CN110940807A

CN110940807A - Method for researching expression of bombyx mori gene for inhibiting BmNPV DNA replication related gene

Info

Publication number: CN110940807A
Application number: CN201911086586.7A
Authority: CN
Inventors: 夏定国; 张园; 赵巧玲; 张业顺
Original assignee: Jiangsu University of Science and Technology
Current assignee: Jiangsu University of Science and Technology
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2020-03-31

Abstract

The invention discloses a method for researching the expression of bombyx mori genes for inhibiting BmNPV DNA replication related genes, which comprises the following steps: (1) designing a primer for synthesizing cloning according to steps required by plasmid cloning; (2) constructing a recombinant plasmid pFastBacDual-polyhedrin-A3-Bmlispase-1 by the recombinant plasmid; (3) constructing recombinant viruses, namely transforming E.coli DH10Bac delta EGT cells by using the recombinant plasmids prepared in the step (2) to construct recombinant Bacmid; (4) verifying and verifying the influence of the recombinant Bacmid prepared in the step (3) on the BmNPV DNA replication related gene; verifying the expression quantity change of DBP; the EMSA method verifies the interaction between the protein and the nucleic acid; analyzing the dbp promoter activity of the deleted Bmlispase-1 binding site. Compared with the prior art, the invention has the following advantages: the invention analyzes the influence of Bmlispase-1 on virus replication related genes in cells on the basis of constructing recombinant viruses capable of expressing the Bmlispase-1, and researches the action site of the Bmlispase-1 by using EMSA, thereby clarifying the mechanism of the BmlnpV replication and proliferation inhibition of the Bmlispase-1.

Description

Method for researching expression of bombyx mori gene for inhibiting BmNPV DNA replication related gene

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a method for researching a mechanism for inhibiting BmNPV amplification by silkworm virus-resistant related genes, in particular to a method for researching the expression of BmNPV DNA replication-related genes inhibited by silkworm genes.

Background

The economic loss of silkworm nuclear polyhedrosis virus (BmNPV) caused by silkworm nuclear polyhedrosis virus infection to Chinese silkworm breeding accounts for about 70-80% of the total loss of silkworm diseases. The identification of resistance mechanism and resistance gene of bombyx mori against BmNPV has been an important research topic in the silkworm industry, and many advances have been made so far. After BmNPV enters the digestive tract of silkworms through the infection route under food, the resistance of silkworms to viruses is divided into 2 aspects: certain proteins such as alkaline protease which are present in the midgut cells and block virus infection activate the blocking mechanism of the midgut cells on virus replication and proliferation; when the virus is replicated and proliferated in midgut cells, so that the midgut cells are destroyed and spread to a body cavity, the silkworm cells start an apoptosis program, wherein some important proteins related to apoptosis in digestive fluid, such as serine protease and the like, play roles in inhibiting the replication and the spread of the virus.

The gene I of the lipase belongs to α/β hydrolase family, the gene I of the silkworm lipase (Bombyx mori lipase-1, Bmlispase-1) plays a dual role in the digestive tract of the silkworm, plays an important role in regulating physiological functions in the feeding stage, improves the resistance of the BmNPV of the silkworm body during a large amount of feeding, belongs to an important component part which is indispensable in an immunity mechanism, the Bmlispase-1 is obtained by first separation and purification in 2003, the gene is specifically expressed in the midgut tissue of the silkworm and has strong BmNPV resistance activity, the Bmlispase-1 in the midgut tissue of a susceptible strain 306 infected with the BmNPV has weak up-regulation (12h), the expression amount of Bmlispase-1 mRNA in the midgut of different strains is positively correlated with the resistance of the silkworm body to the BmNPV, and the Bmlispase-1 can be induced to be regulated within a certain time period after the BmNPV is infected with the silkworm body, and the Bmlnpv resistance of the Bmlispase-1 of the silkworm is obviously increased by about 30 percent.

Currently, some studies have been made on the resistance of insects to baculovirus. The research of silkworm inhibition of BmNPV infection is a hot spot in the silkworm industry. The protein in the midgut and blood of the silkworm can inhibit the infection of BmNPV through immune activation and apoptosis. The silkworm proteins with the activity of resisting BmNPV comprise Bmlispase-1, BmPP2A, BmEPCK-2 and BmATLAStin-n; BmPGRP2-2 can stimulate the replication of virus in cells; the lipase gene of silkworm intestinal bacteria Bacillus pumilus SW41 also has BmNPV resistance activity. However, the mechanism of resistance of these proteins to BmNPV has not been fully elucidated. However, the establishment of the transgenic cell line requires a long time, and the insect cell culture has the characteristics of easy pollution, long culture time and easy degeneration, so the establishment of the transgenic cell line is difficult.

