Method for detecting activity of insect dsRNA degrading enzyme based on dual luciferase report system

Abstract

The invention discloses a method for detecting insect dsRNA degrading enzyme activity based on a dual luciferase report system, and relates to the technical field of biological medicine. The method comprises the following steps: constructing an insect dsRNA degrading enzyme into a pAc5.1 / V5-His B plasmid to form a recombinant plasmid, co-transfecting the recombinant plasmid and a psiCHECKTM-2 plasmid into an insect cell for culturing, then respectively adding dsLuc and dsGFP into the cell, continuing culturing, finally collecting the cell, and detecting the enzyme activity of the cell. The system is constructed based on a dual-luciferase report system, and qualitative and quantitative enzyme activity is realized through three steps of cell culture, transfection and detection. Compared with in-vitro heterologous protein expression, agarose detection after in-vitro degradation reaction or RNA extraction and then fluorescent quantitative PCR (RT-qPCR) detection, the method provided by the invention is faster and more accurate, and is not limited to the first step in which the protein is difficult to obtain.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for detecting insect dsRNA degrading enzyme activity based on a dual luciferase reporter system. Background Art

[0002] RNA interference technology is now widely used in the fields of medicine, agriculture, etc., and is known as the core technology of the fourth generation of insecticides. However, when this technology is applied to the field of pest control, the most important limiting factor encountered is the dsRNA degrading enzyme of insects. Therefore, it is necessary to detect the activity of dsRNA degrading enzymes in insects. In previous studies or applications, the activity of dsRNA degrading enzymes was mainly detected by in vitro expression and purification of proteins, followed by in vitro degradation, and finally by electrophoresis detection. This method is complicated to operate and has a long cycle, and the results can only be qualitative but not quantitative. Therefore, it is urgent to construct a rapid activity screening system to quickly and accurately detect the activity of insect dsRNA degrading enzymes. Summary of the invention

[0003] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for detecting the activity of insect dsRNA degrading enzymes based on a dual luciferase reporter system. The system is constructed based on the dual luciferase reporter system and can not only be used to quickly detect the activity of insect dsRNA degrading enzymes, but also can be applied to the screening of specific inhibitors of this type of enzymes.

[0004] The technical solution of the present invention to solve the above technical problem is as follows: a method for detecting insect dsRNA degrading enzyme activity based on a dual luciferase reporter system is provided, comprising the following steps:

[0005] The insect dsRNA degrading enzyme was constructed into the pAc5.1 / V5-His B plasmid to form a recombinant plasmid. The recombinant plasmid and psiCHECK TM -2 plasmids were co-transfected into insect cells for culture, and then dsLuc and dsGFP were added to the cells respectively, and the culture was continued. Finally, the cells were collected and the dsRNA degradation enzyme activity was detected.

[0006] Furthermore, when preparing the recombinant plasmid, a negative control was also set up, specifically: the full-length 18S ribosomal RNA gene that does not degrade insect dsRNA degrading enzymes was constructed into the pAc5.1 / V5-His B plasmid.

[0007] Furthermore, the primers used to amplify the full length of the 18S ribosomal RNA gene are:

[0008] F: 5'-CGGGGTACCTGATGGGACGAGTGCTTTTATT-3';

[0009] R: 5'-CCGCTCGAGAATGACGACGCCTATTTAGCAG-3'.

[0010] Furthermore, the recombinant plasmid and psiCHECK TM -2 plasmid was co-transfected into insect cells and cultured for 24 h.

[0011] Furthermore, dsLuc and dsGFP were added to the cells respectively, and the cells were cultured for 24 h.

[0012] Furthermore, the primer sequences used to synthesize dsLuc are:

[0013] F: 5'-taatacgactcactataggggTTCGCTAAGAGCACCCTGAT-3';

[0014] R: 5'-taatacgactcactatagggGCTGCAGGATAGACTCC-3'.

[0015] Furthermore, the primer sequences used to synthesize dsGFP are:

[0016] F: 5'-taatacgactcactatagggGACGTAAACGGCCACAAGTT-3';

[0017] R: 5'-taatacgactcactatagggTGTTCTGCTGGTAGTGGTCG-3'.

