CN113845579B - Preparation method of immune lectin for improving killing efficiency of beauveria bassiana on cotton bollworms - Google Patents
Preparation method of immune lectin for improving killing efficiency of beauveria bassiana on cotton bollworms Download PDFInfo
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- CN113845579B CN113845579B CN202111348007.9A CN202111348007A CN113845579B CN 113845579 B CN113845579 B CN 113845579B CN 202111348007 A CN202111348007 A CN 202111348007A CN 113845579 B CN113845579 B CN 113845579B
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- C12N15/09—Recombinant DNA-technology
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- C12N2800/00—Nucleic acids vectors
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- C12N2800/101—Plasmid DNA for bacteria
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Abstract
The invention relates to a preparation method of an immune lectin for improving killing efficiency of beauveria bassiana on cotton bollworms, which utilizes a rapid cloning technology to clone cotton bollworms CTL26 into a pET-28a vector, and obtains active recombinant proteins in vitro. Through a microinjection mode, an infection experiment is carried out on the cotton bollworm larva, the beauveria bassiana can be used for effectively killing cotton bollworms, the beauveria bassiana treated by the recombinant CTL26 is used for obviously improving the killing efficiency of cotton bollworms, and the protein provides a usable target for developing novel biological pesticides. The cotton bollworm immune lectin CTL26 disclosed by the invention can improve the killing efficiency of beauveria bassiana to pests, and has practical economic benefits.
Description
Technical Field
The invention relates to a preparation method of an immune lectin for improving the killing efficiency of beauveria bassiana on cotton bollworms, which can improve the killing efficiency of beauveria bassiana on cotton bollworms and is used as a target for cotton bollworms control, and belongs to the technical field of biology.
Background
Cotton bollworm is a wide feeding lepidopteran pest, and eating more than 200 plants including cotton brings huge losses to agriculture. The traditional chemical pesticide control method can cause the cotton bollworms to generate drug resistance and cause serious environmental problems. Biological control is becoming a pest control strategy of great interest due to its specificity to pests and environmental friendliness. The entomogenous fungi represented by beauveria bassiana are widely used biological control factors at present, can infect more than 700 insects, and are harmless to warm-blooded animals such as environment, human and the like, and have low culture cost and strong pathogenicity. The infection of the insect by beauveria bassiana is firstly completed by attaching the beauveria bassiana to the surface of the insect, the attached spores germinate on the surface of the insect and form attached cells, and then fungi secrete various enzymes (such as chitinase, lipase and protease) to damage the integrity of the insect epidermis, so that the spores enter the insect body (Butt et al, 2016) and propagate therein, and various virulence factors are generated simultaneously, and act on various physiological processes of the immunity, metabolism and the like of the insect to cause disorder and imbalance, and finally cause death of the insect (Cen et al, 2017; valero-Jimez et al, 2016). The immune system of the cotton bollworm can identify pathogenic matters such as fungi and the like, and transmit information to effector cells to generate immune effect. Immunoagglutinin (CTL) is an important class of immune recognition molecules that recognize and bind to "non-hexose" substances through interactions with specific sugar groups on the surface of pathogens, the first step in the initiation of an immune response by the host. The function of the currently known cotton bollworm CTL is mainly represented by reducing pathogenicity of the cotton bollworm CTL by agglutinating pathogens, activating cellular immunity and humoral immunity of a host, inducing encapsulation and phagocytic reaction, inducing expression of antibacterial peptide, and killing the pathogens to a certain extent, thereby reducing harm of the pathogens such as fungi to the host and ensuring survival of the host (Wang et al, 2017; cheng et al, 2017). The immune lectin can obviously improve the killing power of beauveria bassiana to cotton bollworms, enhance the killing effect of the existing biological pesticide to pests under the condition of ensuring safety, reduce the using amount of the pesticide and save the cost of agricultural production.
Disclosure of Invention
Aiming at the problems, the invention utilizes a rapid cloning technology to clone the cotton bollworm CTL26 into a pET-28a vector, obtains active recombinant protein in vitro, and discovers that the recombinant CTL26 can obviously improve the killing efficiency of beauveria bassiana on cotton bollworms through an infection experiment.
