CN114480496B

CN114480496B - Double-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application

Info

Publication number: CN114480496B
Application number: CN202210168172.4A
Authority: CN
Inventors: 耿少雷; 王天云; 赵小杰; 孙秋丽; 米春柳; 樊振林; 曹祥祥
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2022-02-23
Filing date: 2022-02-23
Publication date: 2024-05-07
Anticipated expiration: 2042-02-23
Also published as: CN114480496A

Abstract

The invention belongs to the field of reporter gene vectors, and particularly relates to a double-luciferase reporter gene vector for insect cells, a construction method, a recombinant vector and application thereof. The double luciferase reporter gene vector for the insect cells is obtained by modifying a psiCHECK-2 vector; the engineering includes replacing the original SV40 promoter with OpIE promoter and inserting a synthetic DNA fragment between HSV-TK promoter and SYNTHETIC POLY (A); downstream of the hRluc gene of the psiCHECK-2 vector for insertion of an insect target gene of interest; the nucleotide sequence of the synthesized DNA fragment is shown as SEQ ID NO: 1. The double luciferase reporter gene vector for the insect cells provided by the invention is a powerful research tool for gene transcription and mRNA processing in the insect cells, and is suitable for research of insect gene functions.

Description

Double-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application

Technical Field

The invention belongs to the field of reporter gene vectors, and particularly relates to a double-luciferase reporter gene vector for insect cells, a construction method, a recombinant vector and application thereof.

Background

Insects are the most huge organisms in the world, are numerous and have strong environmental adaptability. The material is easy to obtain, the gene operation is simple, the genetic phenotype is obvious, and the like, so that the gene is favored by researchers and plays an important role in various aspects such as biological medicine, insect pesticides, basic research and the like. With the continuous development of molecular biology technology, more than 800 insect cell lines are established, and are commonly used for research of immunofluorescence, protein interaction, small molecule RNA on target gene regulation and the like.

MiRNAs are non-coding RNA molecules of about 22 nucleotides (nt), which degrade target mRNA or repress translation, and regulate gene expression at posttranscriptional levels in nearly all biological processes. Untranslated region (Untranslated Region, UTR) refers to a fragment that is not translated into a protein at both ends of the coding region of the mRNA strand, and the fragment of mRNA that is located upstream of the coding region is referred to as the 5 'untranslated region (5' -untranslated region,5 '-UTR) and downstream of the coding region is referred to as the 3' untranslated region. Numerous studies have shown that miRNAs induce mRNA degradation or translational silencing by complementarity with the sequence of the 3'-UTR, 5' -UTR, or genome of the target mRNA, and are demonstrated to be evolutionarily conserved during major evolutionary processes in eukaryotes. In recent years, a large number of mirnas have been identified and annotated from eukaryotic organisms including insects. They are thought to be modulators of many biological processes, including cell proliferation differentiation, metabolism, viral infection, cell cycle progression, anti-stress and life prolongation. Potential mirnas or target genes are typically screened by a dual luciferase reporter system.

The luciferase reporter gene system refers to a method for indirectly screening potential regulatory factors affecting the expression of a target gene and quantifying the regulatory activity thereof by quantifying the bioluminescence emitted by luciferase-catalyzed oxidation of luciferin to oxyluciferin. In insect cells, the commonly used luciferase reporter assay is mainly implemented by a cotransfection method, i.e., by a method of transfecting a recombinant luciferase reporter plasmid and an internal reference reporter. If the target promoter sequence is cloned into a PGL3-Basic vector, the activation of the gene promoter by the endogenous transcription factor is investigated by cotransmitting an internal reference plasmid TK. Similarly, when exploring the targeting relationship between 3'/5' -UTR and miRNA, three vectors, namely miRNA over-expression vectors (chemicals), recombinant luciferase reporter vectors containing 3'/5' -UTR gene sequences, and internal reference vectors, were required to be co-transferred. The cotransfer experiment had the following drawbacks: first, the full length 3' -UTR of most genes varies in length from several hundred to several thousand bases, and recombinant reporter vectors are constructed. In the co-transfection system, the size and the addition ratio of each vector can have serious influence on the transfection efficiency of the respective vector, and the accuracy of experimental results is likely to be influenced. Second, co-transfection of multiple vectors increases the amount of transfection reagent used, enhances cell stress and toxic effects, and the parameters detected are often not a reflection of the values in the normal physiological state of the cell.