[0018] Furthermore, the fluorescence data were obtained after the enzyme activity was detected, and the fluorescence difference between the addition of dsLuc and the addition of dsGFP was statistically analyzed, and then the fluorescence rebound value was compared with the negative control group.

[0019] The present invention has the following beneficial effects:

[0020] The system of the present invention is not limited to protein expression and purification. The first step of the traditional method is to express and purify the protein. Not all genes can successfully obtain their proteins in vitro. If not, the next step will not be able to proceed. The system of the present invention does not require in vitro protein expression, but only requires extraction of total cell protein.

[0021] The method of the present invention can quantify the activity of insect dsRNA degrading enzymes. In the traditional method, the purity of the protein, the error in the in vitro degradation experiment operation process, and the observation of the results by agarose gel electrophoresis will affect the experimental results and cannot be quantified. Even if the enzyme activity is quantified by extracting RNA and performing fluorescent quantitative PCR, it will be affected by the in vitro degradation experiment error, RNA extraction error, etc.

[0022] The method of the present invention can be used to detect the ability of different dsRNA degrading enzymes of the same or different insect species, the activity of different mutants of the same enzyme, and the inhibitory effect of inhibitors on specific dsRNA degrading enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results are as follows: the activities of four dsRNA degrading enzymes of locusts were detected using the system of the present invention.

[0024] Figure 2 The results of RT-qPCR analysis of the residual amount of dsGFP in cells. DETAILED DESCRIPTION

[0025] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] Embodiment 1:

[0027] In order to quickly detect the activity of insect dsRNA degrading enzymes, the present invention constructs an insect dsRNA degrading enzyme activity screening system based on the dual luciferase reporter system. The operation process of the system is as follows:

[0028] Step 1: construct the dsRNA degrading enzyme into the pAc5.1 / V5-His B plasmid;

[0029] Step 2: Extraction of pAc5.1-dsRNase and psiCHECK TM -2 plasmid, transfected into Drosophila S2 cells;

[0030] Step 3: Synthesize firefly luciferase (Luc) dsRNA (dsLuc) and green fluorescent protein dsRNA (dsGFP), add 2 μg of dsGFP or dsLuc to the S2 cells transfected 24 hours after the previous step in the control group and the test group, respectively;

[0031] Step 4: After 24 hours, the cells were collected and the enzyme activity was detected using a micro-well multifunctional detector;

[0032] Step 5: Statistical analysis.

[0033] The present invention uses the above method to detect the activities of four dsRNA degrading enzymes of locusts, and the specific process is as follows:

[0034] (I) Constructing the dsRNA degrading enzyme into the pAc5.1 / V5-His B plasmid to obtain the recombinant plasmid pAc5.1-dsRNase

[0035] There are four nucleic acid degrading enzymes (LmdsRNase) in the migratory locust, which are respectively recorded as LmdsRNase1 / 2 / 3 / 4 (the GenBank accession numbers of LmdsRNase1-4 are KY386893, KY274845, KY274844 and KY386894, respectively). Primers were designed based on these four dsRNA degrading enzymes for PCR amplification. The amplification system was: 2×Taq PCR MasterMix 25μL, template 2μL, upstream and downstream primers 1μL each, and ddH2O was added to 50μL; the amplification program was: pre-denaturation at 94℃ for 2min, denaturation at 98℃ for 10s, annealing at 60℃ for 30s, 35 cycles, and finally extension at 68℃ for 1min.

[0036] The above primer sequences are as follows:

[0037] dsRNase1:

[0038] F: 5'-AAATATGCGGCCGCATGTGGACCAGAGGTC-3' (SEQ ID NO. 1);

[0039] R: 5'-GCTCTAGAGCGAGAGTCGGCAGAGAG-3' (SEQ ID NO. 2);

[0040] dsRNase2:

[0041] F: 5'-AAATATGCGGCCGCATGGCCTCCCTACCGCTG-3' (SEQ ID NO. 3);

[0042] R: 5'-GCTCTAGAGCGGTAAGCAGAGAGCTAAC-3' (SEQ ID NO. 4);

[0043] dsRNase3:

[0044] F: 5'-AAATATGCGGCCGCATGCCGTCGCCGCAGC-3' (SEQ ID NO. 5);

[0045] R: 5'-GCTCTAGAGCGGCTCCCGTGAGAGCTAC-3' (SEQ ID NO. 6);

[0046] dsRNase4:

[0047] F: 5'-AAATATGCGGCCGCATGGAGCGGACTGCAGCA-3' (SEQ ID NO.7);

[0048] R: 5'-GCTCTAGAGCGTTATAAGACAGTCGCAA-3' (SEQ ID NO. 8).