The invention is realized by the following technical scheme:
the preparation method of the immune lectin for improving the killing efficiency of beauveria bassiana on cotton bollworms is characterized by comprising the following steps:
step (1), obtaining a pET-28a-CTL26 prokaryotic expression vector;
extracting total fat RNA of cotton bollworms, reversely transcribing the total fat RNA into cDNA, and carrying out PCR (polymerase chain reaction) amplification by taking the cDNA as a template, wherein the upstream primer sequence is GCATGACTGGTGGACAGAAGCCTGATTACTTGTACGA, and the downstream primer sequence is CTAGTTATTGCTCAGCGGTTTCTGTGTAGATCTTCCAAA, thus obtaining a target fragment; determined as CTL26 after sequencing; then, obtaining a pET-28a-CTL26 prokaryotic expression vector by using a seamless cloning method;
and (2) transforming the pET-28a-CTL26 prokaryotic expression vector into competent cells of escherichia coli BL21 for culture, selecting single colony, transferring for culture, adding IPTG for induction, centrifuging to obtain a supernatant containing the CTL26 protein, and purifying by a Ni affinity chromatographic column and ultrafiltering to obtain the denatured CTL26 recombinant protein.
2. The method for preparing an immunoagglutinin for improving killing efficiency of beauveria bassiana to cotton bollworm according to claim 1, wherein in the step (1), the obtained cDNA sequence of CTL26 is as follows: ATGTTAACAATATATTTAATTTGTTTGTCATATTTGTTGATTGCAACTGGTCCCGTGCGATGCAAGCCTGATTACTTGTACGACAGTGAAGTGCAGGGCTGGCTCAAGCTTCACGTGATTCCAGCAACGTGGGAGCAAGCGGTTTTGCGCTGTCACTATGAAGGAGCAGTGCTGGCGTCTCCACTTAACGAAGAGCTGACCAAAGCTCTCCACTCAACAATGACAAAGTTTGGTATCAATCGTTGCATCTTCTTGGGTACCAGTTTGTTGCTCTCCAATGGCGACTTCGTTTCAATAGAAGGCGTCCCACTGTCTGACCAGGAGATACAGTGGAGCCCACAAGGGCCCGATCCAGGACAATGCCTCACCATGGCCATCTCTGGCAGCGAGCACTTCATGTATACCGCGTCATGCGACGAACAACTACCCTACATATGCTATAGAAATCATGACAATAGTACTATGAATGAATGTGGCACATTTGACAACAAATACCACTACAATCAGAAAACTGGCAGCTGTTACAAAGTCCACAACGAGAAACACACCTGGTACCGCGCAAATATGATCTGCTTTGCGGAGGGAGGTCATCTCGTCATCCTGAATGATGACGTGAAAGCTAACATCGTCAAGGATATGTTCCCCGTTCGTACCGATAACACGACGAATTTATGGGAGCAAATCCACATCGGTCTAAAAGCCTGGGACGACCGCATATGGTTTACCATTCACGGTGATAAAATAGACGATGTGTACAACCAATGGGCTGCAGGACAACCAGACAATAAGATGGGCACACAAAATATTGGTACTATTCTTCGGAGTGGCCTCTTGGATGATGCAAATCCTCTCAAACGGAACATGTTTGTGTGCGAGAAAGCAGCTCATAAAATACGCTTCGAATCTCCAAAATTGCGATGGAATGAATTAATTGTACCTTTTGGAAGATCTACACAGAAATAA;
the protein sequences corresponding to the above cDNA sequences are as follows:
MLTIYLICLSYLLIATGPVRCKPDYLYDSEVQGWLKLHVIPATWEQAVLRCHYEGAVLASPLNEELTKALHSTMTKFGINRCIFLGTSLLLSNGDFVSIEGVPLSDQEIQWSPQGPDPGQCLTMAISGSEHFMYTASCDEQLPYICYRNHDNSTMNECGTFDNKYHYNQKTGSCYKVHNEKHTWYRANMICFAEGGHLVILNDDVKANIVKDMFPVRTDNTTNLWEQIHIGLKAWDDRIWFTIHGDKIDDVYNQWAAGQPDNKMGTQNIGTILRSGLLDDANPLKRNMFVCEKAAHKIRFESPKLRWNELIVPFGRSTQK。
the method for detecting the renaturation and activity of the denatured CTL26 recombinant protein comprises the following steps:
firstly, carrying out gradient renaturation on denatured CTL26 recombinant protein for a plurality of times by using a dialysis bag and a protein renaturation solution to obtain soluble protein; finally, the recognition activity of beauveria bassiana is analyzed by agglutination, binding and encapsulation experiments.
