown in SEQ ID NO:1.

[0046] Specifically, the plasmid contains the firefly luciferase gene, which can be recombined with the 3'UTR sequence to be detected to form a dual-luciferase recombinant vector, which is then used to detect the activity of the 3'UTR sequence.

[0047] It is understandable that the 3'UTR sequence to be detected for recombination with the plasmid to form a dual luciferase recombinant vector can be a part of the bases or sequences in the entire 3'UTR sequence of the genome of a species (e.g., pig). Compared with existing plasmids such as pmirGLO, pgl4.23, and pgl3-promoter, the plasmid having the sequence shown in SEQ ID NO: 1 provided in the embodiment of the present application can improve the fluorescence ratio of the dual luciferase recombinant vector constructed according to the plasmid when used to detect the activity of the 3'UTR sequence, thereby overcoming the defects mentioned in the background.

[0048] It is understandable that when the fluorescence ratio of the dual-luciferase recombinant vector is improved, the sensitivity of detection can be further improved and the detection range of sequence activity can be expanded.

[0049] In addition, from the perspective of detecting the SNP effect of the 3'UTR sequence, when the vector constructed according to the plasmid of the embodiment of the present application is used to detect the activity of the 3'UTR sequence, the detection effect is more stable and the difference between the SNP sites is more significant, that is, the vector can more sensitively capture the expression difference of the SNP in the 3'UTR sequence.

[0050] In addition, the commonly used dual luciferase reporter vector for verifying the functional activity of 3'UTR is pmirGLO vector, but pmirGLO vector focuses more on exploring the activity of miRNA bound to the 3'UTR region. There are many problems in applying pmirGLO vector to identify the functional activity of SNP in 3'UTR region. For example, firefly fluorescence and Renilla fluorescence are in the same vector in pmirGLO vector, and both are driven by medium-strength promoters to drive expression, so that the expression values ​​of firefly fluorescence and Renilla fluorescence are similar in the empty vector. When detecting the activity of SNP that affects 3'UTR function, if the sequence to be tested is inserted and its activity is small, it will not cause a large change in the expression of firefly luciferase, and this small change is difficult to capture, that is, the effect of SNP with small activity is difficult to be sensitively detected. Due to the lack of a more suitable dual luciferase vector for functional activity testing of 3'UTR sequences, some research institutes will also select some other commonly used dual luciferase reporter vectors, such as pgl4.23, pgl3-basic, pgl3-promoter, etc., but these vectors are mainly widely used in the research of enhancers, promoters, 5'UTR, etc., and there are many problems such as promoter strength mismatch, fewer restriction sites at the insertion position, and difficulty in selecting, which make it difficult to verify the functional activity of SNP sites in the 3'UTR region. The aforementioned defects can be overcome by using the plasmid with the sequence shown in SEQ ID NO:1 to construct a dual luciferase recombinant vector.

[0051] <New Uses of Plasmids>

[0052] It is understandable that, in the second aspect, the embodiments of the present application may also provide a use of the plasmid having the sequence shown in SEQ ID NO: 1 in preparing a dual luciferase recombinant vector.

[0053] <Recombinant vector>

[0054] In a third aspect, the embodiments of the present application provide a recombinant vector, which can be used as a dual-luciferase recombinant vector for detecting the activity of a 3'UTR sequence.

[0055] The recombinant vector comprises:

[0056] A plasmid having the nucleotide sequence shown in SEQ ID NO: 1; and

[0057] A target recombinant sequence, wherein the target recombinant sequence includes a target gene sequence, wherein the target gene sequence includes a SNP selected from a 3'UTR sequence;

[0058] Wherein, the recombinant sequence is inserted into the plasmid.

[0059] Specifically, the target recombinant sequence (base sequence) can be inserted into a plasmid to form a recombinant vector. The insertion method can be achieved according to existing gene recombination technology.

[0060] It is understandable that when the dual luciferase recombinant vector provided in the third aspect of the embodiment of the present application is used to detect the activity of the 3'UTR sequence, the fluorescence ratio can be increased, thereby making the detection activity range wider.