Disclosure of Invention

The technical problem to be solved is as follows: in order to overcome the defects of the prior art, the invention analyzes the influence of Bmlispase-1 on virus replication related genes in cells on the basis of constructing recombinant viruses capable of expressing the Bmlispase-1, and researches the action site of the Bmlispase-1 by using EMSA, thereby clarifying the mechanism of inhibiting the replication and proliferation of BmNPV by the Bmlispase-1. The research provides a new theoretical basis for preventing and treating lepidoptera insect viruses and provides a new idea for further discussing the inhibition mechanism of silkworm genes on the viruses.

The technical scheme is as follows: a method of studying the expression of bombyx mori genes to inhibit BmNPV DNA replication associated genes, the method comprising the steps of:

(1) primer design

Designing primers for synthesizing and cloning according to steps required by plasmid cloning;

(2) construction of recombinant plasmids

Constructing a recombinant plasmid pFastBacDual-polyhedrin-A3-Bmlispase-1;

(3) recombinant virus construction

Transforming the recombinant plasmid prepared in the step (2) into E.coli DH10Bac delta EGT cells to construct recombinant Bacmid;

(4) result verification

Verifying the influence of the recombinant Bacmid prepared in the step (3) on the BmNPV DNA replication related gene; verifying the expression quantity change of DBP; the EMSA method verifies the interaction between the protein and the nucleic acid; analyzing the dbp promoter activity of the deleted Bmlispase-1 binding site.

Preferably, the specific construction method of the recombinant plasmid pFastBacDual-polyhedrin-A3-Bmlispase-1 in the step (2) is as follows: taking BmNPV genome as a template, cloning polyhedrin by PCR with a primer Polyhe-F/R (the primer sequence is shown in Table 1), carrying out double enzyme digestion by BamHI/hindIII, and connecting the cloned polyhedrin to pFastBacDual to obtain a recombinant vector pFastBacDual-polyhedrin; taking pPIGA3GFP plasmid as a template, carrying out PCR amplification on an A3-egfp fragment by using an Actin3-F/R primer, and carrying out double enzyme digestion through EcoR I/Sph I to connect the fragment to a pMD18-T plasmid to obtain T-A3-egfp; after Sna I/Sph I is used for double digestion of pFastBacDual-polyhedrin and T-A3-egfp, target fragments are recovered by glue and then connected to obtain recombinant plasmid pFastBacDual-polyhedrin-A3-egfp. After the T-A3-egfp is subjected to Nco I/Sph I double enzyme digestion, the T-A3-egfp is connected with MCS-F/MCS-R mixed pairing solution to obtain T-A3-MCS; T-A3-MCS and pFastBacDual-polyhedrin are subjected to Sna I/Sph I double enzyme digestion, target fragments are recovered and are then connected to obtain recombinant plasmid pFastBacDual-polyhedrin-A3-MCS; taking the extracted total RNA of the midgut tissue of the bombyx mori p50 as a template, carrying out reverse transcription to obtain a cDNA library, taking the cDNA library as the template, amplifying a Bmlispase-1 fragment by using a primer pair Bmlispase-F/R, carrying out double enzyme digestion by using Nco I/Sph I, and then connecting the amplified fragment to pFastBacDual-polyhidrin-A3-MCS to obtain pFastBacdual-polyhidrin-A3-Bmlispase-1. All clones were verified by sequencing by Biotechnology engineering (Shanghai) Inc.

The resulting pFastBacDual-polyhedrin-A3-MCS vector was prepared by the method described above.

TABLE 1 primers used for cloning

Preferably, the specific method for constructing the recombinant virus is as follows: the constructed recombinant plasmids pFastBacDual-polyhedrin-A3-egfp, pFastBacDual-polyhedrin-A3-Bmlispase-1 transformation of E.coli DH10Bac delta EGT cells to construct recombinant Bacmid, according to

Expression System instructions for identification, purification and extraction of recombinant Bacmid. The BmN cells were cultured at 27 ℃ in an insect cell culture medium TC-100 containing 10% fetal bovine serum. mu.L of Cellffectin reagent (invitrogen USA) was mixed with 1. mu.g of recombinant Bacmid, BmN cells were transfected, and the condition of the cells was observed with a fluorescence microscope Olympus SZX 12. And collecting virus culture solution on the 4 th day after transfection, infecting the BmN cells again, and collecting the cells and the culture solution on the 4 th day after infection to obtain high-titer virus solution. Viral titers were determined by 50% endpoint dilution.

All the cloned genes were examined for expression: detecting the expression of Bmlispase-1 by using a Western blot method: after SDS-PAGE electrophoresis of cell lysate, transferring a protein band to a PVDF membrane by adopting a wet transfer membrane (100V, 60min), washing for 1 time by TTBS, then sealing for 3 hours at room temperature by using 5% defatted milk powder, washing for three times by TTBS, and adding a goat anti-mouse polyclonal antibody with a His label (1: 10000 (V/V)); GenScript, USA ], 4 ℃ overnight, TTBS 3 times. Then using a goat anti-mouse secondary antibody marked by HRP (horse radish peroxidase) (1: 20000 (v/v); GenScript, USA ] incubation for 1h at RT, three TTBS washes, one TBS wash and visualization with DBA visualization kit (Qiagen). The expression status of egfp and polyhedrin was observed by Olympus SZX12 fluorescence microscope.