The fungus infection experimental method comprises the following specific steps:
three-year-old cotton bollworms with the same size are divided into four groups, PBS, fungal spores, double-stranded RNA aiming at CTL26, fungal spores and fungal spores after incubation of CTL26 recombinant proteins are respectively injected by using a microinjection method, the death condition of larvae is recorded, and the survival rate is analyzed.
The method is advanced and scientific, and the technical scheme adopted by the invention is as follows:
1. the expression and purification method of the CTL26 recombinant protein comprises the specific steps of firstly obtaining a pET-28a-CTL26 prokaryotic expression vector, transforming the prokaryotic expression vector into BL21 competent cells for culture, selecting single colonies, transferring for culture, adding IPTG for induction, centrifuging to obtain a supernatant containing the CTL26 protein, purifying by a Ni affinity chromatography column and ultrafiltering for purification, and obtaining the denatured CTL26 recombinant protein.
2. The method for detecting the renaturation and activity of the recombinant CTL26 comprises the following specific steps: firstly, carrying out gradient renaturation on the denatured CTL26 protein for a plurality of times by using a dialysis bag and a protein renaturation solution to obtain soluble protein; finally, the recognition activity of beauveria bassiana is analyzed by agglutination, binding and encapsulation experiments.
3. The fungus infection experimental method comprises the following specific steps: three-year-old cotton bollworms with the same size are divided into four groups, PBS, fungal spores, double-stranded RNA aiming at CTL26, fungal spores and fungal spores after incubation of CTL26 recombinant proteins are respectively injected by using a microinjection method, the death condition of larvae is recorded, and the survival rate is analyzed.
According to the invention, through an infection experiment on the cotton bollworm larvae in a microinjection mode, beauveria bassiana can be used for effectively killing cotton bollworms, and the killing efficiency of the beauveria bassiana treated by using recombinant CTL26 is obviously improved, so that the protein provides a usable target for developing novel biological pesticides.
The invention utilizes the rapid cloning technology to clone the cotton bollworm CTL26 into the pET-28a vector, and obtains the active recombinant protein in vitro. Through a microinjection mode, an infection experiment is carried out on the cotton bollworm larva, the beauveria bassiana can be used for effectively killing cotton bollworms, the beauveria bassiana treated by the recombinant CTL26 is used for obviously improving the killing efficiency of cotton bollworms, and the protein provides a usable target for developing novel biological pesticides.
The pesticides used in the market at present are mainly chemical pesticides, but the chemical pesticides have proved to have the defect of detail, so that the use of biological pesticides instead of chemical pesticides is a trend of future development. The global bio-pesticide sales in 2020 exceeds 50 hundred million dollars, and the global bio-pesticide sales account for less than 10% of the total pesticide market, and has wide development prospect. In biological pesticides in China, the ratio of microbial (bacteria, fungi and viruses) pesticides in 2020 is 38%, wherein the registration number of beauveria bassiana pesticides is 25, and the beauveria bassiana pesticides are second to bacillus thuringiensis and are second largest microbial pesticides in China. The cotton bollworm immune lectin CTL26 disclosed by the invention can improve the killing efficiency of beauveria bassiana to pests, and has practical economic benefits.
