CN101434955A - Recombinant expression method of insect C-type lectin and use - Google Patents
Recombinant expression method of insect C-type lectin and use Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering and provides a biological medicament that has recombinant gene expression of helicoverpa armigera C-type lectin and two carbohydrate recognizing structural domains of Ha-CRD1 and Ha-CRD2 of the gene. The helicoverpa armigera C-type lectin gene and the two carbohydrate recognizing structural domains of Ha-CRD1 and Ha-CRD2 of the gene have recombinant expression in Escherichia coli. The invention further provides a recombinant gene expression method of the helicoverpa armigera C-type lectin and the two carbohydrate recognizing structural domains of Ha-CRD1 and Ha-CRD2 of the gene. The lectin can reduce the insect fatal rate of pathogenic organisms, improve the pathogenic organism phagocytizing efficiency of blood corpuscle, and reduce the survival number of pathogenic organisms in insect bodies. The biological medicament does not generate drug-fast bacteria, does not have adverse effect to the normal vital activity of insects, can reduce the usage of antibiotic, and can reduce the drug-fast bacteria generation in environments.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to DNA recombination and expression techniques and application in the technical field of bioengineering, be specifically related to the recombinant expressed method for creating and the application of a kind of C-type lectin and functional domain thereof.
Background technology
Animal immune system is divided into congenital and acquired two portions, and the early stage appearance that congenital immunity is evolved multicellular organism does not exist only in ridge impellent, no ridge impellent, be present in the plant, and acquired immunity only is present in the vertebrates yet.Congenital immunity is the unique channel that invertebratess such as insect are resisted pathogeny biotic intrusion and harm.Congenital immunity comprises cellular immunization and two approach of humoral immunization, its primary process is: at first discern external foreign matter by the pattern recognition acceptor, be called the non-own identification of infection, cause the extracellular cascade reaction that activates serine protease and remove serpin subsequently, thereby the signal that will be infected is enlarged into stronger " danger " signal or removes false alarm, then priming signal transduction pathway and cause immunogene transcribe and produce immunne response.The strategy of innate immune response does not lie in them can discern each possible antigen, but can be identified in the conservative apokoinou construction of ubiquitous height in the microorganism.These structures are called as pathogen-associated molecular pattern (pathogen assiciated molecular pattems, PAMPs), and the acceptor that can discern these molecular patterns in the innate immune system be called the pattern recognition acceptor (pattern recognition receptors, PRRs).Identification to pathogenic micro-organism surface unique molecular structure is the congenital immunity base of recognition.Wherein, non-oneself identification of infection is particularly important for the invertebrates that lacks adaptive immunity.Non-oneself identification be because the pattern recognition acceptor can be discerned or in conjunction with those microorganisms surface conservative and in the host non-existent pathogen-associated molecular pattern.The most extensive and effective recognition oneself is exactly the carbohydrate structure on identification pathogenic micro-organism surface with non-own method.
C-type lectin (C-type lectin) is a kind of pattern recognition acceptor in the innate immune response, at Ca
2+There is the glucide on identification pathogenic micro-organism surface that down can be special, thereby causes that a series of immune responses of host resist the invasion of pathogenic micro-organism effectively.C-type lectin family is at immunity high expression level in relevant period in the invertebrates, and reason is to play an important role in immunoreactive non-oneself identification with its distinctive carbohydrate recognition structure territory CRD in conjunction with the microorganism surface carbohydrate.C-type lectin (C-type lectin) was defined since the title from 1988, had had been found that kind more than 1,000.Whole genome sequence studies show that, C-type lectin is from worm, fruit bat is to vertebrates, though it is incomplete same, but carbohydrate recognition structure territory CRD sequence (the carbohydrate recognition domain of different C-type lectins, CRD) has conservative relatively amino-acid residue, the carbohydrate of C-lectin is realized by carbohydrate recognition structure territory in conjunction with activity, therefore, the recombinant expressed lectin full-length gene (containing structural domain) or the carbohydrate recognition structure territory of only expressing lectin all can obtain activated product.
