A recombinant baculovirus and its construction method and application
By amplifying the transmembrane domain in the gp64 gene and constructing the recombinant baculovirus AcMNPV-gp64TMdouble-egFP, the problem of slow proliferation of existing baculovirus insecticides was solved, and the proliferation rate and insecticidal efficiency were significantly improved.
Patent Information
- Application Number
- CN202310050168.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-01
- Publication Date
- 2025-05-02
- Estimated Expiration
- 2043-02-01
AI Technical Summary
Existing baculovirus insecticides proliferate slowly and have slow results, making them unable to quickly kill insects in the short term. Moreover, due to the large genome of the virus, it is difficult to directly insert exogenous genes.
By amplifying a sequence of a transmembrane domain in the gp64 gene, the recombinant baculovirus AcMNPV-gp64TMdouble-egFP was constructed, and the target gene was integrated into the viral genome using the Bac-to-Bac transposal recombination system.
It significantly improves the proliferation rate of viruses in host cells, reduces the half-lethal time to fall armyworm larvae, and improves insecticidal efficiency.
Smart Images
Figure CN116064621B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a recombinant virus, in particular to a recombinant baculovirus and a construction method thereof. The recombinant virus has a strong proliferation ability and can be used to prepare a microbial insecticide for preventing and controlling fall armyworm larvae. Technical Background
[0002] Baculovirus insecticides, as new and safe biological pesticides for crop and forest pest control, have attracted more and more attention. Compared with traditional chemical pesticides, baculovirus is highly pathogenic to host insects, very safe to other non-target organisms, can survive in the environment for a long time, is harmless to humans, livestock and other vertebrates, does not pollute the environment, can be mixed with other chemical pesticides, and has the advantages of long protection time and easy use. Therefore, as early as 1973, it was recommended by the Food and Agriculture Organization of the United Nations as an ideal substitute for chemical pesticides.
[0003] However, baculovirus insecticides also have certain disadvantages. To a certain extent, baculovirus insecticides are less toxic, proliferate slowly, and take a long time to take effect. After the virus enters the insect body, it takes a period of time for it to proliferate in large quantities and kill the pest, which generally takes 7 to 14 days. It is impossible to meet the demand for rapid insecticide in a short period of time. Therefore, increasing the proliferation rate of baculovirus in the host pest and thus improving its insecticide activity has become the development direction of the current research and development of baculovirus biological insecticides.
[0004] Since the baculovirus genome is large, it cannot be directly manipulated to insert foreign genes, so an intermediate vector is needed to obtain a recombinant baculovirus. GP64 is a major glycoprotein on the envelope of the budding virus particle (BV) of the baculovirus. Studies have shown that the transmembrane domain (TM) on GP64 is the key to determining the membrane fusion function of GP64. Therefore, the present invention amplifies a TM domain sequence in the gp64 gene to obtain gp64. TM double Recombinant Autographa californica nuclear polyhedrosis baculovirus AcMNPV-gp64 TM double -egfp. Experiments have shown that the recombinant virus can significantly increase the proliferation rate in host cells and reduce the half-lethal time to the larvae of the fall armyworm. Therefore, it can be used alone or in combination with other insect-resistant or exogenous genes to construct highly efficient recombinant baculovirus insecticides. Summary of the invention
[0005] The object of the present invention is to provide a recombinant baculovirus, which can increase the virus proliferation rate after infecting Spodoptera frugiperda Sf9 cells.
[0006] The present invention also aims to provide a method for constructing the recombinant baculovirus, including a method for constructing an intermediate vector for obtaining the recombinant baculovirus.
[0007] To achieve the above-mentioned purpose of the present invention, the following technical solutions can be used:
[0008] The present invention provides a recombinant baculovirus, which is obtained by integrating the gene shown in SEQ ID No. 1 into the AcMNPV genome through the Bac-to-Bac transposition recombination system. The gene sequence is gp64 of Autographa californica nuclear polyhedrosis virus AcMNPV TM double A tandem gene of the jellyfish enhanced green fluorescent protein egfp gene.
[0009] The recombinant baculovirus can express the fusion protein GP64 in infected Sf9 host cells. TM double -EGFP.