Preferably, the genes involved in viral replication are selected in step (4): lef-3, helicase, lef-1, lef-2, dnapoly, dbp, lef-11, me53, lef-7 and ie-1 were investigated, and qRT-PCR was performed, respectively, using the primers shown in Table 2. The specific method comprises the following steps: equivalent amounts of recombinant viruses egfp, Bmlispase-1 [ MOI (multiplicity of infection) ═ 6]Separately infecting six-well BmN cells in logarithmic growth phase (about 10)⁶/ml), collecting the BmN cells 6h after infection; at the same time, the remaining cell culture solution was removed, the cells were washed with Phosphate Buffered Saline (PBS), and 2ml of TC-100 (containing 10% fetal calf serum) was added to continue the culture. Collecting cells and

culture solution

12h, 24h, 36h, 48h, 60h and 72h after infection, respectively, and ultracentrifuging (25000 rpm/m)in, 1h 30min, 4 ℃), all collected samples were suspended in 1ml TRIzol (invitrogen, USA), and total RNA was extracted by one step. Then digested with DNase I (Takara, China). Total RNA quality was determined by 260/280 absorbance and electrophoresis. cDNA was synthesized using reverse transcriptase (Takara, China), and the concentration of cDNA was totally diluted to 100 ng/. mu.L using

The PrimeScriptTM RT-PCR kit (Takara, China) was used for Real-Time PCR detection, and the experiment was performed on a Bio-Rad Chromo 4 instrument. The experimental procedure was performed as described in the specification, with 3 replicates per sample.

TABLE 2 primers for qRT-PCR of genes associated with viral replication

Northern Blot is adopted to verify the expression change of DBP, and the specific method is as follows: respectively electrophoresing 6 μ g of total RNA extracted from 1.4 in formaldehyde-denatured agarose, and transferring membrane by semidry method (3 mA/cm)²35min), transferring the RNA band onto a nitrocellulose membrane, carrying out ultraviolet crosslinking for 3 min at the wavelength of 254nm, then hybridizing with a DBP probe labeled and combined with a DIG-High Prime (Roche, Switzerland), washing twice by using a washing buffer solution, incubating with an Anti-DIG-AP antibody for 30min, and finally developing by using a newly prepared substrate developing solution.

The specific method for verifying the interaction between the protein and the nucleic acid by the EMSA method is as follows:

(1) vector construction, prokaryotic protein expression and purification

Vector construction: and (3) taking the midgut cDNA of the fifth age of p50 as a template, amplifying the Bmlispase-1 by using a primer pair DBP-F1/R1, carrying out double enzyme digestion by using EcoR I/Not I, and connecting the product to pGEX-4T-1 to obtain the positive recombinant plasmid pGEX-4T-1-Bmlispase-1.

Prokaryotic protein expression: selecting monoclonal thallus, inoculating into a test tube containing 3ml LB culture solution with 50 mug/ml Amp, shaking overnight at 37 ℃; inoculating into 30ml LB culture solution of 50 μ g/ml Amp at a ratio of 1:100, and shaking at 37 deg.C until the thallus OD600 is 0.6-0.8; adding IPTG to the final concentration of 0.5mM, shaking overnight at 20 ℃, and inducing the expression of the fusion protein; centrifuging the bacterial liquid at 4000r/mim for 10min, discarding the supernatant, and resuspending the bacterial precipitate with PBS; after the resuspension was sonicated, 12% SDS-PAGE was carried out and the bands were visualized by Coomassie blue staining.

And (3) purifying an expression product: using a low-pressure chromatography system, loading the supernatant solution to a GST affinity chromatography column pre-balanced by GSTBinding-Buffer at the flow rate of 0.5 ml/min; flushing with GST Binding-Buffer at a flow rate of 0.5ml/min until the effluent OD280 value reaches the baseline; eluting the target protein with GST Elution-Buffer (20mM Tris-HCl, 50mM GSH, 0.15M NaCl, pH8.0) at a flow rate of 1ml/min, and collecting the effluent; adding the collected protein solution into a dialysis bag, and dialyzing overnight by using 20mM Tris-HCl, 0.15M NaCl, pH8.0; 12% SDS-PAGE and WesternBlot analysis were performed.

(2)EMSA

Preparing a probe: the biotin-labeled probe was synthesized according to the Lightshift Cheminimescent EMSA Kit (Thermo USA).

Electrophoresis: preparing 6% non-denatured polyacrylamide gel; pre-electrophoresis at 4 deg.c and 100V for 60 min; reacting the purified protein and the probe in a binding buffer solution, and standing at room temperature for 40 min; adding 5 mu L of Loading Buffer, mixing uniformly and Loading; electrophoresis was stopped at 4 ℃ and 100V until the length of the gel was 2/3.