Drawings
FIG. 1 is an electrophoresis chart of CTL26 amplification in the present invention;
FIG. 2 shows purification and renaturation of recombinant CTL26 of the present invention;
FIG. 3 is an aggregation of recombinant CTL26 of the present invention on beauveria bassiana;
FIG. 4 shows the binding of recombinant CTL26 to Beauveria bassiana in the present invention;
FIG. 5 is a graph showing that recombinant CTL26 promotes encapsulation of chromatographic beads by blood cells in the present invention;
FIG. 6 is a survival analysis of fungal infected cotton bollworms in the present invention.
Detailed Description
The invention is further described with reference to the accompanying drawings.
Embodiment one: the expression and purification method of the CTL26 recombinant protein comprises the following specific steps:
1. obtaining a pET28a-CTL26 prokaryotic expression vector:
1.1 extracting total fat body RNA of cotton bollworms, reversely transcribing the total fat body RNA into cDNA, and carrying out PCR (polymerase chain reaction) amplification by taking the cDNA as a template, wherein the upstream primer and the downstream primer are respectively GCATGACTGGTGGACAGAAGCCTGATTACTTGTACGA and CTAGTTATTGCTCAGCGGTTTCTGTGTAGATCTTCCAAA, and the obtained target fragment is shown in figure 1; after sequencing, it was determined as CTL26.
1.2, obtaining a pET28a-CTL26 prokaryotic expression vector by using a seamless cloning method;
2. prokaryotic expression of pET28a-CTL26 recombinant protein:
2.1 converting the obtained pET28a-CTL26 prokaryotic expression vector into competent cells of escherichia coli BL21, culturing, picking single colony, and amplifying culture.
2.2 When the OD600 value of the culture medium reaches about 0.5, IPTG is added into the culture medium to make the final concentration of the IPTG be 0.3mmol/L, and CTL26 protein expression is induced for 4h at 37 ℃.
2.3 Taking the bacterial liquid prepared by the steps, centrifuging and discarding the supernatant. Adding a certain volume of Binding Buffer into the precipitate, mixing uniformly, performing ultrasonic pyrolysis, and centrifuging to obtain a supernatant containing CTL26 protein.
2.4 Adding the obtained supernatant containing CTL26 protein into a chromatographic column containing Ni-Agarose Resin filler, washing the chromatographic column with 15 times of Binding Buffer to wash off the impurity protein, eluting with a proper amount of absorption Buffer in sequence, collecting the eluent, and detecting the eluent by SDS-PAGE (SDS-PAGE), as shown in figure 2; the single eluent is collected, part of impurities are removed by ultrafiltration, and the protein is concentrated.
Embodiment two: the CTL26 recombinant protein renaturation and activity detection comprises the following specific steps:
1. the recombinant protein CTL26 renaturation specifically comprises the following steps:
1.1 use 2% NaHCO 3 And 1mM EDTA to pretreat the dialysis bag.
1.2 The protein renaturation solution is prepared by the following steps:
tris HCl:20mM; naCl:150mM; reduced glutathione: 2mM; oxidized glutathione: 0.02mM; glycerol: 5% (V/V); tween-20:0.005% (V/V); urea: 3M;
tris HCl:20mM; naCl:150mM; reduced glutathione: 2mM; oxidized glutathione: 0.02mM; glycerol: 5% (V/V); tween-20:0.005% (V/V); urea: 1M;
tris HCl:20mM; naCl:150mM; reduced glutathione: 2mM; oxidized glutathione: 0.02mM; glycerol: 5% (V/V); tween-20:0.005% (V/V); urea: 0M.
1.3 The CTL26 protein obtained above was subjected to dialysis renaturation using a dialysis bag and a protein renaturation solution, and the dialysis was performed in three steps, all at 4 ℃.
1.4 After protein renaturation, protein concentration was measured using BCA, calculated to be about 60% renaturation rate, and whether protein was degraded was observed by SDS-PAGE as shown in fig. 2.
2. The recombinant protein CTL26 activity detection specifically comprises the following steps:
taking beauveria bassiana spores expressing green fluorescent protein GFP, preparing a suspension, and counting by using a blood cell counting plate to obtain a spore suspension with the concentration of 1 multiplied by 10 8 And each mL.
