Anti-PxTsf1 protein polyclonal antibody and application thereof

Abstract

The invention discloses a polyclonal antibody for resisting PxTsf1 protein and application of the polyclonal antibody. According to the present invention, a New Zealand white rabbit is immunized by using the recombinant protein rcp-PxTsf1 of the PxTsf1 protein so as to obtain the polyclonal antibody anti-rcp-PxTsf1 capable of resisting the PxTsf1 protein; when the polyclonal antibody anti-rcp-PxTsf1 is used for treating plutella xylostella pupae, the antagonistic plutella xylostella PxTsf1 protein can delay the ovarian development of female plutella xylostella, reduce the reproductive capacity of the female plutella xylostella, reduce the eclosion rate of the plutella xylostella, improve the sensitivity of the plutella xylostella to metarhizium anisopliae and improve the death rate of the plutella xylostella infected by the metarhizium anisopliae. The PxTsf1 protein inhibitor can prevent and control the plutella xylostella by reducing the reproductive capacity and eclosion rate of the plutella xylostella and the sensitivity of the plutella xylostella to metarhizium anisopliae, and provides a new direction for green prevention and control of plutella xylostella and other plutella xylostella pests.

Description

Technical Field

[0001] The present invention belongs to the technical field of pest control, and more specifically relates to a polyclonal antibody against PxTsf1 protein and its application. Background Art

[0002] The diamondback moth (Plutella xylostella), a representative species of the family Diamondback Moth, is one of the most destructive holometabolous insects in cruciferous crop production systems worldwide. Its population expansion is due to its remarkable reproductive potential (9 to 14 generations per year) and rapid evolution. Especially under the selective pressure of chemical pesticides, the diamondback moth has developed resistance to multiple insecticides, making it a key target for control in plant protection.

[0003] Relying on chemical pesticides to control the diamondback moth not only leads to increased resistance but also kills its natural enemies, disrupting the ecological balance. Developing and utilizing biocontrol fungi with excellent insecticide efficacy, such as Metarhizium anisopliae, to control the diamondback moth can overcome the shortcomings of chemical pesticides and offer environmental benefits. However, enhancing the insecticidal activity of Metarhizium anisopliae against the diamondback moth, or in other words, increasing the moth's sensitivity to Metarhizium anisopliae, is crucial for improving diamondback moth control effectiveness.

[0004] Iron plays a crucial role in insect growth and immune regulation. It's not only an essential trace element but also a cofactor for key enzymes, participating in important physiological processes such as electron transport in the cellular respiratory chain, genetic replication, exogenous toxin metabolism, and neural signaling. Transferrin (Tsf) is responsible for transporting iron absorbed by the digestive tract and iron released by red blood cell degradation. As a protein that binds iron ions, it participates in various physiological processes, including iron homeostasis and immunity. For example, studies have found that when Western honey bees (Apis mellifera) are infected with microsporidia, suppressing the transcription of AmTsf through RNAi can actually increase the survival rate of infected bees. However, there are no reports linking transferrin in the diamondback moth with its sensitivity to Metarhizium anisopliae. Summary of the Invention

[0005] The present invention provides a polyclonal antibody against the PxTsf1 protein to enhance the insecticidal activity of Metarhizium anisopliae against the diamondback moth. Using this polyclonal antibody to treat diamondback moth not only increases the moth's sensitivity to Metarhizium anisopliae, significantly increasing the mortality rate of P. xylostella infected with Metarhizium anisopliae, but also affects the ovarian development and fertility of female P. xylostella moths, reducing their emergence rate. This polyclonal antibody can be used to control P. xylostella and other P. xylostella pests.

[0006] The first object of the present invention is to provide a polyclonal antibody against PxTsf1 protein.

[0007] The second object of the present invention is to provide a use of an inhibitor of PxTsf1 protein in controlling or preparing a product for controlling Plutella xylostella pests.

[0008] The third object of the present invention is to provide a use of an inhibitor of PxTsf1 protein in reducing or preparing a product for reducing the emergence rate of Plutella xylostella pests.

[0009] The fourth object of the present invention is to provide a use of an inhibitor of PxTsf1 protein in reducing or preparing a product for reducing the fertility of female Plutella xylostella pests.

[0010] The fifth object of the present invention is to provide a use of an inhibitor of PxTsf1 protein in improving or preparing a product for improving the sensitivity of Plutella xylostella pests to Metarhizium anisopliae.