[0049] The amplified product was inserted into the pAc5.1 / V5-His B plasmid vector by double enzyme digestion. The enzyme digestion system was as follows: 1 μg of vector or target fragment; 1 μL of endonuclease Not I and Xba I; 5 μL of 10×Cut buffer, and ddH2O was added to 50 μL. The reaction conditions were 37°C for 2 hours.

[0050] The digested products were connected, and the connection system was as follows: 1 μL of the vector recovered by digestion; 3 μL of the target fragment recovered by digestion; 0.5 μL of T4 ligase; 2 μL of 10×T4 buffer; add ddH2O to 20 μL. The reaction conditions were 4°C and 12 h.

[0051] The ligation product was transformed into E. coli competent cells DH5α, cultured overnight, single colonies were picked, and stored after sequencing verification. In addition, a negative control group was designed in this system, and the full-length 18S ribosomal RNA gene that does not degrade dsRNA was constructed into the pAc5.1 / V5-His B plasmid. The primers used to amplify the full length of the gene are as follows:

[0052] F: 5'-CGGGGTACCTGATGGGACGAGTGCTTTTATT-3' (SEQ ID NO.9);

[0053] R: 5'-CCGCTCGAGAATGACGACGCCTATTTAGCAG-3' (SEQ ID NO. 10).

[0054] (ii) Extract plasmid and transfect into Drosophila S2 cells

[0055] The four recombinant plasmids obtained in step (I) were pAc5.1-dsRNase plasmids and psiCHECK TM -2 plasmid was co-transfected into Drosophila S2 cells. The specific process is as follows:

[0056] (1) When Drosophila S2 cells are growing well, the cells that have attached to a culture flask are suspended by blowing and inoculated into a 24-well plate. 1 mL of Schneider's Drosophila Medium containing 10% fetal bovine serum is added to each well, and the 24-well plate is cultured in an incubator for 12 h.

[0057] (2) Prepare solution A in an EP tube: 50 μL serum-free culture medium, 3 μL Lipofectamine 3000 Reagent;

[0058] Solution B: 50 μL serum-free medium, 4 μL P3000 Reagent, 1000 ng psiCHECK TM -2, 1000ngpAc5.1-LmdsRNase.

[0059] (3) Add the prepared solution B to solution A, mix well, and leave at room temperature for 15 minutes. Add the mixed transfection solution to the cells in the 24-well plate and place in an incubator for culture.

[0060] (III) Synthesizing dsRNA for interference experiments

[0061] The T7 RiboMAXTM Express RNAi System kit was used to synthesize dsLuc and dsGFP. The primers used to synthesize dsLuc and dsGFP are as follows:

[0062] The primer sequences used for dsLuc are as follows:

[0063] F: 5'-taatacgactcactatagggTTCGCTAAGAGCACCCTGAT-3' (SEQ ID NO. 11);

[0064] R: 5'-taatacgactcactatagggGCTGCAGGATAGACTCC-3' (SEQ ID NO. 12).

[0065] The primer sequences used for dsGFP are as follows:

[0066] F: 5'-taatacgactcactataggggGACGTAAACGGCCACAAGTT-3' (SEQ ID NO. 13);

[0067] R: 5'-taatacgactcactatagggTGTTCTGCTGGTAGTGGTCG-3' (SEQ ID NO. 14).

[0068] After culturing the S2 cells transfected with the plasmid in step (ii) for 24 h, 2 μg of dsLuc was added to each well of the 24-well plate in the experimental group, and 2 μg of dsGFP was added to the control group.