Film transfer: soaking nylon membrane in 0.5 × TBE for 15min, sequentially placing sponge, filter paper, gel, nylon membrane, filter paper and sponge according to sandwich method, and fixing mesh plate; the gel faces the cathode and is inserted into an electrophoresis tank, and 0.5 xTBE and 300mA constant current are poured into the electrophoresis tank for 30 min; and (4) ultraviolet crosslinking.

Washing the membrane and detecting signals: method is provided with

The Chemilmescent EMSA Kit (Thermo USA) states the procedure. Nylon membrane is sealed by sealing buffer solution, combined by horse radish streptavidin peroxidase, rinsed and balancedAfter buffer equilibration, Substrate work Solution (Substrate work Solution) reaction, the machine was exposed and the results recorded.

Preferably, the specific method for analyzing the dbp promoter activity lacking the Bmlispase-1 binding site in the step (4) is as follows: directly synthesizing a dbp promoter dbp-65bp sequence without a binding site, adding enzyme cutting sites EcoR I and BamH I at two ends, cloning dbp-65bp into pFastBac1-MCS-egfp through double enzyme cutting, constructing a recombinant virus, infecting BmN cells with the same amount of the recombinant virus, observing the cell state 48h after infection, and verifying the expression amount of the egfp by using a Western Blot method.

The materials and sources used in this application are as follows: the tested silkworm varieties are p50 and 54A, and the bombyx mori BmN cells, the vectors pFastBac1 and the pGEX-4T-1 are all stored in a silkworm genetic improvement key laboratory of the department of agriculture. The pPIGA3GFP plasmid was gifted by doctor Tamura and doctor Chavancy.

Silkworm midgut tissue (third day of p50 fifth age), pFastBacDual insect cell expression vector, BmNPV genome, BmNPV cell, pcDNA3.0 (engineered with an Actin3(A3) initiated egfp).

Plasmid vector pMD18-T,

Premix Ex Taq^TMII, Oligo dT, dNTP, LA Taq enzyme, T4DNA ligase, M-MLV reverse transcriptase, Proteinase K, restriction enzyme were purchased from TaKaRa engineering Co., Ltd. Fetal bovine serum, Cellffectin transfection reagent, TRIzol, RNase Inhibitor were purchased from Invitrogen corporation. Insect cell culture medium TC-100 was purchased from PAA, Germany. His Tag goat anti-mouse polyclonal antibody, horseradish peroxidase labeled secondary IgG (H + L).

Has the advantages that: (1) on the basis of constructing recombinant viruses capable of expressing Bmlispase-1, the invention analyzes the influence of Bmlispase-1 on virus replication related genes in cells, and researches the action site of Bmlispase-1 by using EMSA, thereby clarifying the mechanism of inhibiting the replication and proliferation of BmNPV by Bmlispase-1; (2) the method skillfully avoids the defects that the establishment of a transgenic cell line needs a long time, and the culture of insect cells is easily polluted, long in culture time and easy to degenerate; (3) the method of the invention can increase the expression level of the candidate gene along with the proliferation of the virus, and the inhibition effect on the BmNPV is continuously enhanced, so that the target gene information can be rapidly obtained.

Drawings

FIG. 1 is a recombinant plasmid construction diagram, wherein A is a pFastBacDual-polyhedrin-A3-egfp construction diagram, and B is a pFastBacdual-polyhedrin-A3-Bmlispase-1 construction diagram.

FIG. 2 is a PCR identification diagram of recombinant Bacmid capable of expressing Bmlispase-1, egfp, wherein M-1: DL2000 Marker; m-2: lambda-EcoT 14I digest; lane 1-2, recombinant virus egfp; lane 3-4 recombinant virus Bmlispase-1.

FIG. 3 is a gene expression verification diagram in which A: the control group expressed egfp (160 ×); b: recombinant virus formed viral polyhedra (640 ×); c: western blot to detect the expression of Bmlispase-1, lane 1: infection with recombinant virus Bmlispase-1, lane 2: and (4) a control group.

FIG. 4 is a graph showing the change in the expression level of genes involved in viral replication, in which A is a standard curve, B is dbp gene, C is ie-1 gene, D is helicase gene, E is dnapoly gene, F is me53 gene, G is lef-1 gene, H is lef-2 gene, I is lef-3 gene, J is lef-7 gene, and K is lef-11 gene; the differences were very significant.

FIG. 5 is a graph of Northern Blot method for verifying the expression change of DBP at different time points, wherein Lip is Bmlispase-1 recombinant Bacmid, and Control is egfp recombinant Bacmid.

FIG. 6 is a graph showing the variation of copy numbers of genes involved in viral replication, wherein A is ie-1 gene and B is a standard curve; the differences were very significant.

FIG. 7 is a SDS-PAGE photograph showing the expression of Bmlispase-1 gene; wherein M is the protein molecular mass standard; 1, no induction; 2, after induction at 20 ℃; 3, inducing crushing at 20 ℃ and then clearing the supernatant; precipitating after induced crushing at the temperature of 4:20 ℃.