The Chinese patent with the publication number of CN111440823B researches the regulation mechanism of the Ccdc124 gene and miRNA thereof by constructing a recombinant vector comprising the double luciferase reporter gene of the Ccdc124 gene of the brown planthopper. The double luciferase reporter gene vector used is Psicheck-2 vector, and the cell line used is Drosophila S2 cell (embryo cell). In strict sense, drosophila S2 cells are model species cells, have strong compatibility, and the physicochemical properties, common vectors and expression elements of the Drosophila S2 cells are more similar to those of higher animal cells; in addition, most of the technical approaches for S2 cell lines are not applicable in conventional insect cell lines.

In the prior art, the Psicheck-2 vector is designed, and the change of gene expression can be monitored by fusing firefly and sea cucumber luciferase reporter genes, but the vector has little activity in insect cells due to the specific reason of promoter species, and can not normally start the expression of luciferase genes on the vector, so that the vector has limited use in the insect cells.

Disclosure of Invention

The invention aims to provide a double-luciferase reporter gene vector for insect cells, which is designed aiming at the insect cells, ensures the transcription and mRNA processing of target genes, and is suitable for detecting the targeting relationship between an untranslated region of an insect gene 3' and miRNA.

The second object of the invention is to provide a method for constructing the double-luciferase reporter gene vector.

The third object of the present invention is to provide a recombinant vector based on the above-mentioned dual luciferase reporter vector.

It is a fourth object of the present invention to provide the use of the recombinant vector described above.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

A double luciferase reporter gene vector for insect cells, which is obtained by modifying a psiCHECK-2 vector; the engineering includes replacing the original SV40 promoter with OpIE promoter and inserting a synthetic DNA fragment between HSV-TK promoter and SYNTHETIC POLY (A); downstream of the hRluc gene of the psiCHECK-2 vector for insertion of an insect target gene of interest; the nucleotide sequence of the synthesized DNA fragment is shown as SEQ ID NO: 1.

The double luciferase reporter gene vector for insect cells provided by the invention comprises OpIE promoter, transcription terminator SV40 polyA late signal, synthetic DNA element sequence, sea cucumber and firefly luciferase reporter gene and resistance marker ampicillin. Wherein, the combination of OpIE2 and SV40 poly A ensures that the gene of interest is transcribed while ensuring that transcription termination is tightly regulated; the synthetic DNA element can attenuate the effect of OpIE promoter on firefly luciferase, ensuring powerful gene transcription and mRNA processing in insect cells.

Preferably, the nucleotide sequence of the OpIE promoter is shown in SEQ ID NO: 2. Further preferably, the OpIE promoter fragment is obtained by PCR amplification of pIZ/V5-His vector. Experiments prove that OpIE promoter matched with SV40 polyA late signal can ensure that target genes are transcribed in insect cells, and simultaneously ensure that transcription termination is strictly regulated.

Still further preferably, the upstream primer and the downstream primer of the PCR amplification are respectively set forth in SEQ ID NO: 3. SEQ ID NO: 4.

The technical scheme of the construction method of the double-luciferase reporter gene vector is as follows:

The construction method of the double-luciferase reporter gene vector for the insect cells comprises the following steps: after double cleavage of psiCHECK-2 plasmid by Kpn I and Nhe I, cleavage products are recovered, opIE gene fragment is ligated to the cleavage products, and the synthesized DNA fragment is then ligated between HSV-TK promoter and SYNTHETIC POLY (a).