[0011] A sixth object of the present invention is to provide a preparation for controlling Plutella xylostella pests.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The present invention uses the amino acid sequence 105-277 of the PxTsf1 protein (i.e., the amino acid sequence shown in SEQ ID NO. 3) as a target fragment. Prokaryotic expression of this target fragment yields the recombinant protein rcp-PxTsf1. New Zealand white rabbits were immunized with the recombinant protein rcp-PxTsf1 as an antigen to generate the anti-PxTsf1 polyclonal antibody. Treatment of diamondback moth pupae with the polyclonal antibody revealed that injection of the anti-rcp-PxTsf1 polyclonal antibody to antagonize PxTsf1 delayed ovarian development in female diamondback moths, reduced their fecundity, and lowered their emergence rate. It also significantly increased their susceptibility to Metarhizium anisopliae, significantly increasing mortality in diamondback moths infected with Metarhizium anisopliae. In other words, antagonizing or inhibiting PxTsf1 can control P. diamondback moth and other Plutella xylostella pests by reducing their emergence rate and susceptibility to Metarhizium anisopliae. Therefore, the present invention claims protection for the polyclonal antibody anti-rcp-PxTsf1.

[0014] The present invention provides a polyclonal antibody against PxTsf1 protein. The polyclonal antibody is obtained by immunizing an animal with a recombinant protein having an amino acid sequence as shown in SEQ ID NO.3.

[0015] In a specific embodiment of the present invention, the amino acid sequence of the recombinant protein is shown as SEQ ID NO.4.

[0016] In a specific embodiment of the present invention, the animal is a New Zealand white rabbit.

[0017] Given that antagonizing PxTsf1 protein can delay ovarian development in female diamondback moths, reduce their fertility, decrease their emergence rate, and significantly increase their susceptibility to Metarhizium anisopliae, the present invention seeks to protect the following applications of inhibitors of PxTsf1 protein:

[0018] The present invention claims protection for the use of an inhibitor of PxTsf1 protein in controlling or preparing a product for controlling Plutella xylostella pests.

[0019] The present invention also claims the use of the inhibitor of PxTsf1 protein in reducing or preparing a product for reducing the emergence rate of Plutella xylostella pests.

[0020] The present invention also claims to protect the use of the inhibitor of PxTsf1 protein in reducing or preparing a product for reducing the fertility of female Plutella xylostella pests.

[0021] The present invention also claims the use of the inhibitor of PxTsf1 protein in delaying or in preparing a product for delaying the ovarian development of female Plutella xylostella pests.

[0022] The present invention also claims to protect the use of an inhibitor of PxTsf1 protein in improving or preparing a product for improving the sensitivity of Plutella xylostella pests to Metarhizium anisopliae.

[0023] Specifically, the amino acid sequence of the PxTsf1 protein is shown in SEQ ID NO.1.

[0024] Optionally, the inhibitor of PxTsf1 protein is an antibody against PxTsf1 protein.

[0025] Specifically, the antibody is a polyclonal antibody.

[0026] More specifically, the polyclonal antibody is the polyclonal antibody anti-rcp-PxTsf1 of the present invention.

[0027] Specifically, the Plutella xylostella pest is Plutella xylostella.

[0028] The present invention also provides a preparation for preventing and controlling Plutella xylostella pests, which contains an inhibitor of PxTsf1 protein and Metarhizium anisopliae.

[0029] Specifically, the amino acid sequence of the PxTsf1 protein is shown in SEQ ID NO.1.

[0030] Specifically, the inhibitor of PxTsf1 protein is an antibody against PxTsf1 protein.

[0031] Specifically, the antibody is a polyclonal antibody.

[0032] More specifically, the polyclonal antibody is the polyclonal antibody anti-rcp-PxTsf1 of the present invention.

[0033] Specifically, the green muscardine is Metarhizium anisopliae.

[0034] Specifically, the Plutella xylostella pest is Plutella xylostella.

[0035] The present invention also provides a method for preventing and controlling Plutella xylostella pests, which comprises treating the Plutella xylostella pests with an inhibitor of PxTsf1 protein during their pupal stage.

[0036] Specifically, the Plutella xylostella pest is Plutella xylostella.

[0037] The present invention has the following beneficial effects:

[0038] The present invention utilizes the recombinant protein rcp-PxTsf1 of the PxTsf1 protein to immunize New Zealand white rabbits, generating a polyclonal antibody, anti-rcp-PxTsf1, capable of resisting the PxTsf1 protein. Using the resulting polyclonal antibody, anti-rcp-PxTsf1, to treat diamondback moth pupae revealed that antagonizing the PxTsf1 protein delayed ovarian development in female diamondback moths, reduced their fertility, and simultaneously decreased their emergence rate, increasing their susceptibility to Metarhizium anisopliae, and increasing the mortality rate of P. diamondback moths infected with Metarhizium anisopliae. In other words, PxTsf1 protein inhibitors can control P. diamondback moth by reducing its fertility, emergence rate, and susceptibility to Metarhizium anisopliae, providing a new direction for the green control of P. diamondback moths and other Plutella decidua pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The PCR amplification results of the gene sequence encoding the target fragment; lane M in the figure is a 2000 bp DNA marker; lane 1 is the PCR amplification product.