2.1 Agglutination experiments:
10. Mu.L of the prepared spore suspension was taken, 10. Mu.g of CTL26 recombinant protein was added thereto, and CaCl was then added at a final concentration of 2mM 2 Or CaCl 2 The mixture with CTL26 polyclonal antibody, while replacing target protein with Bovine Serum Albumin (BSA), as control group, adding PBS to 50 μl, standing at room temperature for 30min, observing beauveria bassiana with fluorescence microscopeAggregation. As shown in FIG. 3, BSA did not agglutinate spores, but did not agglutinate CaCl 2 CTL26 had little agglutination on spores in the presence, while CaCl was added 2 post-CTL 26 produces a pronounced agglutination of spores, while antibodies to CTL26 neutralize its agglutination on to fungi.
2.2 Binding experiments:
after 100 mu L of beauveria bassiana bacterial suspension is added with 100ug of CTL26 recombinant protein and incubated for 45min at room temperature, unbound protein is washed off by using sterile PBS, then the CTL26 recombinant protein bound on the surface of the bacterial body is eluted by using 1% SDS, and the CTL26 antibody is used for detecting whether the corresponding recombinant protein exists in the eluent. The results showed that CTL26 was able to bind efficiently to fungal spores, as shown in figure 4.
2.3 Encapsulation experiments:
small amount of chromatographic beads were taken, washed three times with TBS, suspended in TBS, and CaCl was added 2 To a concentration of 10mM; after adding 10. Mu.g of CTL26 recombinant protein or BSA overnight at 4℃the cells were washed 5 times with TBS; taking 5-year-old larva blood cells, and re-suspending with TBS to 3×10 cells per ml 6 And mixing with recombinant protein or BSA coated chromatographic beads, and incubating overnight at room temperature. The beads were collected and after resuspension with TBS, the encapsulation of the beads was observed under a microscope and showed little encapsulation of the BSA coated beads by the blood cells, whereas the CTL26 coated beads had a significant encapsulation of about 66% as shown in FIG. 5.
Embodiment III: the beauveria bassiana infection cotton bollworm experiment comprises the following specific steps:
1. double-stranded RNA for CTL26 and GFP was synthesized using in vitro transcription, and 2ug of double-stranded RNA for CTL26 or GFP was injected to end-2-year larvae.
After 2.24 hours, 10nL beauveria bassiana spores (1×10) 7 individual/mL) or PBS to the above bollworm, 24 per group, and larval death at various time points was recorded.
3. In 50uL beauveria bassiana (1×10) 7 And b) adding CTL26 recombinant protein 10ug into the bacterial liquid, standing for 1h at room temperature, and injecting the suspension until reaching the cotton boll of the GFP double-stranded RNA injected in advanceIn the insects, larval death was recorded and the survival rates of the different groups were analyzed. As shown in fig. 6, interfering with the expression of CTL26 reduces mortality of cotton bollworms, while recombinant CTL26 increases mortality of cotton bollworms.