The construction method of the double-luciferase reporter gene vector for the insect cells is simple and convenient to operate and high in stability, and can be used for researching the specific regulation mechanism of the insect miRNA on the target gene 3' UTR in the cells.

The technical scheme of the insect cell double luciferase reporter gene recombinant vector is as follows:

an insect cell double-luciferase reporter gene recombinant vector is characterized in that an insect target gene is inserted into the downstream of the hRluc gene of the double-luciferase reporter gene vector.

The double-luciferase reporter gene recombinant vector provided by the invention is used for detecting the targeting relationship between a target gene 3' untranslated region and miRNA in insect cells; the whole detection process is short in time consumption, good in repeatability and high in accuracy.

Preferably, the insect target gene is an insect gene 3' utr sequence.

Further preferably, the insect gene 3' utr sequence is selected from the trehalase gene 3' utr sequence or the glucose dehydrogenase gene 3' utr sequence. Experiments prove that the double-luciferase reporter gene vector can be used for detecting the targeting relationship between miRNA and the target genes, so that the double-luciferase reporter gene vector is used for researching related regulation and control mechanisms.

The application of the insect cell double-luciferase reporter gene recombinant vector in the aspect of detecting the targeting relationship between the insect gene 3' UTR and miRNA.

The double-luciferase reporter gene vector can be used for researching a specific regulation mechanism of insect miRNA on target gene 3' UTR in cells; the method can also be used for screening candidate miRNA to predict target genes and researching the regulation mechanism of the miRNA to regulate and predict the target genes at the cellular level.

Preferably, the insect gene 3' UTR is trehalase gene 3' UTR sequence or glucose dehydrogenase gene 3' UTR sequence, and the miRNA is miRNA-317-5p or miRNA-34-5p-2.

Drawings

FIG. 1 is a prior art plasmid map of the vector psiCHECK-2;

FIG. 2 is a map of a vector pIZ/V5-His plasmid according to the prior art;

FIG. 3 is a map of a vector pPIZ/3UTR constructed in accordance with the present invention;

FIG. 4 is a diagram showing the analysis of pPIZ/3UTR dual-fluorescein reporter gene activity in the examples of the present invention; (A) The bar graph sequentially comprises No Mimic, control, miRNA-317-5P 100nM, miRNA-317-5P 200nM from left to right; (B) The bar graph sequentially comprises No Mimic, control, miRNA-34-5p-2 nM, miRNA-317-5P 200nM from left to right; * Represents p <0.01; n.s. stands for p >0.05.

Detailed Description

The Psicheck-2 vector can be designed to rapidly and quantitatively detect the regulation and control effect of miRNA on target genes. In this vector, the change in gene expression is monitored by fusing firefly and sea cucumber luciferase reporter genes, which are the primary reporter genes, with the exogenous gene of interest cloned downstream. When miRNA is combined with a target gene, the regulation and control function is started, and the luciferase activity of the sea cucumber is correspondingly changed. The vector realizes the expression of the exogenous fragment and the internal reference gene on the same vector, and has been widely applied to research of RNAi, target gene 3' -UTR and miRNA targeting relationship in mammalian cells. However, the psicheck-2 vector is not functional in insect cells due to the specificity of the promoter species, limiting the use of this vector.

In the invention, a primer OpIE, a primer1 and a primer OpIE, namely primer2 are designed, a complete segment of a OpIE promoter is obtained through amplification, and is connected into a psiCheck-2 vector through double enzyme digestion to replace the original vector partial sequence, and meanwhile, a synthetic DNA original sequence is inserted to construct a double luciferase reporter gene vector pPIZ/3UTR.

The main function of the synthesized DNA fragment is to isolate the influence of the promoter on hluc reporter genes. If the fragment is not inserted, hluc reporter gene detection values will be too high. A high value of this number directly affects the evaluation of whether the miRNA has an inhibitory effect on the target site. Especially when the binding of miRNA to the target site is weak, the miRNA can be covered up, and the result is affected.