Economic domestic insect such as silkworm suffers various pathogeny biological hazard in breeding process, silkworm is already produced cause serious financial loss, and the medicine that presses for effective, harmless is prevented and treated disease.Though the lectin clone of insects such as silkworm and the report of functional study are arranged, so far not about the recombinant expressed of insect lectin and the research report that is used for the infectious diseases control.Particularly the bollworm agglutinin gene is that we clone first and obtain and carry out recombinant expressed and functional study.Be used for bollworm and under the selection of long-term natural selection and the various microbial pesticides of artificial use, formed extremely strong resistance, aspect immunogene, be evolved into the immunologic function stronger than breeding insects such as silkworms, and bollworm and silkworm all are to belong to lepidopterous insects, the similarity of gene is higher, therefore the bollworm C-type lectin of producing by biotechnology has better resistance against diseases from the lectin of other biogenetic derivation, can be used for the disease control of breeding insects such as silkworm.
Summary of the invention
At the deficiencies in the prior art, the purpose of this invention is to provide the bio-pharmaceutical that a kind of domestic insect infection venereal disease evil is used, be used for the infectious disease control of culturing economic insects such as silkworm.
Two carbohydrate recognition structure territory (carbohydrate-recognition domains of the present invention recombinant expressed bollworm C-type lectin (Ha-lectin) gene and this gene in intestinal bacteria, CRD) Ha-CRD1 and Ha-CRD2, the function of experimental result proof recombinant expressed bollworm C-type lectin (Ha-lectin) gene and two structural domain Ha-CRD1 and the helpful insect opposing of Ha-CRD2 pathogeny biological attack in intestinal bacteria can be used for the disease control in breeding production of economic insects such as silkworm.
The invention provides two carbohydrate recognition structure territory Ha-CRD1 of dna recombinant expression bollworm C-type lectin and this gene and the bio-pharmaceutical of Ha-CRD2, it is characterized in that, with two the carbohydrate recognition structure territory Ha-CRD1 and the Ha-CRD2 of bollworm C-type agglutinin gene and this gene, recombinant expressed in intestinal bacteria.
Preferably, described bollworm (Helicoverpa armigera) C-type lectin (Ha-lectin) gene is that (the gene complete sequence is submitted to GenBank by us to Ha-Lect, and http://www.ncbi.nlm.nih accepts number: DQ533877).Recombinant expressed bollworm C-type lectin (Ha-lectin) full-length gene, functional domain and modify on the basis of said gene or gene that base mutation produces in intestinal bacteria obtain recombinant protein lectin or lectin functional domain.156 the amino acid whose sequences of the 30th amino acid to the DQ533877)) and Ha-CRD2 (total length Ha-Lect (acceptance number: 307 the amino acid whose sequences of the 68th amino acid to the DQ533877)) (total length Ha-Lect (accepts number: carbohydrate recognition structure territory Ha-CRD1
The present invention also provides the method for two the carbohydrate recognition structure territory Ha-CRD1 and the Ha-CRD2 of a kind of dna recombinant expression bollworm C-type lectin and this gene, comprises the steps:
[1] designs the primer of band restriction enzyme site respectively at the gene order of the Ha-Lect, the Ha-CRD1 that insert expression vector and Ha-CRD2, with PCR method increase respectively Ha-Lect and Ha-CRD1 and Ha-CRD2;
[2] amplification and extraction expression vector-Ha-Lect, expression vector-Ha-CRDl and expression vector-Ha-CRD2 plasmid in intestinal bacteria;
[3] expression vector-Ha-Lect, expression vector-Ha-CRD1 and expression vector-Ha-CRD2 plasmid are changed over to respectively in the host, use the inductor abduction delivering
[4] use Ni
2+Post or other filler purification of Recombinant rHa-Lect, rHa-CRD1 and rHa-CRD2.