[0010] The present invention provides a method for constructing a recombinant baculovirus, comprising the following steps:
[0011] (1) Using the AcMNPV genome as a template, PCR was used to identify gp64 TM double UF (SEQ ID No. 2) and gp64 TM double DR (SEQ ID No.5) was used as primer to amplify the gp64 gene fragment; gp64 gene was used as template and gp64 TM double UF (SEQ ID No. 2) and gp64 TM double UR (SEQ ID No.3) was used as primer to amplify gp64 TM double U fragment; gp64 TM double DF (SEQ ID No. 4) and gp64 TM double DR (SEQ ID No.5) was used as primer to amplify gp64 TM double Then, gp64 was obtained by bridge PCR TM double Then, the gene was constructed into the plasmid pFastBacDual-egfp by restriction endonuclease digestion and ligation reaction to obtain the recombinant plasmid pFastBacDual-gp64 TMdouble -egfp, the recombinant plasmid contains the gene of SEQ ID No.1;
[0012] (2) The recombinant plasmid pFastBacDual-gp64 TM double -egfp transformed DH10B competent cells, target gene Ac-gp64 TM double -egfp was transposed to the Bacmid in DH10B competent cells through Tn7 transposition recombination, and then the transformed DH10B cells were plated on LB plates;
[0013] (3) Screening of Bacmid-gp64 containing successful recombinant DNA by PCR TM double Extract the recombinant bacmid-gp64 from a single DH10B colony expressing -egfp TM double -egfp;
[0014] (4) Recombinant bacmid-gp64 TM double -EGFP and MirusBioMIR – INSECT Transfection Reagent transfection reagent was mixed and transfected into Sf9 cells to obtain recombinant baculovirus AcMNPV-gp64 TM double -egfp.
[0015] After the recombinant baculovirus of the present invention infects Sf9 cells, the first, second and third generation viruses gradually increase the expression of green fluorescent protein in the cells; TCID 50 The experimental results showed that AcMNPV-gp64 TM double -egfp significantly increased the proliferation rate in Sf9 cells compared with AcMNPV-egfp; the fourth-instar larvae of fall armyworm were used as the test insects, and the virus infection concentration was 2.8×10 8 vp / μL and 2.8×10 9 vp / μL, AcMNPV-gp64 TM double -egfp compared with the AcMNPV-egfp treated group 50 1.12 days and 0.628 days in advance respectively.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] 1. The recombinant baculovirus of the present invention can promote the proliferation of progeny viruses compared with wild-type viruses;
[0018] 2. The recombinant baculovirus of the present invention can improve the insecticidal efficiency against the larvae of the fall armyworm compared to the wild-type virus. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Recombinant plasmid pFastBacDual-gp64 TM double -egfp construction flow chart
[0020] Figure 2 Fluorescence microscopy examination of the first, second and third generation recombinant viruses AcMNPV-gp64 TM double -EGFP emits green fluorescence in Sf9 cells
[0021] Figure 3 TCID 50 Detection of recombinant virus AcMNPV-gp64 TM double Proliferation efficiency of -egfp and control virus AcMNPV-egfp
[0022] Table 1 Recombinant virus AcMNPV-gp64 TM double Analysis of the insecticidal efficiency of AcMNPV-egfp and the control virus AcMNPV-egfp against the larvae of Spodoptera frugiperda DETAILED DESCRIPTION
[0023] Example 1 Recombinant virus AcMNPV-gp64 TM double -EGFP construction
[0024] (1)Ac-gp64 TM double Cloning and identification of genes and intermediate plasmid pFastBacDual-gp64 TM double -egfp construction (see the construction process Figure 1 )
[0025] Design primers gp64 TM double UF (SEQ ID No. 2), gp64 TM double DR (SEQ ID No.5), the gp64 gene was amplified by PCR using the AcMNPV genome as a template. TM doubleUF (SEQ ID No. 2) and gp64 TM double UR (SEQ ID No.3) was used as primer to amplify gp64 TM double U fragment; gp64 TM double DF (SEQ ID No. 4) and gp64 TM double DR (SEQ ID No.5) was used as primer to amplify gp64 TM double Then, gp64 was obtained by bridge PCR TM double The gene was cleaved with Sam I restriction sites at both ends, and the PCR product was recovered after restriction digestion; at the same time, the constructed intermediate vector pFastBacDual-egfp was digested with Sam I, and the digestion product was recovered; the digested PCR product and the intermediate vector were connected with T4 ligase at 16°C overnight, the ligation product was transformed into Escherichia coli DH5α, coated on LB solid culture medium plates containing ampicillin, and cultured at 37°C overnight, a single colony was picked and cultured in LB liquid culture medium in large quantities, the plasmid was purified for PCR identification and restriction digestion identification, and the successfully identified plasmid pFastBacDual-gp64 TM double -egfp was sequenced and confirmed. The plasmid contained the gene of SEQ ID No.1.