In examples one, two and three, the cDNA sequence of CTL26 obtained was as follows: ATGTTAACAATATATTTAATTTGTTTGTCATATTTGTTGATTGCAACTGGTCCCGTGCGATGCAAGCCTGATTACTTGTACGACAGTGAAGTGCAGGGCTGGCTCAAGCTTCACGTGATTCCAGCAACGTGGGAGCAAGCGGTTTTGCGCTGTCACTATGAAGGAGCAGTGCTGGCGTCTCCACTTAACGAAGAGCTGACCAAAGCTCTCCACTCAACAATGACAAAGTTTGGTATCAATCGTTGCATCTTCTTGGGTACCAGTTTGTTGCTCTCCAATGGCGACTTCGTTTCAATAGAAGGCGTCCCACTGTCTGACCAGGAGATACAGTGGAGCCCACAAGGGCCCGATCCAGGACAATGCCTCACCATGGCCATCTCTGGCAGCGAGCACTTCATGTATACCGCGTCATGCGACGAACAACTACCCTACATATGCTATAGAAATCATGACAATAGTACTATGAATGAATGTGGCACATTTGACAACAAATACCACTACAATCAGAAAACTGGCAGCTGTTACAAAGTCCACAACGAGAAACACACCTGGTACCGCGCAAATATGATCTGCTTTGCGGAGGGAGGTCATCTCGTCATCCTGAATGATGACGTGAAAGCTAACATCGTCAAGGATATGTTCCCCGTTCGTACCGATAACACGACGAATTTATGGGAGCAAATCCACATCGGTCTAAAAGCCTGGGACGACCGCATATGGTTTACCATTCACGGTGATAAAATAGACGATGTGTACAACCAATGGGCTGCAGGACAACCAGACAATAAGATGGGCACACAAAATATTGGTACTATTCTTCGGAGTGGCCTCTTGGATGATGCAAATCCTCTCAAACGGAACATGTTTGTGTGCGAGAAAGCAGCTCATAAAATACGCTTCGAATCTCCAAAATTGCGATGGAATGAATTAATTGTACCTTTTGGAAGATCTACACAGAAATAA;
the protein sequences corresponding to the above cDNA sequences are as follows:
MLTIYLICLSYLLIATGPVRCKPDYLYDSEVQGWLKLHVIPATWEQAVLRCHYEGAVLASPLNEELTKALHSTMTKFGINRCIFLGTSLLLSNGDFVSIEGVPLSDQEIQWSPQGPDPGQCLTMAISGSEHFMYTASCDEQLPYICYRNHDNSTMNECGTFDNKYHYNQKTGSCYKVHNEKHTWYRANMICFAEGGHLVILNDDVKANIVKDMFPVRTDNTTNLWEQIHIGLKAWDDRIWFTIHGDKIDDVYNQWAAGQPDNKMGTQNIGTILRSGLLDDANPLKRNMFVCEKAAHKIRFESPKLRWNELIVPFGRSTQK。
sequence listing
<110> university of Yangzhou
<120> method for preparing an immune lectin for improving killing efficiency of beauveria bassiana on cotton bollworms
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> Cotton bollworm (Helicoverpa armigera)
<400> 1
atgttaacaa tatatttaat ttgtttgtca tatttgttga ttgcaactgg tcccgtgcga 60
tgcaagcctg attacttgta cgacagtgaa gtgcagggct ggctcaagct tcacgtgatt 120
ccagcaacgt gggagcaagc ggttttgcgc tgtcactatg aaggagcagt gctggcgtct 180
ccacttaacg aagagctgac caaagctctc cactcaacaa tgacaaagtt tggtatcaat 240
cgttgcatct tcttgggtac cagtttgttg ctctccaatg gcgacttcgt ttcaatagaa 300
ggcgtcccac tgtctgacca ggagatacag tggagcccac aagggcccga tccaggacaa 360
tgcctcacca tggccatctc tggcagcgag cacttcatgt ataccgcgtc atgcgacgaa 420
caactaccct acatatgcta tagaaatcat gacaatagta ctatgaatga atgtggcaca 480
tttgacaaca aataccacta caatcagaaa actggcagct gttacaaagt ccacaacgag 540
aaacacacct ggtaccgcgc aaatatgatc tgctttgcgg agggaggtca tctcgtcatc 600
ctgaatgatg acgtgaaagc taacatcgtc aaggatatgt tccccgttcg taccgataac 660
acgacgaatt tatgggagca aatccacatc ggtctaaaag cctgggacga ccgcatatgg 720
tttaccattc acggtgataa aatagacgat gtgtacaacc aatgggctgc aggacaacca 780
gacaataaga tgggcacaca aaatattggt actattcttc ggagtggcct cttggatgat 840
gcaaatcctc tcaaacggaa catgtttgtg tgcgagaaag cagctcataa aatacgcttc 900
gaatctccaa aattgcgatg gaatgaatta attgtacctt ttggaagatc tacacagaaa 960
taa 963
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<212> PRT
<213> Cotton bollworm (Helicoverpa armigera)
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Gly Pro Val Arg Cys Lys Pro Asp Tyr Leu Tyr Asp Ser Glu Val Gln
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Gly Trp Leu Lys Leu His Val Ile Pro Ala Thr Trp Glu Gln Ala Val
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Leu Arg Cys His Tyr Glu Gly Ala Val Leu Ala Ser Pro Leu Asn Glu