FIG. 8 is an analysis of Bmlispase-1 fusion protein; wherein A is SDS-PAGE analysis and B is Western Blot analysis.

FIG. 9 is a graph showing the results of EMSA analysis; wherein, A is the length of dbp promoter and the design site of probe, B is exposure for 1 minute, and C is exposure for 3 minutes.

FIG. 10 is a graph of the activity analysis of the dbp promoter lacking the binding site; wherein A is a cell fluorescence image, B is a Western Blot analysis, 1 is dbp-75bp, and 2 is dbp-65 bp.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 construction and verification of recombinant plasmid and recombinant virus capable of expressing Bmlispase-1 and egfp

Respectively transforming the recombinant plasmids into DH10Bac cells to obtain recombinant Bacmid according to

Expression System instructions for identification, purification and extraction of recombinant Bacmid. Recombinant Bacmid identification was performed with M13-F/R. The results show (fig. 2): positive recombinant Bacmid was obtained, lane 1 is recombinant Bacmid-egfp (control group), and lane 3 is recombinant Bacmid-Bmlispase-1 (test group). And (3) transfecting the recombinant Bacmid into the BmN cell to obtain a recombinant virus, and determining the virus titer.

BmN cells are infected by the recombinant viruses Bmlispase-1 and egfp respectively, and the expression conditions of the egfp and polyhedrin are observed by an Olympus SZX12 fluorescence microscope. At 72h, a lot of green fluorescence appeared in Bacmid-egfp infected BmN cells, indicating that egfp could be expressed (FIG. 3. A); a large number of viral polyhedra appeared in and outside BmN cells infected with Bacmid-Bmlispase-1 (FIG. 3. B); cells were harvested 96h after infection, proteins were extracted, and expression of Bmlispase-1 was confirmed by Western Blot (FIG. 3. C). The above results indicate that all gene clones were successful and that further experiments can be carried out.

Example 2 qRT-PCR detection of the Effect of Bmlispase-1 on Virus replication-related genes and changes in the viral genome

The BmNPV needs the participation of the following enzymes in the processes of replication and proliferation: DNA polymerase (dnapoly) which extends DNA from the 3' -OH of the RNA primer in the 5' to 3' direction; helicase having the biochemical properties of ATPase and Helicase and the ability to bind to ssDNA and dsDNA; DNA primer enzyme, LEF-1 and primer enzyme related factor, LEF-2; a single-stranded DNA binding protein (SSB) LEF-3 having an interaction with an alkaline nuclease and capable of regulating the function of the enzyme and also capable of unwinding and annealing DNA according to its concentration or redox state; the second SSB is the DNA Binding Protein (DBP) necessary for the production of the nucleocapsid and pathogenic matrix; IE-1 is transcribed early in infection and continues to be transcribed at a later stage, IE1 is essential for transient DNA replication and is an essential gene, since baculovirus with this gene deletion cannot survive; LEF-11 stimulates late gene transcription, but is not required for transient DNA replication. However, the lef-11 knockout AcMNPV baculovirus, when transfected into Sf-9 cells, is unable to synthesize DNA or undergo late gene transcription; the deletion me53 prevents the virus from forming mature virus particles, thereby influencing the replication effect of the virus; lef-7 stimulated transient DNA replication, and when deleted, infection in Tn368 cells was unaffected, but in Sf21 and s.exigua cells, DNA replication was 10% of controls. A reduction in DNA synthesis was also observed in the absence of lef-7 in BmNPV.

Ie-1 was ligated into the vector pMD18-T, plasmid concentration was determined to be 247.3 ng/. mu.L, and plasmid copy number was determined to be 5.08X 10¹⁰copies/mL, the plasmid was diluted 10-fold and q-PCR was performed. The results are shown in FIG. 4. A: the standard curve equation is that y is-3.6857 x +43.827, R²＝0.9997。

Equivalent recombinant viruses egfp and Bmlispase-1 respectively infect the BmN cells. Collecting cells and

culture solution

6, 12, 24, 36, 48, 60 and 72 hours after infection, extracting total RNA after ultracentrifugation, carrying out reverse transcription to synthesize cDNA, detecting the expression levels of lef-3, helicase, lef-1, lef-2, dnapoly, dbp, lef-11, me53, lef-7 and ie-1 according to the same method, and calculating the copy number of the expression level of the gene to be detected according to a formula. The results show that: at all sampling time points, the expression level of dbp in the cells of the recombinant virus expressing Bmlispase-1 was lower than that of the control (egfp), wherein Bmlip of 24, 36, 48 and 60 hoursThe expression level of dbp in BmN cells infected with ase-1 was 1.70X 10⁷、6.45×10⁶、2.52×10⁵、2.45×10⁷(copy number), 33.93%, 59.16%, 12.41%, 50.43% of the controls, respectively, all achieved very significant differences (fig. 4. B). The expression level measurements of the remaining 9 genes revealed (FIG. 4.A, C-K) that the gene expression levels in the test group were slightly lower than those in the control group, and that the trend of the curve was consistent with dbp.