[0061] In addition, from the perspective of detecting the SNP effect of the 3'UTR sequence, when the dual luciferase recombinant vector according to the embodiment of the present application is used to detect the activity of the 3'UTR sequence, the detection effect is more stable and the difference between the SNP sites is more significant, that is, the vector can more sensitively capture the expression difference of the SNP in the 3'UTR sequence.

[0062] In some embodiments, the 3'UTR sequence is selected from the 3'UTR sequence of the porcine genome.

[0063] In some embodiments, the target gene sequence also includes the upstream and downstream sequences of the SNP. The upstream and downstream sequences include the upstream sequence and the downstream sequence of the SNP.

[0064] Optionally, the sequence numbers (number of bases) of the upstream sequence and the downstream sequence are equal or unequal.

[0065] Optionally, the sequence number (number of bases) of the upstream and downstream sequences is 1 to 400bp. It should be understood that the number of bases of the upstream sequence and the downstream sequence are both 1 to 400bp, and the upstream and downstream sequences of the SNP include the sequence of 1 to 400bp upstream of the SNP and the sequence of 1 to 400bp downstream of the SNP.

[0066] Optionally, the sequence length of the upstream and downstream sequences is 20 to 300 bp.

[0067] Optionally, the sequence length of the upstream and downstream sequences is 50 bp, and the target gene sequence includes a 50 bp upstream sequence and a 50 bp downstream sequence.

[0068] In some embodiments, the target sequence also includes a first common sequence and a second common sequence; wherein the first common sequence is CGAGCTCGCTAGCCT, with a length of 15 bp; and the second common sequence is AGATCGGAAGAGCGTCG, with a length of 17 bp.

[0069] Furthermore, the first common sequence, the target gene sequence and the second common sequence are connected in sequence.

[0070] Furthermore, in the structure of the target recombinant sequence: the first common sequence, the target gene sequence, and the second common sequence are sequentially distributed along the direction from the 5' end to the 3' end, and the first common sequence, the target gene sequence, and the second common sequence are located between the 5' end and the 3' end. Specifically, the synthetic sequence structure of the target recombinant sequence is: 5' end CGAGCTCGCTAGCCT [target gene sequence] AGATCGGAAGAGCGTCG 3' end).

[0071] In some embodiments, the recombinant vector is formed by inserting the target recombinant sequence into the downstream of the firefly luciferase gene of the plasmid having the sequence shown in SEQ ID NO: 1.

[0072] In some embodiments, the recombinant vector further comprises a reference recombinant sequence, wherein the reference recombinant sequence comprises a reference sequence. The reference sequence is a reference genome sequence containing the coordinates of the mutation site of the target gene sequence, for reference.

[0073] In some embodiments, the reference recombinant sequence also includes a first common sequence and a second common sequence; wherein the first common sequence is CGAGCTCGCTAGCCT, with a length of 15 bp; and the second common sequence is AGATCGGAAGAGCGTCG, with a length of 17 bp.

[0074] <Transformants>

[0075] In a fourth aspect, the present invention provides a transformant, comprising:

[0076] The recombinant vector according to the third aspect; and

[0077] The host cell is selected from Escherichia coli cells.

[0078] More specifically, the host cell is selected from Escherichia coli competent cells.

[0079] Introducing the recombinant vector into the host cell (E. coli competent cell) to form a transformant can achieve the propagation of the recombinant vector and facilitate subsequent fluorescence detection.

[0080] The specific introduction method can be achieved according to the existing technology. For example, in some embodiments, the host cells can be treated with calcium chloride to increase the permeability of the cell membrane, so that the exogenous recombinant vector DNA can easily enter the host cells.

[0081] <Transfectants>

[0082] In a fifth aspect, the present application provides a transfectant, comprising:

[0083] The recombinant vector according to the third aspect; and

[0084] The transfected cells are selected from C2C12 cells; C2C12 cells are mouse myoblasts.

[0085] The recombinant vector described in the third aspect is introduced into the transfected cells (C2C12 cells) to obtain transfected cells, namely transfectants, which are convenient for fluorescence detection and further used for the detection of 3'UTR sequence activity.