[0040] Figure 2 The results of enzyme digestion identification of the recombinant expression vector pET-28a-PxTsf1 are shown; lane M is a 10kb DNA marker; lane 1 is an undigested pET-28a circular vector; lane 2 is a single-digested pET-28a linear vector; and lane 3 is a single-digested pET-28a-rcp-PxTsf1 linear recombinant vector.

[0041] Figure 3Figure 1 is the SDS-PAGE and Western blot results of the recombinant protein rcp-PxTsf1; Figure A is the SDS-PAGE detection result, stained with Coomassie Brilliant Blue; Figure B is the Western blot detection result, the primary antibody is anti-His; lane M is the 10-180kDa protein marker; lane 1 is the uninduced bacterial solution; lane 2 is the whole bacteria after induction; lane 3 is the supernatant after induction; lane 4 is the precipitate after induction; lane 5 is the purified recombinant protein rcp-PxTsf1.

[0042] Figure 4 Figure 1 is the titer test result of the polyclonal antibody anti-rcp-PxTsf1 (anti-PxTsf1) and the analysis results of Western blot analysis of recombinant protein rcp-PxTsf1 and Plutella xylostella PxTsf1 protein using it as the primary antibody; Figure A is the titer test result of the polyclonal antibody anti-rcp-PxTsf1; Figure B is the analysis result of Western blot analysis of recombinant protein rcp-PxTsf1 using the polyclonal antibody anti-rcp-PxTsf1 as the primary antibody, lane M in the figure is a 10-180kDa protein marker, lane 1 is an uninduced bacterial solution, and lane 2 is the purified recombinant protein rcp-PxTsf1; Figure C is the Western blot analysis of Plutella xylostella PxTsf1 protein using the polyclonal antibody anti-rcp-PxTsf1 as the primary antibody The results of blot analysis: lane M in the figure is a 10-180 kDa protein marker, and lane 1 is the fat body tissue protein of the fourth instar larvae of the diamondback moth.

[0043] Figure 5 Figure 3 shows the development of the ovaries of the diamondback moth in normal condition and after antagonism of the diamondback moth PxTsf1 protein; Figure A shows the development of the ovaries of the diamondback moth in normal condition; Figure B shows the development of the ovaries of the diamondback moth after injection of anti-rcp-PxTsf1 to antagonize the diamondback moth PxTsf1 protein; PBS: blank control injected with phosphate buffered saline (PBS); IgG: negative control injected with rabbit immunoglobulin G; anti-PxTsf1: treatment group injected with anti-rcp-PxTsf1; Pupae: pupal stage; Adult: adult.

[0044] Figure 6 The figure shows the effect of anti-rcp-PxTsf1 injection on the emergence time of Plutella xylostella pupae. The emergence rate was analyzed by Cox regression method. Significant differences are indicated by *, ***P<0.001.

[0045] Figure 7Figure 3 shows the effect of anti-rcp-PxTsf1 injection on the reproductive capacity of female diamondback moth; Figure A shows the statistical results of the total egg production of the diamondback moth in the first three days; Figure B shows the statistical results of the number of larvae hatched from eggs; Figure C shows the statistical results of the egg hatching rate; different lowercase letters indicate significant differences.

[0046] Figure 8 Figure 3 shows the effect of injection of anti-rcp-PxTsf1 on the immunity of diamondback moth to Metarhizium anisopliae; Figure A shows the survival curves of diamondback moth pupae with different injection treatments infected with Metarhizium anisopliae; Figure B shows the emergence rate of diamondback moth pupae with different injection treatments infected with Metarhizium anisopliae; PBS+Ma represents diamondback moths infected with Metarhizium anisopliae spore suspension after injection of phosphate buffer; IgG+Ma represents diamondback moths infected with Metarhizium anisopliae spore suspension after injection of rabbit immunoglobulin G; anti-PxTsf1+Ma represents diamondback moths infected with Metarhizium anisopliae spore suspension after injection of anti-rcp-PxTsf1 antibody; survival curves were analyzed by Cox regression method, significance analysis was indicated by *, ***P<0.001; emergence rate data were analyzed by one-way analysis of variance, different lowercase letters indicate significant differences. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0048] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0049] Example 1 Preparation of polyclonal antibodies against PxTsf1 protein

[0050] The PxTsf1 protein of the present invention is a Plutella xylostella transferrin protein containing a PBP2_transferrin domain, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO.2.