We verified the change in expression of dbp at different time points using the Northern Blot method. Total RNA from BmN cells at various time points after infection with the recombinant virus was bound to dbp-DNA digoxigenin-labeled probes by northern Blot and visualized with NBT/BCIP (FIG. 5): the expression level of all dbps in the test group is obviously lower than that of the Control group, and the change trend is basically consistent with the qRT-PCR result.

On the other hand, cells were infected and collected using the above method, and genomic DNA was extracted, and the copy value of ie-1 was detected using a standard curve method (FIG. 6. B). The copy number of ie-1 was lower in the cells of the test group at all time points than in the control (FIG. 6.A), wherein the copy numbers of ie-1 were 7.09X 10 for 12, 24, 36, 48, 60h, respectively⁶、3.63×10⁸、3.59×10⁹、5.56×10⁹、4.70×10⁹Significant differences were achieved at 38.58%, 20.04%, 42.81%, 53.74%, 65.13% of the controls, respectively. ie-1 exists in a single copy in the BmNPV genome, and changes in copy value represent changes in the BmNPV genome. DBP (Bm16), known as DNA Binding Protein (DBP). It has properties similar to LEF-3, and is capable of melting and annealing DNA. DBP also competes with LEF-3 for binding sites on ssDNA templates and protects ssDNA from hydrolysis by the baculovirus Alkaline Nuclease (AN)/LEF-3 complex. DBP is not a virion structural protein, but it is an integral part of the pathogenic matrix and is also necessary for the production of nucleocapsids. Furthermore, although viral DNA synthesis occurs in cells transfected with the virus constructed with the knock-out DBP, the levels are lower than those of the control virus, indicating that DBP may be necessary for normal DNA synthesis levels or nascent viral DNA stability. DBP can protect the mature viral genome from nuclease attack and prior to packaging into virionsIt is stabilized. On the other hand, the processing of replicative intermediates requires the unwinding and renaturation activities of DBP, which may involve annealing and strand invasion reactions of DNA recombination, which is critical for the complete replication and processing of the baculovirus genome. In conclusion, the inhibition of the expression of the virus dbp is probably the key of the Bmlispase-1 in inhibiting the replication and proliferation of the virus.

Example 3 Bmlispase-1 analysis of the site of action of dbp upstream promoter

(1) Bmlispase-1 prokaryotic expression and purification

The middle intestine cDNA of bombyx mori p50 fifth-instar day 3 is used as a template, Bmlispase-1 is amplified by PCR, and is connected to pGEX-4T-1 after double enzyme digestion to obtain a positive recombinant plasmid pGEX-4T-1-Bmlispase-1. The recombinant plasmid was transformed into BL21 cells, monoclonal cells were selected and inoculated into LB medium, expression of the fusion protein was induced with IPTG at 20 ℃ and the bacterial solution was disrupted by ultrasonication and then subjected to 12% SDS-PAGE, and bands were visualized by Coomassie blue staining (FIG. 7). The results show that: in the thalli which are not induced by IPTG, the expression quantity of Bmlispase-1 is very little; the Bmlispase-1 content in the supernatant and the sediment of the induced cells is increased, and the molecular weight of the fusion protein is about 59.63KD (the molecular weight of the Bmlispase-1 is 31.73KD, and the molecular weight of GST-tag is about 27.90 KD).

The fusion protein constructed on the pGEX-4T-1 vector and induced by low temperature is expressed in the form of supernatant, and the GST affinity purification of the supernatant of the fusion protein is carried out in the next step.

Using a low-pressure chromatography system to sample the supernatant solution to a GST Binding-Buffer pre-balanced GST affinity chromatography column; eluting the target protein by GST Elution-Buffer when the effluent OD280 value reaches a baseline, and collecting the effluent; adding the collected protein solution into a dialysis bag for dialysis overnight; 12% SDS-PAGE and Western Blot analysis were performed. The results are shown in FIG. 8: in the sample after the cell is ultrasonically crushed and the effluent after chromatography, besides Bmlispase-1 fusion protein, a large amount of other proteins exist; in contrast, only the Bmlispase-1 fusion protein was present in the dialysis eluate (FIG. 8. A). Western Blot analysis shows that the Bmlispase-1 fusion protein is a monomeric protein. The above results illustrate that: the purified protein after the Bmlispase-1 prokaryotic expression has high purity and can be used for EMSA experiments.

(2) EMSA analysis

In the BmNPV genome, the sequence size between an open reading frame of a dbp gene and an adjacent orf _17 gene is 75bp, promoter activity analysis is carried out on a promoter region (dbp-75bp) of the dbp gene and an upward extension promoter region (dbp-126bp) (figure 9.A), the two sequences are cloned into pFastBac1-MCS-EGFP, and after Bacmid is constructed by a Bac-to-Bac system, silkworm BmN cells are transfected, green fluorescence is observed, but the fluorescence difference is not obvious.