The double-luciferase reporter vector obtained by the method reduces the number of vectors during cotransfection, improves the transfection efficiency, has good experimental repeatability, and can be used for detecting the targeting relationship between a 3' untranslated region and miRNA compared with the traditional luciferase reporter plasmid, and the method can greatly help to accelerate the research process of miRNA on a target gene regulation mechanism in insect cells.

Specifically, the OpIE promoter gene fragment digested with the restriction endonuclease was ligated into the double luciferase reporter vector digested with the same restriction endonuclease by ligase, and OpIE/3 UTR was selected. Further, the double luciferase reporter gene vector of the 3' -untranslated region of the insect gene is inserted with a synthetic DNA sequence, such as SEQ ID NO: 1. Further, the fragment was seamlessly cloned to connect the sequence of the synthesized DNA fragment between HSV-TK promoter and SYNTHETIC POLY (A) in the vector. The vector was designated pPIZ/3UTR.

Further, the OpIE promoter gene fragment was obtained by PCR amplification using primers comprising: the primer is OpIE primer (1/2), and contains restriction sites of Kpn I and Nhe I restriction enzymes respectively.

Further, the dual-luciferase reporter vector is psiCHECK-2 subjected to Kpn I and Nhe I restriction enzyme dual-cleavage.

Further, the ligase is T4 DNA ligase provided that ligation is performed at 16℃for 3 hours or more.

The targeting relationship between insect miRNAs and the gene 3'-UTR can be detected by using the insect gene 3' UTR double-luciferase reporter gene vector. In the following examples, the regulatory mechanisms of miRNAs (miRNA-317-5 p and miRNA-34-5 p-2) on insect trehalase and glucose dehydrogenase genes were verified using the above-described dual-luciferase reporter gene vector.

The following describes the practice of the invention in detail with reference to specific examples.

Example 1 double luciferase reporter vector and construction procedure thereof

The double luciferase reporter gene vector of the insect gene 3' UTR of the embodiment is obtained by modifying a psiCHECK-2 vector, and specifically comprises the steps of replacing an original SV40 promoter by a OpIE promoter and inserting a synthesized DNA fragment. The sequence of the synthesized DNA fragment is connected between HSV-TK promoter and SYNTHETIC POLY (A) of the psiCHECK-2 vector; the insect gene 3' UTR sequence is inserted into the hRluc downstream of psiCHECK-2 vector; the nucleotide sequence of the synthesized DNA fragment is shown as SEQ ID NO: 1. OpIE2 promoter sequence is shown in SEQ ID NO: 2.

The above double luciferase reporter gene vector (pPIZ/3 UTR) is constructed by selecting the vector pIZ/V5-His and the vector psiCHECK-2, and the construction method is as follows:

(1) Plasmid maps of the vectors psiCHECK-2 and pIZ/V5-His are shown in fig. 1 and 2. According to pIZ/V5-His vector sequence analysis, primer OpIE primer1 and OpIE primer2 of OpIE2 fragment were synthesized, kpn I and Nhe I restriction sites and protecting bases were added to the upstream and downstream primers of the gene, respectively, and primer oligo sequences of OpIE2 promoter gene amplification are shown in the following Table 1.

TABLE 1 OpIE2 promoter Gene amplification primer sequences

OpIE2primer1(SEQ ID NO：3)	GGGGTACCTCATGATGATAAACAATGTATGGTGCTAATG
		OpIE2primer2(SEQ ID NO：4)	CTAGCTAGC TAAATTCGAACAGATGCTGTTCAACGTT

The primer is synthesized by Hongsun biotechnology limited company; the primer dry powder synthesized in Table 1 was prepared into 10. Mu.M working solution, and PCR amplification was performed, and the reaction system was as follows in Table 2:

TABLE 2 PCR amplification reaction System

PCR Mix	10μL
		OpIE2primer1	1μL
OpIE2primer2	1μL
		DNA template	1μL
ddH₂O	7μL

The amplification procedure is shown in table 3 below:

TABLE 3 PCR amplification procedure

After the PCR amplification reaction was completed, opIE fragment was recovered by Agarose electrophoresis and gel cutting. The OpIE gene fragment and vector were digested with KpnI and Nhe I, respectively, at 37℃for 3 hours, and the double digestion system was as shown in Table 4 below:

Table 4 double enzyme digestion System

10xBuffer	5μL
		DNA fragment/vector	25μL/1μg
KpnI	2μL
		Nhe I	2μL
ddH₂O	Make up to 50 mu L

Electrophoresis, and recovering OpIE gene fragment and carrier psiCHECK-2 by using DNA gel recovery kit; the OpIE gene fragment obtained by double digestion was ligated with the linearized vector psiCHECK-2 using T4 DNA LIGASE for more than 3 hours at 16℃and the ligation system was as follows in Table 5:

table 5 connection system

10xBuffer	2μL
		psiCHECK-2	2μL
DNA fragment	8μL
		T4 DNA ligase	0.5μL
ddH₂O	Make up to 20 mu L

Transforming the ligation product into DH5 alpha competent cells; picking clone colony from the plate, extracting plasmid and identifying to pick positive clone; sequencing verifies that the vector is named OpIE2/3UTR.

(2) The synthesized DNA fragment (SEQ ID NO: 2) was connected between HSV-TK promoter and SYNTHETIC POLY (A) in OpIE/3 UTR vector by seamless cloning, positive monoclonal colonies were picked and sequenced for verification, the vector was named pPIZ/3UTR, and the vector map is shown in FIG. 3.

Example 2 recombinant vector and application experiments

The insect cell double luciferase reporter gene recombinant vector and the application of the embodiment are specifically described as follows:

2.1 construction of recombinant vectors

The pPIZ/3UTR vector is respectively connected with 3' UTR sequences of insect trehalase gene and glucose dehydrogenase gene to construct recombinant plasmids pPIZ/3UTR-Tre and pPIZ/3UTR-GDH.

(1) 3' UTR sequence:

The sequence of the trehalase gene 3' UTR is shown in SEQ ID NO: shown at 5.

The glucose dehydrogenase gene 3' UTR sequence is shown in SEQ ID NO: shown at 6.

(2) And (3) constructing a carrier:

the construction methods of recombinant plasmids pPIZ/3UTR-Tre and pPIZ/3UTR-GDH were similar to those in example 1, and trehalase gene and glucose dehydrogenase gene 3' UTR fragments were obtained by PCR amplification, respectively. The fragments were then digested and ligated into pPIZ/3UTR vectors, respectively, with the restriction enzymes Pme I (F) and Not I (R).

2.2 Cell transfection experiments

Sf9 cells with good growth status were seeded at a density of 5 x 10 ⁴ into 96-well plates and the prepared recombinant dual luciferase reporter vector was co-transfected with MIRNA MIMICS (100/200 nM) or negative control (200 nM) following transfection reagent instructions.

Human synthesis miR-317-5p：5-CGGGUGCCACGCUGUGCUCUCU-3(SEQ ID NO：7);miR-34-5p：5-UGGCAGUGUGGUUAGCUGGUUGUG-3(SEQ ID NO：8) and NC controls, nucleic acid sequences were as follows, NC controls: 5-GCGACGAUCUGCCUAAGAUTT-3 (SEQ ID NO: 9), MIRNA MIMICS was synthesized by Shanghai Ji Ma pharmaceutical technologies Co., ltd.

The method comprises the following specific steps:

(1) The Sf9 cells are proliferated to 80-90%, washed for 3 times by PBS, added with a complete culture medium, blown uniformly, inoculated in 96-well plates, and cultured for 24 hours with 0.1mL per well; miR-317-5p or miRNA-34-5p-2mimics is diluted to 10. Mu.M.