[0051] The present invention analyzes the sequence of the PxTsf1 protein and selects amino acids 105 to 277 of the PxTsf1 protein, i.e., the amino acid sequence shown in SEQ ID NO. 3, as the target fragment. A corresponding prokaryotic expression vector is constructed and the recombinant protein rcp-PxTsf1 is obtained through prokaryotic expression. The amino acid sequence of the recombinant protein is shown in SEQ ID NO. 4. New Zealand white rabbits were immunized with the recombinant protein rcp-PxTsf1 as an antigen to obtain a polyclonal antibody, anti-rcp-PxTsf1, which can block the PxTsf1 protein.

[0052] The specific preparation process of the polyclonal antibody anti-rcp-PxTsf1 is as follows:

[0053] 1. PCR amplification of the gene sequence encoding the target fragment

[0054] Based on the gene sequence shown in SEQ ID NO.2, the present invention designed a pair of PCR primers for amplifying the gene sequence encoding the target fragment, and simultaneously introduced restriction enzyme sites BamH I and Xho I into the primers, respectively, to construct a recombinant expression vector of rcp-PxTsf1.

[0055] The nucleotide sequences (5'→3') of the PCR primers are as follows:

[0056] Primer F: cgcggatccgcgCGGACTGATGAGGAGCCAGAT (SEQ ID NO.5)

[0057] Primer R: gtggtggtggtggtgctcgagGTAGGCGAAGTTGTCCGGG (SEQ ID NO.6)

[0058] Fourth-instar larvae of the diamondback moth were disinfected with 75% ethanol and washed with phosphate-buffered saline (PBS). The washed larvae were placed in a 1.5 mL RNase-free centrifuge tube, and total RNA from the diamondback moth was extracted using Trizol reagent (Novagen). The quality and concentration of the obtained total RNA were measured using an ultramicro spectrophotometer and stored at -80°C. 1 μg of the extracted total RNA from the diamondback moth was reverse transcribed into first-strand cDNA using the Color Reverse Transcription Kit (with gDNA Remover) (EZB), and this was used as a template for PCR amplification of the gene sequence encoding the antigen rcp-PxTsf1. The reaction system was 25 μL and contained the following reagents: 2× SuperNova PCR Mix; 12.5 μL, 0.75 μL each of primers F and R, 1 μL of cDNA template, and 10 μL of sterile water; PCR reaction conditions are as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, for a total of 35 cycles; 72°C extension for 5 min; cooling to 4°C; after the completion of PCR amplification, the amplified products were analyzed by gel electrophoresis and the electrophoresis results were observed using a gel imager. The results are as follows Figure 1 As shown. Figure 1It can be seen that the PCR amplification product is a single band, and the size is consistent with the expectation (the length of the gene sequence encoding the target fragment is 519 bp), indicating that the present invention successfully amplified the gene sequence encoding the target fragment, and the PCR amplification product was purified and recovered.

[0059] 2. Construction of recombinant expression vector pET-28a-PxTsf1

[0060] The purified and recovered PCR amplification product and vector pET-28a were double-digested with restriction endonucleases BamH I and Xho I, respectively. The digested PCR amplification product and vector pET-28a were ligated and transformed into DH5α competent cells. The competent cells were spread on LB plates containing 50 μg / mL kanamycin sulfate. The LB plates were placed in a 37°C incubator for overnight culture. Single colonies were selected for streaking and preservation. The recombinant plasmids in the preserved single colonies were extracted for enzyme digestion identification and sequencing. The recombinant plasmid was extracted using the FastPure Plasmid Mini Kit (Novozymes), and polyacrylamide gel electrophoresis was performed after single enzyme digestion. The electrophoresis results were observed using a gel imager. The results are as follows: Figure 2 The full length of the vector pET-28a is 5369 bp, and the full length of the recombinant expression vector pET-28a-PxTsf1 is 5854 bp. Figure 2 Lane 1 is the undigested pET-28a circular vector, lane 2 is the single-digested pET-28a linear vector, and lane 3 is the single-digested pET-28a-rcp-PxTsf1 linear recombinant vector. Figure 2 It can be seen that the present invention has successfully constructed the recombinant expression vector. The positive clones with the correct electrophoresis band size were sent to Qingke Company for sequencing to verify the correctness of the sequence.