Described inductor is IPTG (abbreviation of isopropylthio-), methyl alcohol or other compound.
Wherein, expression vector is selected from coli expression carrier, Yeast expression carrier or insect baculovirus carrier described in the step [1].Preferably, coli expression carrier is selected from pET serial carrier or pGEX serial carrier, and Yeast expression carrier is selected from the pPIC serial carrier.
Wherein, Ha-Lect, Ha-CRD1 and Ha-CRD2 described in the step [1] are that (GenBank, http://www.ncbi.nlm.nih accept number the Ha-Lect gene: DQ533877) and modify on the basis or gene that base mutation produces.
Wherein, escherichia coli expression bacterial strain described in the step [2] is any escherichia coli expression bacterial strain, preferred DH5 α.
Wherein, host described in the step [3] is that intestinal bacteria, yeast or other expression are biological.Described intestinal bacteria, preferred BL21 (DE3); Described yeast, preferred pichia spp (Pichia pastoris); Described other expressed biological, preferred insect cell.
The present invention also provides the application of the bio-pharmaceutical of two carbohydrate recognition structure territory Ha-CRD1 of described dna recombinant expression Ha-Lect and this gene and Ha-CRD2, it is characterized in that, is used to prevent and treat domestic insect infection venereal disease evil.Described domestic insect is selected from silkworm, bollworm, beet armyworm.
Utilize gene recombination rHa-lectin, rHa-CRD1 and the rHa-CRD2 lectin of method initiative of the present invention, can be used for the infectious disease control of domestic economic insects such as silkworm.
RHa-lectin, rHa-CRD1 and rHa-CRD2 and pathogeny biology are expelled in the insect larvae body jointly, can reduce the larval mortality that is caused by the pathogeny biology, the minimizing amplitude is 50～70%.
RHa-lectin, rHa-CRD1 and rHa-CRD2 and pathogeny biology are expelled in the insect larvae body jointly, can increase the phagocytic activity of larva hemocyte to the pathogeny biology, increasing degree is 40～60%.
RHa-lectin, rHa-CRD1 and rHa-CRD2 and pathogeny biology are expelled in the insect larvae body jointly, can reduce pathogeny biology survival quantity in vivo, the minimizing amplitude is 50～70%.
Described insect is selected from bollworm, silkworm, beet armyworm.Described pathogeny biology is selected from Bacillus thuringiensis Bacillus thuringiensis (Bacillus thuringiensis), klebsiella (Klebsiella pneumoniae), Candida albicans (Candidaalbicans), green muscardine fungus Metarhizium. anisopliae, muscardine Beauveria bassiana.
RHa-lectin, rHa-CRD1 and rHa-CRD2 are added in the feed of the insect of propagating artificially that sterilization is cooled to 37 ℃, 1mg/L, the control microorganism is propagated the infection of insect artificially to silkworm etc.The described insect of propagating artificially is selected from bollworm, silkworm, beet armyworm.Described microorganism is selected from Bacillus thuringiensis (Bacillus thuringiensis), green muscardine fungus (Metarhiziumanisopliae), muscardine (Beauveria bassiana).
Gene recombination rHa-lectin, the rHa-CRD1 that this invention is formulated and thinking, gene and the method for rHa-CRD2 lectin all are by our reported first.
Gene recombination rHa-lectin of the present invention, rHa-CRD1 and rHa-CRD2 lectin can reduce the lethality rate of pathogeny biology to insect, increase the engulf efficient of hemocyte to the pathogeny biology, reduce the existence quantity of pathogeny biology in polypide.The traditional method of control insect bacteriosis mainly is to use microbiotic, as adds food paraxin, erythromycin, terramycin, and such method can cause resistant organism to produce, and microbiotic also can have a negative impact to the normal activities of insect.We belong to bio-pharmaceutical by the insect lectin of invention, and the insect lectin is the crude substance of resisting the microorganism dip-dye that insect itself exists, the deficiency that can replenish the intravital lectin of insect by the insect lectin of the recombinant expressed production of biotechnological means.Thereby recombinant expressed lectin increases hemocyte engulfing bacterium by the aggegation invading bacteria by getting after food or breathing enter polypide, can not cause resistant organism to produce, normal activities to insect itself does not have detrimentally affect, use this medicine can reduce antibiotic use, reduce the generation of resistant organism in the environment.