[0086] The specific transfection method can be implemented according to the existing technology. For example, in some embodiments, the transfected cells can be treated with calcium chloride to increase the permeability of the cell membrane, so that the exogenous recombinant vector DNA can easily enter the interior of the transfected cells.

[0087] In some embodiments, the transfectant further comprises:

[0088] A Renilla luciferase plasmid containing the Renilla luciferase gene.

[0089] The Renilla luciferase gene-containing Renilla fluorescent plasmid is introduced into transfected cells (C2C12 cells) to obtain transfectants.

[0090] In some embodiments, the mass ratio of the recombinant vector to the Renilla luciferase gene-containing Renilla fluorescence plasmid is 40:1.

[0091] <Preparation of plasmid>

[0092] Artificial synthesis was performed according to the nucleotide sequence shown in SEQ ID NO: 1 to obtain the SCP1 plasmid.

[0093] <Construction of recombinant sequence>

[0094] In the following, a SNP on the 3'UTR of the pig genome is selected as the SNP to be tested, and the corresponding target gene sequence to be tested and the recombinant sequence to be inserted into the plasmid are determined to construct a dual luciferase vector and verify the use of the SCP1 plasmid in the examples of this application.

[0095] The construction of the recombinant sequence includes the following steps S101-102:

[0096] Step S101, determining at least one SNP to be tested on the 3'UTR of the pig genome, and determining the corresponding target gene sequence and the reference sequence corresponding to the target gene sequence according to each SNP to be tested.

[0097] The target gene sequence is the corresponding SNP to be tested and the 50 bp sequence upstream and downstream thereof, and the target gene sequence length is 101 bp. The reference sequence is the pig reference genome sequence containing the mutation site coordinates corresponding to the target gene sequence, which is used as a control.

[0098] In the embodiment of the present application, the resequencing data of 234 oriental pigs and western pigs in the early stage of the laboratory were analyzed, and the GATK software was used to identify the genomic SNPs of the resequencing data, and a total of 26,641,976 SNP sites were identified. Subsequently, the annovar software was used to annotate the SNP sites of eastern and western pigs and screen out the SNPs located in the 3'UTR region based on the annotation information. A total of 220,082 SNP sites located in the 3'UTR range were found, and 3 SNPs related to meat production traits in the pig genome were randomly selected as the SNPs to be tested. Accordingly, it can be determined that there are 3 target gene sequences and corresponding reference sequences.

[0099] Specifically, the three target gene sequences are the first target sequence, the second target sequence and the third target sequence, and the three reference sequences are the first reference sequence, the second reference sequence and the third reference sequence. For specific sequence information, see Table 1.

[0100] The first target sequence can be obtained from the genome of Duroc pigs, Yucatan miniature pigs, Luchuan pigs and other pigs.

[0101] The second target sequence can be obtained from the genome of Tongcheng pig, Luchuan pig, Tibetan pig and other pigs.

[0102] The third target sequence can be obtained from the genomes of Korean Native black pig, Chinese wild boar, Tongcheng pig and other pigs.

[0103] Table 1. Sequence information

[0104]

[0105]

[0106] Step S102: synthesize the corresponding target recombinant sequence and reference recombinant sequence according to the target gene sequence and the reference sequence.

[0107] In order to better amplify the target gene sequence, 15bp and 17bp common sequences were introduced at both ends of the target gene sequence to form the corresponding target recombinant sequence.

[0108] Specifically, the target recombinant sequence is synthesized by the first common sequence, the second common sequence and the corresponding target gene sequence; wherein the first common sequence is CGAGCTCGCTAGCCT, with a length of 15 bp; the second common sequence is AGATCGGAAGAGCGTCG, with a length of 17 bp;

[0109] The synthetic sequence structure of the target recombinant sequence is: 5' end CGAGCTCGCTAGCCT [target gene sequence] AGATCGGAAGAGCGTCG 3' end).

[0110] It can be understood that the target recombinant sequences corresponding to the first to third target gene sequences also include three, specifically the first target recombinant sequence, the second target recombinant sequence, and the third target recombinant sequence.