(2) Mu.L of Grace medium and 10ng pPIZ/3UTR-Tre or pPIZ/3UTR-GDH vector were added to a 1.5mL centrifuge tube. Then, negative control, 100, 200nM (final concentration) single strand miR-317-5p/miRNA-34-5p-2mimics were added sequentially, and a new 1.5mL centrifuge tube was added with 20. Mu.l Grace medium and 2. Mu.lHD transfection reagent, mix evenly separately, stand for 5min, mix the above-mentioned two kinds of components subsequently, stand for 15min again at room temperature; the old culture medium in the plate hole is replaced by a new antibiotic-free culture medium, the well-standing transfection system is gently dripped into the plate hole, the plate hole is gently shaken, and the plate hole is replaced by a complete culture medium after 5 hours.

Each set of experiments was repeated 3 times. Transfected cells were grouped as follows: ① No mic group: transfection pPIZ/3UTR-Tre or pPIZ/3UTR-GDH; ② (-) Control group: co-transfection pPIZ/3UTR-Tre+NC or pPIZ/3UTR-GDH+NC (negative control); ③ 100 group 100: co-transfection pPIZ/3UTR-Tre+100nM mimics or pPIZ/3UTR-GDH+100nM mimics; ④ 200 group 200: co-transfection pPIZ/3UTR-Tre+ nM mimics or pPIZ/3UTR-GDH+ nM mimics.

2.2 Dual luciferase Activity assay and statistical analysis

Cells were collected 48h after transfection and tested for intracellular dual luciferase reporter activity. Each set of experiments was repeated 3 times.

The method comprises the following specific steps: removing old cell culture medium, washing with PBS for 2 times, adding 100 μL of 1 XPLB (lysis buffer), and shaking at room temperature for 30min to allow cells to be fully lysed; 50 μl of the lysate and 100uL LARII (luciferase detection Reagent II) are added into an ELISA plate, after mixing uniformly, the activity of firefly luciferase is detected, after detection, 100 μl of Stop & Glo Reagent is rapidly added into a raw tube, after mixing uniformly, the luciferase activity of sea cucumber is detected, and the ratio of the two is calculated.

All statistical analyses, p <0.05 was significant. All statistical significance was analyzed by one-way ANOVA using SPSS software and each set of experiments was repeated 3 times. * Represents p <0.01; n.s. stands for p >0.05.

As can be seen from the results of the dual luciferase activity assay of FIG. 4, the Sf9 cell group luciferase activity of the co-transformed MIRNA MIMICS group was significantly reduced by about 50% (P < 0.01) compared with the other groups, and there was a statistically significant difference between the control groups.

<110> New Country medical college

<120> Double luciferase reporter gene vector for insect cells, construction method, recombinant vector and application

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 368

<212> DNA

<213> Artificial sequence

<221> Synthetic DNA fragment

<400> 1

ctcccctgtg aggaactact gtcttcacgc agaaagcgtc tagccatggc gttagtatga 60

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tgagtacacc ggaattgcca ggacgaccgg gtcctttctt ggatcaatcc cgctcaatgc 180

ctggagattt gggcgtgccc ccgcgagact gctagccgag tagtgttggg tcgcgaaagg 240

ccttgtggta ctgcctgata gggtgcttgc gagtgccccg ggaggtctcg tagaccgtgc 300

accatgagca caaatcctaa acctcaaaga aaaaccaaac gtaacaccaa ccgccgccca 360

caggacgt 368

<210> 2

<211> 559

<212> DNA

<213> Moth nuclear polyhedrosis virus (Orgyia pseudotsugata multiple nucleopolyhedrovirus)