[0069] (IV) Collecting cells for enzyme activity detection

[0070] After culturing the S2 cells transfected with plasmids in the incubator for 24 hours, the cells were collected. The original culture medium in the 24-well plate was discarded, and 500 μL of 1×PBS was slowly added to the wells; PBS was discarded, 100 μL of cell lysis solution was added, and the cells were lysed by shaking at 300 rpm for 20 minutes; 20 μL of cell lysis solution was transferred to the 96-well plate dedicated to the micro-well multifunctional detector; the left substrate was configured: Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer Ⅱ. The right substrate was configured: Buffer and The substrate was configured in a ratio of 49: 1. The enzyme activity was detected by a microwell multifunctional detector to obtain the fluorescence value.

[0071] 5. Statistical analysis

[0072] The obtained fluorescence data were plotted and analyzed in Excel. First, the fluorescence difference between the dsLuc and dsGFP groups was counted, and then the fluorescence rebound value was compared with the negative control group (pAc5.1-18S group). The two fluorescence differences were subjected to t-test, p<0.05 was marked as *, p<0.01 was marked as **, and p>0.05 was marked as no significant difference as NS. The results are shown in Figure 1 .

[0073] Depend on Figure 1 It can be seen that the activities of the four dsRNA degrading enzymes of the migratory locust are different, among which dsRNA degrading enzymes 1 and 2 are active (34.5% and 34.9%), while dsRNA degrading enzymes 3 and 4 are inactive. This is consistent with the results of qualitative detection using traditional methods before, and compared with traditional methods, quantitative results are obtained.

[0074] The present invention constructs an insect dsRNA degradation enzyme activity screening system based on a dual luciferase reporter system. The plasmid psiCHECK TM -2 contains the firefly luciferase (Luc) and sea cucumber luciferase (RLuc) genes. pAc5.1-LmdsRNase2 enters S2 cells through transient transfection and is expressed in the cells. Addition of dsLuc will silence psiCHECK TM -2, if LmdsRNase2 degrades dsLuc, the RNAi efficiency of dsLuc will be reduced, and the firefly luciferase activity will rebound. In the control group, Of18S-ribosomal has no degradation effect on dsRNA. Therefore, if the luciferase activity of the experimental group rebounds compared with the control group, it proves that the dsRNA degrading enzyme in the experimental group has the activity of degrading dsRNA.

[0075] The present invention relates to a pAc5.1-LmdsRNase plasmid expressing a dsRNA enzyme and a psiCHECK containing a luciferase gene. TM -2 plasmid transfection, the present invention screened the co-transfection and separate transfection and transfection time, and the screening results showed that pAc5.1-LmdsRNase and psiCHECK TM -2 was co-transfected into S2 cells and dsRNA was added for RNA interference 24 hours later, and fluorescence detection was performed 24 hours later. This system can better detect the degradation activity of the enzyme.

[0076] Comparative Example 1:

[0077] The degradation activity of dsRNA degrading enzymes was quantitatively analyzed using RT-qPCR, and the process was as follows:

[0078] (I) Construction of recombinant plasmid pAc5.1-dsRNase (also referred to as pAc5.1-LmdsRNase), the process is the same as in Example 1

[0079] (II) Transient transfection of S2 cells

[0080] Lipofectamine 3000 kit was used to transfect cells, and psiCHECK was co-transfected in the experimental group. TM -2 and pAc5.1-dsRNase, and the control group was co-transfected with psiCHECK TM -2 and Of18S-ribosomal.

[0081] (III) RNA interference in S2 cells

[0082] Drosophila S2 cells transfected with plasmids were cultured in an incubator for 24 h, 4 μg of dsGFP was added to each well of a 12-well plate, and cells were collected after 0 h, 6 h, and 24 h.

[0083] (IV) Extraction of total RNA and synthesis of cDNA template

[0084] (V) Detecting the degradation activity of dsRNA degrading enzymes using RT-qPCR, specifically using Green Real-time PCR Master Mix kit was used, and the reaction system was: SYBR Green Real Master Mix 10 μL, primers 0.8 μL each, cDNA 4 μL, ddH2O 4.4 μL; the reaction program was: 95 °C for 10 min, 95 °C for 15 s, 55 °C for 31 s, for a total of 40 cycles.