[0026] (2) Recombinant AcMNPV genome bacmid-gp64 TM double -egfp build
[0027] pFastBacDual-gp64 TM double -egfp was transformed into E. coli DH5α competent cells, the plasmid was extracted and transformed into E. coli DH10B to cause Tn7 transposition recombination, and the target gene gp64 TM double -egfp was recombined on the AcMNPV genome bacmid, then spread on a solid culture medium plate containing kanamycin and gentamicin, cultured overnight at 37°C, and a single large white colony was picked and cultured in 5 mL LB liquid culture medium to extract and purify the bacmid-gp64 TM double -egfp was used for PCR identification.
[0028] (3) Recombinant virus AcMNPV-gp64 TMdouble -egfp construct and propagation (see Figure 2 )
[0029] Sf9 cells were inoculated into a 24-well cell culture plate. When the cells were more than 90% confluent, the culture medium was removed and the residual culture medium was washed away with PBS. TM double -egfp (μg) and transfection reagent MirusbioMIR –INSECT Transfection Reagent (μl) was added to 100μl serum-free medium at a volume of 1μg / 3μl. After vigorous shaking for 15s, it was allowed to stand for 15min and added to Sf9 cells. It was incubated at 27℃ for 4h, and then 400μl of serum-free medium was added. Four days after transfection, green fluorescence was observed under a fluorescence microscope. The supernatant and cells were collected and repeatedly frozen and thawed to break the cells. The supernatant was collected by centrifugation to obtain the first-generation virus. Sf9 cells were infected with the first-generation virus. Three days later, the cells and supernatant were collected, and the cells were repeatedly frozen and thawed to break the cells. The supernatant was collected by centrifugation to obtain the second-generation virus. By analogy, the third-generation virus was obtained and stored at 4℃. Figure 2 Fluorescence microscopy for the examination of the first, second and third generation recombinant viruses AcMNPV-gp64 TM double -Green fluorescence emitted by EGFP in Sf9 cells.
[0030] Example 2 Sf9 cells were plated in a 12-well plate and infected with a virus solution at 5 MOI. After 2 hours, all the virus solution was removed, and then 1 ml of cell culture medium was added. The supernatant containing BV was collected at 24 hours, 48 hours, 72 hours, and 96 hours, and the collected supernatant was diluted to determine the BV titer. Figure 3 TCID 50 Detection of recombinant virus AcMNPV-gp64 TM double -egfp and the control virus AcMNPV-egfp.
[0031] Example 3 The concentration is 2.8×10 8 vp / μL and 2.8×10 9 vp / μL AcMNPV-gp64 TM double -egfp and AcMNPV-egfp virus solutions were injected into the 4th instar larvae of Spodoptera frugiperda. Ten larvae were injected at each concentration and repeated three times. The number of dead insects was recorded every day, and the half-lethal time (LT) of each treatment group was calculated using SPSS software. 50 , data and analysis results are shown in Table 1.
[0032] Table 1
[0033]
Claims
1. A gp64 TMdouble -egfp gene, characterized by The sequence is SEQ ID No.
1.
2. A recombinant baculovirus, characterized in that The gp64 of claim 1 was transformed into TMdouble The -egfp gene was integrated into the genome of Autographa californica nuclear polyhedrosis virus AcMNPV.
3. The method for constructing a recombinant baculovirus according to claim 2, wherein: The steps include: a) Using the AcMNPV genome as a template, amplify the gp64 gene; b) Using the gp64 gene as a template, the sequence encoding the transmembrane domain (TM) was doubled by PCR to obtain gp64 TMdouble ; c) Constructing gp64 TMdouble Recombinant plasmid pFastBacDual-gp64 containing gene and enhanced green fluorescent protein egfp gene TMdouble -egfp; d) The recombinant plasmid pFastBacDual-gp64 TMdouble -egfp was transformed into DH10B competent cells to obtain recombinant bacmid-gp64 TMdouble -egfp; e) Recombinant bacmid-gp64 TMdouble -egfp was transfected into Spodoptera frugiperda Sf9 cells to obtain the recombinant baculovirus AcMNPV-gp64 TMdouble -egfp.
4. The recombinant baculovirus according to claim 2 is used for preparing a microbial insecticide for controlling fall armyworm larvae.