[0111] Exemplarily, the structure of the first target recombinant sequence is: 5' end CGAGCTCGCTAGCCT [first target sequence (2_4295650_C_alt)] AGATCGGAAGAGCGTCG 3' end).

[0112] The structures of the second target recombinant sequence and the third target recombinant sequence can be similarly understood and constructed with reference to the first target recombinant sequence, and will not be described in detail.

[0113] It should also be understood that the structure of the reference recombinant sequence is similar to that of its corresponding target recombinant sequence, and therefore can be similarly understood and constructed in structure.

[0114] Therefore, the reference recombinant sequence is synthesized from the first common sequence, the second common sequence and the corresponding reference sequence; wherein the first common sequence is CGAGCTCGCTAGCCT, with a length of 15 bp; the second common sequence is AGATCGGAAGAGCGTCG, with a length of 17 bp;

[0115] The synthetic sequence structure of the reference recombinant sequence is: CGAGCTCGCTAGCCT [reference sequence] 5' end AGATCGGAAGAGCGTCG 3' end).

[0116] It can be understood that the reference recombinant sequences corresponding to the reference sequence also include three, specifically a first reference recombinant sequence corresponding to the first target recombinant sequence, a second reference recombinant sequence corresponding to the second target recombinant sequence, and a third reference recombinant sequence corresponding to the third target recombinant sequence.

[0117] Exemplarily, the structure of the first reference recombinant sequence is: 5' end CGAGCTCGCTAGCCT [first reference sequence (2_4295650_C_alt)] AGATCGGAAGAGCGTCG 3' end).

[0118] The structures of the second reference recombinant sequence and the third reference recombinant sequence can be similarly understood and constructed with reference to the first reference recombinant sequence, and will not be described in detail.

[0119] <Construction of recombinant vector>

[0120] The purpose of constructing the vector is to insert the target recombinant sequence to be tested into the plasmid to form a recombinant vector, and then transfect the cells to detect the regulatory activity of the target gene sequence. Specifically, the target recombinant sequence is inserted downstream of the firefly luciferase gene of the plasmid to form a corresponding recombinant vector.

[0121] In order to detect the superiority of the SCP1 plasmid in the present example in verifying the SNP activity in the 3'UTR sequence, three commonly used dual luciferase vectors were selected as controls in the present example, namely, recombinant vectors constructed based on the pgl4.23 plasmid, pmirGLO plasmid and pgl3-promoter plasmid.

[0122] For details on the construction of each recombinant vector, please refer to the following examples and comparative examples. .

[0123] Example 1

[0124] The first target recombinant sequence and the first reference recombinant sequence are inserted into the SCP1 plasmid to form the SCP1 vector 1.

[0125] Example 2

[0126] The second target recombinant sequence and the second reference recombinant sequence are inserted into the SCP1 plasmid to form SCP1 vector 2.

[0127] Example 3

[0128] The third target recombinant sequence and the third reference recombinant sequence are inserted into the SCP1 plasmid to form SCP1 vector 3.

[0129] Comparative Example 1

[0130] The first target recombinant sequence and the first reference recombinant sequence are inserted into the pgl4.23 plasmid to form the pgl4.23 vector 1.

[0131] Comparative Example 2

[0132] The second target recombinant sequence and the second reference recombinant sequence are inserted into the pgl4.23 plasmid to form pgl4.23 vector 2.

[0133] Comparative Example 3

[0134] The third target recombinant sequence and the third reference recombinant sequence are inserted into the pgl4.23 plasmid to form pgl4.23 vector 3.

[0135] Comparative Example 4

[0136] The first target recombinant sequence and the first reference recombinant sequence are inserted into the pmirGLO plasmid to form a pmirGLO vector 1.

[0137] Comparative Example 5

[0138] The second target recombinant sequence and the second reference recombinant sequence are inserted into the pmirGLO plasmid to form pmirGLO vector 2.

[0139] Comparative Example 6

[0140] The third target recombinant sequence and the third reference recombinant sequence are inserted into the pmirGLO plasmid to form pmirGLO vector 3.

[0141] Comparative Example 7

[0142] The first target recombinant sequence and the first reference recombinant sequence are inserted into the pgl3-promoter plasmid to form the pgl3-promoter vector 1.