[0061] 3. Obtaining the recombinant protein rcp-PxTsf1

[0062] The recombinant expression vector pET-28a-PxTsf1 verified to be correct by sequencing was transformed into Escherichia coli Rosetta (DE3) competent cells, spread on LB plates containing 50 μg / mL kanamycin sulfate, and placed the LB plates in a 37°C incubator for overnight culture; single transformed colonies were picked and placed in 50 mL liquid LB medium containing 50 μg / mL kanamycin sulfate and 25 μg / mL chloramphenicol, and placed in a 37°C shaker at 200 rpm for overnight culture; 10 mL of overnight cultured seed bacterial liquid was added to 1 L liquid LB medium containing 50 μg / mL kanamycin sulfate and 25 μg / mL chloramphenicol, and placed in a 37°C shaker at 200 rpm for 4 h; when the bacterial liquid was at 6 When the absorbance at 00nm was about 0.6, IPTG solution was added to LB liquid culture medium to a final concentration of 0.5mM, and the culture was placed in a shaker at 16°C and induced for 12h at 150rpm. After induction, the culture was centrifuged at 4000rpm for 20min at 4°C, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS, centrifuged at 10000rpm for 3min at 4°C, and the precipitate was washed three times. The precipitate was resuspended in Resupension buffer and placed in an ultrasonic crusher (60hz, run for 10s, stop for 10s, 6min) for one time, centrifuged at 4°C, 12000rpm for 15min, the supernatant was discarded, and the inclusion body precipitate was collected. Lysis Buffer was added to dissolve the inclusion bodies overnight, and the centrifuge tube was placed on a small shaker and dissolved overnight at 4°C; centrifugation was carried out at 8000 rpm for 30 min at 4°C, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the target protein solution, which was stored at -80°C for later use; the target protein solution was added to a nickel ion affinity chromatography column, and impurities were eluted using 50 mL Wash Buffer A, 30 mL Wash Buffer B, and 20 mL Wash Buffer C in sequence. The protein was collected using 10 mL Elution Buffer A and 6 mL Elution Buffer B in sequence to obtain the purified protein. The purified protein solution was stored at -20°C and a small amount was taken for SDS-PAGE and Western blot detection.

[0063] In addition to the purified recombinant protein rcp-PxTsf1, the present invention also took the bacterial solution not induced by IPTG, the whole bacteria after IPTG induction, the supernatant after induction and the precipitate after induction to perform SDS-PAGE and Western blot detection. The results are as follows: Figure 3 As shown; Figure 3 A in the figure is the result of SDS-PAGE detection. Figure 3 B in Figure 2 is the result of Western blot analysis. The predicted molecular weight of the recombinant protein rcp-PxTsf1 is 23.99 kDa. Figure 3It can be seen that the size of the recombinant protein purified by the present invention is consistent with the expectation and there is a single band, indicating that the present invention successfully obtained the recombinant protein rcp-PxTsf1, which was stored at -80°C for future use.

[0064] 4. Acquisition of polyclonal antibody anti-rcp-PxTsf1

[0065] The present invention uses the obtained recombinant protein rcp-PxTsf1 as an antigen to prepare a polyclonal antibody anti-rcp-PxTsf1 by immunizing New Zealand white rabbits. In the first immunization, the recombinant protein rcp-PxTsf1, i.e., the antigen concentration is 1 mg / mL, and each rabbit is injected with 0.5 mL. The amount of antigen is halved for the second to fourth immunizations. The adjuvant and antigen are mixed in a 1:1 volume ratio, with a complete adjuvant used for the first immunization and an incomplete adjuvant used for the second to fourth immunizations. Multiple subcutaneous injections are performed, with 0.2 mL injected at each site. The second immunization is performed 14 days after the first immunization, with a 7-day interval between the second and third immunizations. Seven days after the third immunization, a small serum sample is collected from the rabbit's middle ear artery for testing. After the test is qualified, a booster immunization is performed seven days later. Seven days after the booster immunization, whole blood can be collected and the serum is purified to obtain a purified polyclonal antibody anti-rcp-PxTsf1.

[0066] 5. Detection of the effect of polyclonal antibody anti-rcp-PxTsf1

[0067] The ELISA method was used to detect the titer of rabbit serum and purified antibody. The antigen was diluted to 1 μg / mL with coating buffer (Na2CO3 and NaHCO3 buffer), 50 μL was added to each reaction well of the polystyrene plate, and the plate was incubated at 4°C overnight. The solution in the well was discarded and the plate was washed once with 1×PBST washing buffer at 180 μL per well; 150 μL of 1% BSA (prepared with PBST) was added to each well for blocking, and the plate was incubated at 37°C for 1 hour before the blocking solution was discarded; the sample to be tested was diluted according to a certain ratio and 50 μL was added to the blocked reaction well. At the same time, a negative control well (1% BSA), incubated at 37°C for 30 minutes; washed three times with 1×PBST washing buffer at 150 μL per well, added freshly diluted secondary antibody-HRP (diluted with 1% BSA) to the ELISA plate at 50 μL / well, incubated at 37°C for 45 minutes, and washed three times with 1×PBST buffer at 150 μL per well; added 50 μL of temporarily prepared TMB substrate solution to each reaction well, reacted at 37°C for 5 minutes; added 50 μL of 1 M sulfuric acid to each reaction well to terminate the reaction; placed the ELISA plate in a preheated ELISA reader (450 nm) for reading, saved the data and analyzed.