3 biotin-labeled probes were designed and synthesized from the sequence (dbp-126bp) with 10bp repeats between adjacent probes (FIG. 9. A). The 3 probes are respectively mixed with Bmlispase-1 protein, and the combination of the probes and the protein is detected by non-denaturing polyacrylamide gel electrophoresis, membrane transfer, ultraviolet crosslinking, membrane washing and signal detection. The results are shown in FIG. 9. B: binding bands exist between the

probes

2 and 3 and the Bmlispase-1 protein, and the

probes

2 and 3 are supposed to be mutually bound with the Bmlispase-1.

Since a repeated sequence exists between the

probes

2 and 3, the sequence is analyzed by a promoter binding element by using GeneQuest software, and a binding site of the c-Myb transcription factor (TTCAAT) exists in the repeated sequence region. To determine whether this binding site is a binding region of the Bmlispase-1 protein. We performed a second EMSA experiment.

Biotin-labeled probe 4 was synthesized (FIG. 9.A) and mixed with Bmlispase-1 for EMSA analysis (FIG. 9. C). The results show that: probe 4, which lacks the c-Myb transcription factor binding site, has no binding band and can be determined to be the binding region of Bmlispase-1.

Example 4 Activity analysis of dbp promoter lacking binding site

Synthesizing a dbp promoter (dbp-65bp) sequence lacking a binding site, adding restriction enzyme sites (EcoRI and BamH I) at two ends, cloning dbp-65bp into pFastBac1-MCS-EGFP by double restriction enzyme, constructing a recombinant virus, infecting cells after transfection, observing weak fluorescence 48h after infecting the cells, and having obvious difference compared with the activity of the promoter (dbp-75bp) (figure 10. A).

And (3) infecting BmN cells with recombinant viruses carrying promoters of dbp-75bp and dbp-65bp, collecting the cells on the 5 th day, extracting total protein, and performing Western Blot experiment verification by using equivalent protein. The experimental results show that: the expression level of green fluorescent protein in dbp-65bp was significantly lower than that in dbp-75bp (FIG. 10. B). The above results illustrate that: the Bmlispase-1 competitively binds with the dbp upstream promoter binding element, so that the expression quantity of dbp is reduced, the virus titer is reduced, and the effect of inhibiting virus replication and proliferation is achieved.

And (4) conclusion:

silkworm hemopus diseases are extremely harmful to the silkworm industry in China. The breeding of antiviral silkworm varieties and the research of silkworm genes for resisting BmNPV have been always concerned by the industry. By using the technologies of transgenosis, overexpression and the like, a plurality of genes are determined to have the BmNPV resistance activity. The invention reports a method for constructing a recombinant virus capable of expressing Bmlispase-1, researches the interaction of the Bmlispase-1 and the virus, and reduces the expression quantity of dbp due to the combination of the Bmlispase-1 and a dbp upstream promoter binding element, thereby achieving the research result of inhibiting the replication and proliferation of the virus. Provides a new method for researching the mechanism of inhibiting virus replication and proliferation by silkworm gene.

The research finds that: bmlispase-1 can inhibit the expression of dbp of BmNPV. The expression level of dbp 48h after infection of BmN cells with the recombinant virus was 12.41% of that of the control, and reached the lowest expression level, which was also the lowest difference (FIG. 4. B). While the expression levels of the other 9 genes were slightly lower than the control and the trend of the curve was consistent with dbp (FIG. 4.A, C-K). In the test group, the copy number of the viral genome (24h) was 20.04% of the control, reaching the difference minimum, but the viral genome increased continuously throughout the infection and reached equilibrium at 48h (fig. 6). The difference between these 2 curves may be related to the dbp function. The viral DNA synthesis in cells transfected with DBP-deficient viruses is lower than that of normal viruses, and DBP is required for normal DNA synthesis levels or for nascent viral DNA stability. Additionally, DBP can protect the mature viral genome from nuclease attack. The processing of DNA replication intermediates requires unwinding and renaturation activities of DBP, which is critical for the complete replication and processing of the baculovirus genome. The replication, assembly and Bmlispase-1 expression of the recombinant virus exist in the sameIn the environment, the expression level of Bmlispase-1 is increased along with the replication and proliferation of the virus, and the inhibition effect on BmNPV is also enhanced. However, the expression profiles of all the genes involved in viral replication were identical, with 0-48 h being high first and low later, which is likely to be the same as under the present experimental conditions (about 10 infection with MOI 6 virus)⁶BmN cells/ml, medium renewal after 6 h) release of virions, lysis of cells.

The binding site of Bmlispase-1 contains a 6 base (TTCAAT) motif, which is the site of action of the c-Myb transcription factor. MyB transcription factor is widely distributed in plants, animals and fungi, c-Myb is necessary for normal hematopoiesis, and mice with knockout of c-Myb cause embryonic death due to hematopoietic failure; c-Myb plays a crucial role in the activation process of human NK cells (natural killercells); in addition, the OsMYB2 can obviously enhance the cold resistance, salt resistance and drought resistance of rice. Whether the Bmlispase-1 has the related function of the MyB family or not is further researched.