[0143] Comparative Example 8

[0144] The second target recombinant sequence and the second reference recombinant sequence are inserted into the pgl3-promoter plasmid to form the pgl3-promoter vector 2.

[0145] Comparative Example 9

[0146] The third target recombinant sequence and the third reference recombinant sequence are inserted into the pgl3-promoter plasmid to form the pgl3-promoter vector 3.

[0147] It should be understood that after constructing SCP1 vectors 1 to 3, pmirGLO vectors 1 to 3, pgl4.23 vectors 1 to 3, and pgl3-promoter vectors 1 to 3, if expansion is required to facilitate subsequent cell transfection operations, it can be achieved through transformation to obtain a larger number of recombinant vectors.

[0148] In addition, since the SCP1 vector contains the suicide gene CCDB, the suicide gene CCDB can be used as a marker to verify whether the cloning is successful during transformation; the restriction sites SphI and NdeI are used for restriction digestion, the linearized vector after restriction digestion is recovered, and the target fragment is cloned. Specifically, after the recombinant vector is constructed, it can be transformed with E. coli DH5α, and the clones grown after plating contain the sequence of the successful introduction of the target fragment; if an empty plasmid appears after transformation, the empty plasmid will express the CCDB gene, which will cause the E. coli cells to die or growth to be inhibited, and the purpose of propagation cannot be achieved.

[0149] <Cell Transfection with Recombinant Vector>

[0150] After the construction of the recombinant vector was completed, SCP1 vectors 1 to 3, pmirGLO vectors 1 to 3, pgl4.23 vectors 1 to 3, and pgl3-promoter vectors 1 to 3 were taken for cell transfection respectively.

[0151] In order to ensure a single variable in the experiment, for each vector, the host cells to be transfected were inoculated at the same time, the same amount of recombinant vector was used, and three wells were transfected at the same time to eliminate the systematic error of the experiment. The specific operation steps are as follows:

[0152] The host cells to be transfected were inoculated in a 24-well plate. The host cells were C2C12 cells. Cell transfection was performed when the cell confluence was 70%. It should be noted that in the cell transfection, for the pgl4.23 vector, the pgl3-promoter vector, and the SCP1 vector, it was necessary to additionally transfect the Renilla fluorescence plasmid (TK plasmid) as an internal reference. Therefore, the ratio of the amount of the plasmid carrying the firefly luciferase gene (SCP1 vector 1 to 3, pgl4.23 vector 1 to 3, and pgl3-promoter vector 1 to 3) and the plasmid carrying the Renilla luciferase gene was 40:1, that is, the amount of TK in each well cell was 17.5 ng; and for the pmirGLO vectors 1 to 3, since the vectors carried both the firefly luciferase gene and the Renilla luciferase gene, it was not necessary to additionally transfect the TK plasmid. In cell transfection, 700 ng of each vector was transfected into each well of a 24-well plate using jetprime cell transfection reagent. After 24 hours, the cells were collected to obtain transfectants (transfected cells) transfected with the corresponding exogenous vectors (recombinant vectors).

[0153] <Experimental Example, Dual Luciferase Detection>

[0154] Obtain transfected cells corresponding to SCP1 vectors 1 to 3, pmirGLO vectors 1 to 3, pgl4.23 vectors 1 to 3, pgl3-promoter vectors 1 to 3, etc. 24 hours after transfection, detect the fluorescence value, and record the results.

[0155] Specifically, the vector-transfected cells were tested according to the instructions in the luciferase assay kit. The luciferase assay kit was selected from the Dual- Assay System kit.

[0156] Experimental results and analysis

[0157] The fluorescence ratios of the first target recombinant sequence, the second target recombinant sequence, the third target recombinant sequence, the first reference recombinant sequence, the second reference recombinant sequence, and the third reference recombinant sequence were measured using different recombinant vectors. For details, please refer to Table 2-4.