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Glu Leu Thr Lys Ala Leu His Ser Thr Met Thr Lys Phe Gly Ile Asn
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Arg Cys Ile Phe Leu Gly Thr Ser Leu Leu Leu Ser Asn Gly Asp Phe
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Val Ser Ile Glu Gly Val Pro Leu Ser Asp Gln Glu Ile Gln Trp Ser
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Pro Gln Gly Pro Asp Pro Gly Gln Cys Leu Thr Met Ala Ile Ser Gly
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Ser Glu His Phe Met Tyr Thr Ala Ser Cys Asp Glu Gln Leu Pro Tyr
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Ile Cys Tyr Arg Asn His Asp Asn Ser Thr Met Asn Glu Cys Gly Thr
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Phe Asp Asn Lys Tyr His Tyr Asn Gln Lys Thr Gly Ser Cys Tyr Lys
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Val His Asn Glu Lys His Thr Trp Tyr Arg Ala Asn Met Ile Cys Phe
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Ala Glu Gly Gly His Leu Val Ile Leu Asn Asp Asp Val Lys Ala Asn
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Ile Val Lys Asp Met Phe Pro Val Arg Thr Asp Asn Thr Thr Asn Leu
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Trp Glu Gln Ile His Ile Gly Leu Lys Ala Trp Asp Asp Arg Ile Trp
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Phe Thr Ile His Gly Asp Lys Ile Asp Asp Val Tyr Asn Gln Trp Ala
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Ala Gly Gln Pro Asp Asn Lys Met Gly Thr Gln Asn Ile Gly Thr Ile
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Leu Arg Ser Gly Leu Leu Asp Asp Ala Asn Pro Leu Lys Arg Asn Met
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Phe Val Cys Glu Lys Ala Ala His Lys Ile Arg Phe Glu Ser Pro Lys
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Leu Arg Trp Asn Glu Leu Ile Val Pro Phe Gly Arg Ser Thr Gln Lys
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<213> Artificial sequence (Artificial Sequence)
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gcatgactgg tggacagaag cctgattact tgtacga 37
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<213> Artificial sequence (Artificial Sequence)
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ctagttattg ctcagcggtt tctgtgtaga tcttccaaa 39
Claims (2)
1. The preparation method of the immune lectin for improving the killing efficiency of beauveria bassiana on cotton bollworms is characterized by comprising the following steps:
step (1), obtaining a pET-28a-CTL26 prokaryotic expression vector;
extracting total fat RNA of cotton bollworms, reversely transcribing the total fat RNA into cDNA, and carrying out PCR (polymerase chain reaction) amplification by taking the cDNA as a template, wherein the upstream primer sequence is GCATGACTGGTGGACAGAAGCCTGATTACTTGTACGA, and the downstream primer sequence is CTAGTTATTGCTCAGCGGTTTCTGTGTAGATCTTCCAAA, thus obtaining a target fragment; determined as CTL26 after sequencing; then, obtaining a pET-28a-CTL26 prokaryotic expression vector by using a seamless cloning method;
step (2), converting the pET-28a-CTL26 prokaryotic expression vector into competent cells of escherichia coli BL21 for culture, selecting single colony, transferring for culture, adding IPTG for induction, centrifuging to obtain a supernatant containing the CTL26 protein, and purifying by a Ni affinity chromatographic column and ultrafiltering to obtain a denatured CTL26 recombinant protein;