[0068] The test results of rabbit serum titer and purified polyclonal antibody anti-rcp-PxTsf1 titer are as follows Figure 4 As shown in A. As can be seen from the figure, the antibody titer of the serum of rabbits immunized with the recombinant protein rcp-PxTsf1 reaches 1:64000, indicating that the recombinant protein rcp-PxTsf1 prepared by the present invention has high immunogenicity.

[0069] In addition, the present invention used the recombinant protein rcp-PxTsf1 and the fat body tissue of diamondback moth (the PxTsf1 protein is highly expressed in this tissue) to test the purification effect of the obtained polyclonal antibody anti-rcp-PxTsf1 by Western blot. The results are as follows: Figure 4 As shown in B and C in FIG. As shown in FIG. , the polyclonal antibody anti-rcp-PxTsf1 purified by the present invention can specifically recognize the recombinant protein rcp-PxTsf1 ( Figure 4 The purified polyclonal antibody anti-rcp-PxTsf1 can also specifically recognize the PxTsf1 protein in the fat body of Plutella xylostella, which is 76.50 kDa ( Figure 4 C).

[0070] The above results showed that the polyclonal antibody anti-rcp-PxTsf1 obtained by immunizing New Zealand white rabbits with the recombinant protein rcp-PxTsf1 as an antigen can specifically recognize the PxTsf1 protein in the fat body tissue of Plutella xylostella.

[0071] Example 2 Effect of polyclonal antibody anti-rcp-PxTsf1 on ovarian development of Plutella xylostella

[0072] 1. Rearing of diamondback moth

[0073] The diamondback moth (Plutella xylostella) was cultured in an artificial climate chamber at 25 ± 1°C, 65 ± 5% relative humidity, and a 14 L:10 D light-dark cycle. Larvae were fed an artificial diet and transferred to insect cages during the pupal stage. After emerging as adults, they were fed a diet containing 20% ​​honey water. After mating, blank egg cards were placed in the cages. These egg cards were made by streaking Parafilm M laboratory sealing film and soaking it in vegetable juice to attract female insects to lay eggs.

[0074] The artificial feed comprises: 40g yeast powder, 75g wheat germ powder, 2g multivitamins, 2g sorbic acid, 2g paraben, 2g ascorbic acid, 20g sucrose, 6g radish seeds, 12g agar, 2mL rapeseed oil, 3-4 drops of linoleic acid and 500mL water.

[0075] 2. Ovarian development of diamondback moth

[0076] The ovarian tissues of Plutella xylostella from the 1st to 3rd day of the pupal stage and the female adults after eclosion were observed using an MC170 HD Microscope camera. Photos were taken and saved.

[0077] The results of observation on the ovarian development of Plutella xylostella are as follows: Figure 5 As shown in Figure A, at 24 hours into the pupal stage, the ovary is small, the ovarioles appear slender and transparent, and lack distinct egg chamber differentiation, marking the early vitellogenesis stage. By 48 hours into the pupal stage, the ovarioles begin to form distinct egg chamber structures, accompanied by initial yolk deposition, marking the entry into the vitellogenesis stage. By 60 hours into the pupal stage, the ovarioles increase in diameter, with significant yolk deposition, marking the vitellogenesis stage. By 72 hours into the pupal stage, the ovarioles continue to expand, with significantly increased yolk deposition and the formation of a small number of mature eggs. After adult emergence, multiple fully mature eggs are visible within the ovarioles, accompanied by eggshell formation, marking the chorion stage. In summary, ovarian development in Plutella xylostella is synchronized with oogenesis. As the ovarioles lengthen and thicken, yolk protein gradually deposits, the eggs expand and enlarge, and ovarian development gradually completes.