[0085] In the RT-qPCR test, the S2 cells in the experimental group were transfected with the pAc5.1-LmdsRNase2 plasmid that has a cleavage effect on dsRNA degrading enzymes, and the control group was transfected with the pAc5.1-Of18S-ribosomal plasmid that does not have dsRNA cleavage activity. The transfected S2 cells were cultured in an incubator for 24 hours, and then dsGFP was added. The cells were collected at 0h, 6h, and 24h after the addition of dsRNA. The total cell RNA was first extracted, then reverse transcribed, and finally the residual amount of dsGFP was quantitatively detected by RT-qPCR. The experimental results are shown in Figure 2 .Depend on Figure 2 It can be seen that there is no significant difference between the residual dsGFP in the experimental group and the residual dsGFP in the control group, which shows that it is not feasible to use the RT-qPCR method to construct an activity screening system for dsRNA degrading enzymes.

[0086] In summary, the present invention has established a system for rapid screening of insect dsRNA degrading enzyme activity, preparing for the subsequent development of RNA biopesticides. This system is constructed based on a dual luciferase reporter system, and the qualitative and quantitative analysis of the enzyme activity is achieved through three steps of cell culture, transfection, and detection. Compared with in vitro heterologous protein expression, after in vitro degradation reaction, agarose detection or RNA extraction and then fluorescence quantitative PCR (RT-qPCR) detection is faster and more accurate, and is not limited to the first step of difficult protein acquisition.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting insect dsRNA degrading enzyme activity based on a dual luciferase reporter system, characterized in that the following step: The insect dsRNA degrading enzyme was constructed into the pAc5.1 / V5-His B plasmid to form a recombinant plasmid. The recombinant plasmid and psiCHECK TM -2 plasmids were co-transfected into insect cells for culture, and then dsLuc and dsGFP were added to the cells respectively, and the culture was continued. Finally, the cells were collected and the dsRNA degradation enzyme activity was detected.

2. The method for detecting insect dsRNA degrading enzyme activity based on the dual luciferase reporter system according to claim 1, characterized in that: When preparing the recombinant plasmid, a negative control was also set up, specifically: the full-length 18S ribosomal RNA gene that does not degrade insect dsRNA degrading enzymes was constructed into the pAc5.1 / V5-His B plasmid.

3. The method for detecting insect dsRNA degrading enzyme activity based on the dual luciferase reporter system according to claim 2, characterized in that: The primers used to amplify the full length of the 18S ribosomal RNA gene are: F: 5'-CGGGGTACCTGATGGGACGAGTGCTTTTATT-3'; R: 5'-CCGCTCGAGAATGACGACGCCTATTTAGCAG-3'.

4. The method for detecting insect dsRNA degrading enzyme activity based on the dual luciferase reporter system according to claim 1, characterized in that: The recombinant plasmid and psiCHECK TM -2 plasmid was co-transfected into insect cells and cultured for 24 h.

5. The method for detecting insect dsRNA degrading enzyme activity based on the dual luciferase reporter system according to claim 1, characterized in that: After adding dsLuc and dsGFP to the cells, the cells were cultured for 24 h.

6. The method for detecting insect dsRNA degrading enzyme activity based on the dual luciferase reporter system according to claim 1 or 5, characterized in that: The primer sequences used to synthesize dsLuc are: F: 5'-taatacgactcactataggggTTCGCTAAGAGCACCCTGAT-3'; R: 5'-taatacgactcactatagggGCTGCAGGATAGACTCC-3'.

7. The method for detecting insect dsRNA degrading enzyme activity based on the dual luciferase reporter system according to claim 1 or 5, characterized in that: The primer sequences used to synthesize dsGFP are: F: 5'-taatacgactcactatagggGACGTAAACGGCCACAAGTT-3'; R: 5'-taatacgactcactatagggTGTTCTGCTGGTAGTGGTCG-3'.

8. The method for detecting insect dsRNA degrading enzyme activity based on the dual luciferase reporter system according to claim 1, characterized in that: After detecting the enzyme activity, the fluorescence data was obtained. The fluorescence difference between adding dsLuc and adding dsGFP was statistically analyzed, and then the fluorescence rebound value was compared with the negative control group.

Images