The binding of Bmlipitase-1 to the binding site is weak, which is probably related to the fact that Bmlipitase-1 is obtained through prokaryotic expression. Although the protein expressed by the pronucleus has high efficiency and high synthesis speed, the protein expressed by the pronucleus cannot generate glycosylation, phosphorylation and other post-translational modifications, and the biological activity and the function of the synthetic protein are influenced.

At present, the research on the inhibition mechanism of silkworm virus-resistant related genes on BmNPV amplification is not reported yet. However, the establishment of the transgenic cell line requires a long time, and the insect cell culture has the characteristics of easy pollution, long culture time and easy degeneration, so the establishment of the transgenic cell line is difficult. The method for constructing the recombinant virus capable of expressing the Bmlipiase-1 is innovatively adopted, so that the problems are ingeniously avoided; furthermore, this research approach enables the expression level of the candidate gene to increase with the proliferation of the virus, and the effect of inhibiting BmNPV to be enhanced, thereby enabling the target gene information to be obtained quickly. The research provides a new theoretical basis for preventing and treating lepidoptera insect viruses and provides a new research idea for further discussing the inhibition mechanism of silkworm genes on the viruses.

Sequence listing

<110> university of Jiangsu science and technology

<120> method for studying expression of gene related to inhibition of BmNPV DNA replication by silkworm gene

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Claims

1. A method for studying the expression of a bombyx mori gene for inhibiting BmNPV DNA replication associated genes, the method comprising the steps of:

(1) primer design

(2) construction of recombinant plasmids

Constructing a recombinant plasmid pFastBacDual-polyhedrin-A3-Bmlispase-1;

(3) recombinant virus construction

(4) result verification

2. The method for researching the expression of the bombyx mori gene for inhibiting BmNPV DNA replication related gene according to claim 1, wherein the specific construction method of the recombinant plasmid pFastBacDual-polyhedrin-A3-Bmlispase-1 in the step (2) is as follows: taking BmNPV genome as a template, cloning polyhedrin by using a primer Polyhe-F/R PCR, carrying out double enzyme digestion by BamHI/hindIII, and connecting the cloned polyhedrin to pFastBacDual to obtain a recombinant vector pFastBacDual-polyhedrin; taking pPIGA3GFP plasmid as a template, carrying out PCR amplification on an A3-egfp fragment by using an Actin3-F/R primer, and carrying out double enzyme digestion through EcoR I/Sph I to connect the fragment to a pMD18-T plasmid to obtain T-A3-egfp; after the T-A3-egfp is subjected to Nco I/Sph I double enzyme digestion, the T-A3-egfp is connected with MCS-F/MCS-R mixed pairing solution to obtain T-A3-MCS; T-A3-MCS and pFastBacDual-polyhedrin are subjected to Sna I/Sph I double enzyme digestion, target fragments are recovered and are then connected to obtain recombinant plasmid pFastBacDual-polyhedrin-A3-MCS; taking the extracted total RNA of the midgut tissue of the bombyx mori p50 as a template, carrying out reverse transcription to obtain a cDNA library, taking the cDNA library as the template, amplifying a Bmlispase-1 fragment by using a primer pair Bmlispase-F/R, carrying out double enzyme digestion by using Nco I/Sph I, and then connecting the amplified fragment to pFastBacDual-polyhidrin-A3-MCS to obtain pFastBacdual-polyhidrin-A3-Bmlispase-1.

3. The method for studying the expression of bombyx mori genes for inhibiting BmNPV DNA replication-related genes according to claim 1, wherein the recombinant plasmid of reporter gene egfp, specifically pFastBacDual-polyhedrin-A3-egfp, is used as a control group in the step (2).

4. The method for researching the inhibition of expression of BmNPV DNA replication-related genes by silkworm genes as claimed in claim 1, wherein the genes related to virus replication are selected in the step (4): lef-3, helicase, lef-1, lef-2, dnapoly, dbp, lef-11, me53, lef-7 and ie-1 are the research objects.

5. The method for researching the expression of the bombyx mori gene for inhibiting BmNPV DNA replication related gene according to claim 1, wherein the specific method for analyzing the dbp promoter activity of the deleted Bmlispase-1 binding site in the step (4) is as follows: directly synthesizing a dbp promoter dbp-65bp sequence without a binding site, adding enzyme cutting sites EcoR I and BamH I at two ends, cloning dbp-65bp into pFastBac1-MCS-egfp through double enzyme cutting, constructing a recombinant virus, infecting BmN cells with the same amount of the recombinant virus, observing the cell state 48h after infection, and verifying the expression amount of the egfp by using a Western Blot method.

6. The pFastBacDual-polyhedrin-A3-MCS vector prepared by the method of claim 2.