[0078] 3. Effects of polyclonal antibody anti-rcp-PxTsf1 on ovarian development of Plutella xylostella

[0079] Female diamondback moths at 0h in the pupal stage were microinjected with 0.2μL PBS, IgG (purchased from Biyuntian Biotechnology Co., Ltd., as an antibody negative control, 9.24mg / mL) and 9.24mg / mL of polyclonal antibody anti-rcp-PxTsf1, respectively, and then placed in an incubator for further culture. Thirty female pupae were treated in each experimental group, and the ovaries were dissected 72h after treatment and 66h after untreated pupal stage. The ovarian tissue was photographed using an MC170 HD Microscope camera and the images were saved. At the same time, the area of ​​the first 10 egg chambers of each ovarian tube was measured using Image J software, and the plotting and difference significance analysis were performed using Graphpad Prism 8 software. The results are shown in Figure 2. Figure 5 As shown in Figure B, ovarian development was significantly different in the groups treated 72 hours after polyclonal antibody anti-rcp-PxTsf1 injection compared with the controls 72 hours after PBS or IgG injection (P<0.05). However, there was no significant difference in ovarian development in the groups treated 72 hours after anti-rcp-PxTsf1 injection compared with the untreated control group at 66 hours of the pupal stage. These results indicate that polyclonal antibody anti-rcp-PxTsf1 delays ovarian development in Plutella xylostella.

[0080] Example 3 Effect of polyclonal antibody anti-rcp-PxTsf1 on the emergence rate of Plutella xylostella

[0081] The rearing of diamondback moth was the same as in Example 2. Female diamondback moths at the pupal stage 0 h were microinjected with PBS, IgG, and anti-rcp-PxTsf1, respectively, and then placed in an incubator for further culture. 100 female pupae were treated in each experimental group. Starting from 72 h after treatment, the emergence of diamondback moths in each experimental group was observed and recorded every 6 h until 120 h after treatment. The results are shown in Table 1. Figure 6 shown.

[0082] Depend on Figure 6 It can be seen that 72 hours after injection treatment, the eclosion rate of the PBS group was 62.5%, the eclosion rate of the IgG group was 61.7%, and the eclosion rate of the anti-rcp-PxTsf1 group was only 19.17%; 78 hours after injection treatment, the eclosion rate of the PBS group was 83.72%, the eclosion rate of the IgG group was 77.24%, and the eclosion rate of the anti-rcp-PxTsf1 group was only 45.82%; 84 hours after injection treatment, the eclosion rate of the PBS group was 89.15%, the eclosion rate of the IgG group was 88.27%, and the eclosion rate of the anti-rcp-PxTsf1 group was only 68.63%; 120 hours after injection treatment, the eclosion rate of the PBS group was 100%, the eclosion rate of the IgG group was 98.62%, and the eclosion rate of the anti-rcp-PxTsf1 group was only 77.5%. Survival analysis results showed that there was a significant difference in the adult eclosion time between the anti-rcp-PxTsf1 group and the control group (P<0.05), the average eclosion time was delayed by 6 hours, and the eclosion rate was also significantly reduced (P<0.0001).

[0083] Example 4 Effect of polyclonal antibody anti-rcp-PxTsf1 on the reproductive capacity of Plutella xylostella

[0084] The rearing of diamondback moths was conducted in the same manner as in Example 2. Female diamondback moths at the pupal stage of 0 h were microinjected with PBS, IgG, and anti-rcp-PxTsf1, respectively. The moths were then placed in an incubator for further culture. After the female adults emerged from their pupae, they were paired with newly emerged males. Thirty female pupae were treated in each experimental group. Each pairing device contained one female adult, two male adults, a 2 cm x 2 cm egg card, and a cotton ball soaked in 20% honey solution. The egg cards were replaced every 24 h, and the egg production during the first 72 h and the hatching rate during the last 72 h were recorded.

[0085] The results of the effect of polyclonal antibody anti-rcp-PxTsf1 on the reproductive capacity of diamondback moth are as follows Figure 7 As shown; Figure 7 A in the figure is the total number of eggs laid by the diamondback moth in the first three days; B in the figure is the number of larvae hatched from the eggs; and C in the figure is the egg hatching rate. Figure 7When females treated with anti-rcp-PxTsf1 were mated with untreated males, their egg production decreased by 45% (from 162 eggs per female to 73 eggs per female) compared to the negative control (IgG-treated females mating with untreated males). The egg hatching rate also decreased significantly from 72.5% to 36.0%. There were no significant differences in egg production or hatching rate between the blank control (PBS-treated females mating with untreated males) and the negative control. These results suggest that blocking PxTsf1 during the pupal stage through antibody immunotherapy can reduce the reproductive capacity of female diamondback moths.