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<400> 2

ggatcatgat gataaacaat gtatggtgct aatgttgctt caacaacaat tctgttgaac 60

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tcgtagacta ttttacataa atagtctaca ccgttgtata cgctccaaat acactaccac 240

acattgaacc tttttgcagt gcaaaaaagt acgtgtcggc agtcacgtag gccggcctta 300

tcgggtcgcg tcctgtcacg tacgaatcac attatcggac cggacgagtg ttgtcttatc 360

gtgacaggac gccagcttcc tgtgttgcta accgcagccg ggacgcaact ccttatcgga 420

acaggacgcg cctccatatc agccgcgcgt tatctcatgc gcgtgaccgg acacgaggcg 480

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cagcatctgt tcgaattta 559

<210> 3

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ctagctagct aaattcgaac agatgctgtt caacgtt 37

<210> 5

<211> 419

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aattcttcca tttactgcgg cggggatctc ctccggactg tgcagctcgc tagaatattt 60

cccgactcga agactttcgt tgatctgaga cttgcaaaca gcgaaattga aactttggcc 120

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gatatcatca gtatatggcc aggactcggc aggaaagtgt cgccagatgt tcatcgtaac 360

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<210> 6

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<213> Cotton bollworm (Helicoverpa armigera)

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ctgtggaagt caggaacagc tttggtggaa ctgagaccat ttttgctaac aatgagatta 60

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<210> 7

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<213> Artificial sequence

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<400> 7

cgggugccac gcugugcucu cu 22

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<213> Artificial sequence

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<400> 8

uggcagugug guuagcuggu ugug 24

<210> 9

<211> 21

<212> RNA

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gcgacgaucu gccuaagaut t 21

Claims

1. The double luciferase reporter gene vector for the insect cells is characterized in that the double luciferase reporter gene vector is obtained by modifying a psiCHECK-2 vector; the modification comprises replacing the original SV40 promoter with OpIE promoter and inserting a synthetic DNA fragment between the upstream of HSV-TK promoter and the downstream of hRluc gene; downstream of the hRluc gene of the psiCHECK-2 vector for insertion of an insect target gene of interest; the nucleotide sequence of the synthesized DNA fragment is shown as SEQ ID NO: 1.

2. The dual luciferase reporter vector for insect cells according to claim 1, wherein the OpIE promoter has a nucleotide sequence as set forth in SEQ ID NO: 2.

3. The dual luciferase reporter vector for insect cells according to claim 2, wherein the OpIE promoter fragment is obtained by PCR amplification of pIZ/V5-His vector.

4. The dual luciferase reporter vector for insect cells according to claim 3, wherein the upstream primer and the downstream primer of the PCR amplification are respectively set forth in SEQ ID NO: 3. SEQ ID NO: 4.

5. A method for constructing a double luciferase reporter vector for insect cells according to any one of claims 1 to 4, comprising the steps of: after double cleavage of psiCHECK-2 plasmid by Kpn I and Nhe I, cleavage products are recovered, opIE gene fragment is ligated to the cleavage products, and the synthesized DNA fragment is then ligated between HSV-TK promoter and SYNTHETIC POLY (a).

6. An insect cell double luciferase reporter recombinant vector comprising an insect target gene inserted downstream of the hRluc gene of the double luciferase reporter vector according to any one of claims 1 to 4.

7. The insect cell dual luciferase reporter gene recombinant vector according to claim 6, wherein the insect target gene is an insect gene 3' utr sequence.

8. The insect cell dual luciferase reporter gene recombinant vector according to claim 7, wherein said insect gene 3' utr sequence is selected from the group consisting of trehalase gene 3' utr sequence and glucose dehydrogenase gene 3' utr sequence.

9. Use of the insect cell dual-luciferase reporter recombinant vector according to any one of claims 6-8 for detecting targeting relationship of insect gene 3' UTR and miRNA.

10. The use of claim 9, wherein the insect gene 3' UTR is a trehalase gene 3' UTR sequence or a glucose dehydrogenase gene 3' UTR sequence, and the miRNA is miRNA-317-5p or miRNA-34-5p-2.