[0158] In addition, based on the fluorescence ratio, a comparative schematic diagram of the fluorescence ratios of the first target recombinant sequence, the second target recombinant sequence, the third target recombinant sequence, the first reference recombinant sequence, the second reference recombinant sequence, and the third reference recombinant sequence is drawn using different recombinant vectors. For details, please refer to Figures 1 to 3 .

[0159] Table 2

[0160]

[0161] Table 3

[0162]

[0163]

[0164] Table 4

[0165]

[0166] Based on the above dual luciferase fluorescence value detection results, it can be seen that for sequences such as 2_4295650_C_alt, 2_4295650_C_ref, 2_41360435_T_alt, 2_41360435_T_ref, when the SCP1 vector is used for detection, the fluorescence ratio is much higher than that of the pmirGLO vector, pgl4.23 vector, and pgl3-promoter vector; for sequences such as 14_16934435_A_alt and 14_16934435_A_ref, when the SCP1 vector is used for detection, the fluorescence ratio is much higher than that of the pmirGLO vector, pgl4.23 vector, etc., and is basically equivalent to that of the pgl3-promoter vector.

[0167] It can be seen that: on the one hand, without considering SNP, from the perspective of a single sequence, the fluorescence ratio of the SCP1 vector provided in the embodiment of the present application is substantially higher than that of other vectors, that is, the activity range of the SCP1 vector in detecting 3'UTR sequences is wider than that of other vectors.

[0168] On the other hand, from the perspective of detecting the SNP effect of the 3'UTR sequence, the detection effect of the SCP1 vector is more stable and the differences between the SNP sites are more significant, that is, the SCP1 vector can more sensitively capture the expression differences of SNPs in the 3'UTR sequence.

[0169] In summary, the recombinant vectors (i.e., SCP1 vectors 1 to 3) provided in the examples of the present application have a higher fluorescence ratio when used for detection; they are vectors that can detect a wide range of 3'UTR sequence activity and more sensitively detect differences in SNP activity within 3'UTR.

[0170] For the nucleotide sequence shown in SEQ ID NO: 1, please refer to the sequence listing provided in this application, or refer to the sequence shown below.

[0171] SEQ ID NO: 1:

[0172]

[0173] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. Use of a plasmid as a vector for detecting the activity of a 3'UTR sequence, wherein: The plasmid has the nucleotide sequence shown in SEQ ID NO:

1.

2. Use of the plasmid in preparing a dual-luciferase recombinant vector, wherein: The plasmid has the nucleotide sequence shown in SEQ ID NO:

1.

3. Dual luciferase recombinant vector, characterized in that: include: A plasmid having a nucleotide sequence as shown in SEQ ID NO: 1; and A target recombinant sequence, wherein the target recombinant sequence includes a target gene sequence, wherein the target gene sequence includes a SNP selected from a 3'UTR sequence; Wherein, the target recombinant sequence is inserted into the plasmid.

4. The dual luciferase recombinant vector according to claim 3, characterized in that: The 3'UTR sequence is selected from the 3'UTR sequence of the pig genome.

5. The dual luciferase recombinant vector according to claim 3, characterized in that: The target gene sequence also includes the upstream and downstream sequences of the SNP, and the upstream and downstream sequences are sequences of 1 to 400 bp upstream and downstream of the SNP.

6. The dual luciferase recombinant vector according to claim 3, characterized in that: The target recombinant sequence also includes a first common sequence and a second common sequence; wherein the first common sequence is CGAGCTCGCTAGCCT; the second common sequence is AGATCGGAAGAGCGTCG; In the structure of the target recombinant sequence: the first common sequence, the target gene sequence, and the second common sequence are arranged in sequence from the 5' end to the 3' end.

7. The dual luciferase recombinant vector according to claim 3, characterized in that: The target recombinant sequence is inserted into the downstream of the firefly luciferase gene of the plasmid.

8. A transformant, characterized in that include: The dual luciferase recombinant vector according to claim 2; and The host cell is selected from Escherichia coli competent cells.

9. A transfectant, characterized in that include: The dual luciferase recombinant vector according to claim 2; and The transfected cells are selected from C2C12 cells.

10. Use of the dual luciferase recombinant vector according to claim 3 in preparing transfected cells for detecting the activity of 3'UTR sequences.

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