in step (1), the cDNA sequence of CTL26 is obtained as follows: ATGTTAACAATATATTTAATTTGTTTGTCATATTTGTTGATTGCAACTGGTCCCGTGCGATGCAAGCCTGATTACTTGTACGACAGTGAAGTGCAGGGCTGGCTCAAGCTTCACGTGATTCCAGCAACGTGGGAGCAAGCGGTTTTGCGCTGTCACTATGAAGGAGCAGTGCTGGCGTCTCCACTTAACGAAGAGCTGACCAAAGCTCTCCACTCAACAATGACAAAGTTTGGTATCAATCGTTGCATCTTCTTGGGTACCAGTTTGTTGCTCTCCAATGGCGACTTCGTTTCAATAGAAGGCGTCCCACTGTCTGACCAGGAGATACAGTGGAGCCCACAAGGGCCCGATCCAGGACAATGCCTCACCATGGCCATCTCTGGCAGCGAGCACTTCATGTATACCGCGTCATGCGACGAACAACTACCCTACATATGCTATAGAAATCATGACAATAGTACTATGAATGAATGTGGCACATTTGACAACAAATACCACTACAATCAGAAAACTGGCAGCTGTTACAAAGTCCACAACGAGAAACACACCTGGTACCGCGCAAATATGATCTGCTTTGCGGAGGGAGGTCATCTCGTCATCCTGAATGATGACGTGAAAGCTAACATCGTCAAGGATATGTTCCCCGTTCGTACCGATAACACGACGAATTTATGGGAGCAAATCCACATCGGTCTAAAAGCCTGGGACGACCGCATATGGTTTACCATTCACGGTGATAAAATAGACGATGTGTACAACCAATGGGCTGCAGGACAACCAGACAATAAGATGGGCACACAAAATATTGGTACTATTCTTCGGAGTGGCCTCTTGGATGATGCAAATCCTCTCAAACGGAACATGTTTGTGTGCGAGAAAGCAGCTCATAAAATACGCTTCGAATCTCCAAAATTGCGATGGAATGAATTAATTGTACCTTTTGGAAGATCTACACAGAAATAA;
the protein sequences corresponding to the above cDNA sequences are as follows:
MLTIYLICLSYLLIATGPVRCKPDYLYDSEVQGWLKLHVIPATWEQAVLRCHYEGAVLASPLNEELTKALHSTMTKFGINRCIFLGTSLLLSNGDFVSIEGVPLSDQEIQWSPQGPDPGQCLTMAISGSEHFMYTASCDEQLPYICYRNHDNSTMNECGTFDNKYHYNQKTGSCYKVHNEKHTWYRANMICFAEGGHLVILNDDVKANIVKDMFPVRTDNTTNLWEQIHIGLKAWDDRIWFTIHGDKIDDVYNQWAAGQPDNKMGTQNIGTILRSGLLDDANPLKRNMFVCEKAAHKIRFESPKLRWNELIVPFGRSTQK。
2. the method for preparing the immunoagglutinin for improving the killing efficiency of beauveria bassiana to cotton bollworms according to claim 1, further comprising the steps of renaturation and activity detection of the obtained denatured CTL26 recombinant protein, wherein the steps are as follows:
firstly, carrying out gradient renaturation on denatured CTL26 recombinant protein for a plurality of times by using a dialysis bag and a protein renaturation solution to obtain soluble protein; finally, the recognition activity of beauveria bassiana is analyzed by agglutination, binding and encapsulation experiments.
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| CN101492681A (en) * | 2009-01-09 | 2009-07-29 | 华中农业大学 | Cotton bollworm calcium mucoprotein gene fragment hacad1 with insect disinfestation building action and uses thereof |
| CN110759983A (en) * | 2019-09-10 | 2020-02-07 | 华南农业大学 | Recombinant fungus expressed by targeted silent pest pattern recognition protein GNBP3 gene and application thereof in pest control |
| CN110914429A (en) * | 2017-05-01 | 2020-03-24 | 唐纳德丹佛斯植物科学中心 | RNAi method for crop pest protection |
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| CN101492681A (en) * | 2009-01-09 | 2009-07-29 | 华中农业大学 | Cotton bollworm calcium mucoprotein gene fragment hacad1 with insect disinfestation building action and uses thereof |
| CN110914429A (en) * | 2017-05-01 | 2020-03-24 | 唐纳德丹佛斯植物科学中心 | RNAi method for crop pest protection |
| CN110759983A (en) * | 2019-09-10 | 2020-02-07 | 华南农业大学 | Recombinant fungus expressed by targeted silent pest pattern recognition protein GNBP3 gene and application thereof in pest control |
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