[0086] Example 5 Polyclonal Antibody Anti-rcp-PxTsf1 Reduces Susceptibility of Plutella xylostella to Metarhizium Infection

[0087] 1. Cultivation of Metarhizium anisopliae and Preparation of Spore Suspension

[0088] In a clean bench, use a sterile toothpick to pick up conidia from a plate of Metarhizium anisopliae (MaqS1902) and tap the plate five times on a new PDA plate. After sealing, place the plate in an incubator for incubation. After ~2 days of incubation, white hyphae growth can be observed, and after two weeks, a large number of conidia should be produced on the plate. Subsequently, in the clean bench, pour an appropriate amount of spore germination solution into a sterile conical flask, use a spatula to scrape conidia from the M. anisopliae plate, and collect them in the conical flask. After sealing, place the conical flask on a shaker and incubate at room temperature for 1 hour. After the spore suspension in the conical flask is thoroughly mixed, filter it through four layers of gauze in the clean bench into a new sterile conical flask. Finally, place the filtered suspension on a shaker and incubate it overnight at room temperature for subsequent infection experiments.

[0089] 2. Effects of polyclonal antibody anti-rcp-PxTsf1 on the survival and emergence rates of diamondback moth pupae infected with Metarhizium anisopliae

[0090] The diamondback moth at the pupal stage of 0 h was selected and injected with PBS, IgG and anti-rcp-PxTsf1 respectively using a microinjector, and then with a concentration of 1×10 6 Pupae were infected with a spore suspension of Metarhizium anisopliae at a concentration of CFU / mL and cultured in an incubator; 60 pupae were treated in each experimental group, and the mortality of each experimental group was observed every 12 hours starting from 0 hours after treatment until the diamondback moth emerged.

[0091] Effects of polyclonal antibody anti-rcp-PxTsf1 on the survival and emergence rates of diamondback moth pupae infected with Metarhizium anisopliae Figure 8 As shown; Figure 8Figure A shows the effect on the survival rate of diamondback moth pupae infected by Metarhizium; Figure B shows the effect on the emergence rate of diamondback moth pupae infected by Metarhizium. Figure 8 As shown in A, the survival rate of the diamondback moth group infected with Metarhizium anisopliae after injection of anti-rcp-PxTsf1 was significantly lower than that of the negative control group (IgG+Ma). Figure 8 As shown in Figure B, the emergence rate of P. xylostella pupae infected with Metarhizium anisopliae after injection of anti-rcp-PxTsf1 was significantly lower than that of the negative control group (IgG+Ma) and the negative control group (PBS+Ma), with a highly significant difference (P<0.0001). However, the emergence rate of P. xylostella pupae treated with PBS+Ma and IgG+Ma was not significantly different (P>0.05). These results indicate that antagonizing PxTsf1 protein significantly increases the sensitivity of P. xylostella pupae to Metarhizium anisopliae infection.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A polyclonal antibody against PxTsf1 protein, characterized in that: The polyclonal antibody is obtained by immunizing animals with a recombinant protein having an amino acid sequence as shown in SEQ ID NO.

3.

2. Use of an inhibitor of PxTsf1 protein in controlling or preparing a product for controlling Plutella xylostella pests, characterized in that: The amino acid sequence of the PxTsf1 protein is shown in SEQ ID NO.

1.

3. Use of an inhibitor of PxTsf1 protein in reducing or preparing a product for reducing the emergence rate of Plutella xylostella pests, characterized in that: The amino acid sequence of the PxTsf1 protein is shown in SEQ ID NO.

1.

4. Use of an inhibitor of PxTsf1 protein in reducing or preparing a product for reducing the reproductive capacity of female Plutella xylostella pests, characterized in that: The amino acid sequence of the PxTsf1 protein is shown in SEQ ID NO.

1.

5. Use of an inhibitor of PxTsf1 protein in increasing or preparing a product for increasing the sensitivity of Plutella xylostella pests to Metarhizium anisopliae, characterized in that: The amino acid sequence of the PxTsf1 protein is shown in SEQ ID NO.

1.

6. The use according to any one of claims 2 to 5, characterized in that: The inhibitor of PxTsf1 protein is an antibody against PxTsf1 protein.

7. The application according to claim 6, characterized in that The antibody is the polyclonal antibody according to claim 1.

8. A preparation for controlling Plutella xylostella pests, characterized in that: An inhibitor containing PxTsf1 protein and Metarhizium anisopliae; the amino acid sequence of the PxTsf1 protein is shown in SEQ ID NO.

1.

9. The preparation according to claim 8, characterized in that The inhibitor of PxTsf1 protein is an antibody against PxTsf1 protein.

10. The preparation according to claim 8, characterized in that The Metarhizium anisopliae is Metarhizium anisopliae.

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