Embodiment
Below in conjunction with experiment, the effect that utilizes the gene recombination insect lectin that method of the present invention obtains is further described.
Method (1): design 1 pair of primer amplification C-type agglutinin gene mature peptide (84-1013bp) that has restriction enzyme site, promptly remove the signal peptide part.Primer is as follows: LectEco:5 '-TACTCA
GAATTCGCGTTTACATGCGACTAC-3 ' (EcoRI); LectXho:5 '-TACTCA
CTCGAGTTATTCAACTTTGTTATT-3 ' (Xho I), the PCR reaction, condition is 94 ℃ of 30sec, 60 ℃ of 45sec, 72 ℃ of 1min, 72 ℃ of 10min.PCR fragment and pET-30a (+) carrier are cut with EcoRI and XhoI enzyme respectively, by T
4Dna ligase is connected the cDNA fragment of purifying with pET-30a (+) carrier of purifying, to connect product Transformed E .coli DH5 α, screening positive clone, extract plasmid, get the expression plasmid that 1 μ L builds, dilute 10 times, transform expression strain E.coli BL21 (DE3) competent cell, cultivate in the LB/Kan substratum, the adding final concentration is that the IPTG of 0.5mM induces the insertion expression of gene, get the bacterium liquid after inducing, the centrifugal 5min of 6000rpm collects thalline, with 3ml PBS (140mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 1.8mM KH
2PO
4, pH7.4) with 30 μ l, 20% Triton X-100, abundant mixing, the broken 10min of ice-bath ultrasonic, 4 ℃ of broken good thalline, 12, the centrifugal 20min of 000rpm collects upward cleer and peaceful precipitation respectively, uses nickel affinity column purification of recombinant proteins.
Method (2): design 1 couple of primer amplification rHa-CRD1 that has restriction enzyme site, primer is as follows: CRD1Eco:5 '-TACTCA
GAATTCTGCGACTACAAATACAGTCTA-3 ' EcoRI, CDR1Xho5 '-TACTCA
CTCGAGAAAGCAGATGTAAGGTCTGGG-3 ' XhoI, the same method of the step of back (1).
Method (3): design 1 couple of primer amplification rHa-CRD2 that has restriction enzyme site, primer is as follows: CRD2Eco5 '-TACTCA
GAATTCTGTGGTACTCCTGATGATGGA-3 ' EcoRI, CDR2Xho5 '-TACTCA
CTCGAGCTCACAAATGAATGGAGCTGG-3 ' XhoI, the same method of the step of back (1).
Method (4): with rHa-lectin, rHa-CRD1 and rHa-CRD2 (every larva 0.5 μ g, 3 μ g or, 15 μ g) mix with Bt (30/larva), be expelled in insect larvae (as the bollworm) body, add up mortality ratio after 24 and 48 hours, with the control group comparison of injecting normal saline.
Method (5): rHa-lectin, rHa-CRD1 and rHa-CRD2 (every larva 3 μ g) are mixed with Bt (30/larva), be expelled in insect larvae (as the bollworm) body, take out hemolymph after 24 hours, with being coated on LB flat board (1% peptone after the physiological saline dilution, 0.5% yeast extract, 1% NaCl, 1.5% agar, pH7.0), counting statistics bacterium number.
Method (6): with 70% Ethanol Treatment 10 minutes, collecting cell added Sumitomo Acridine Orange RK conc (1 μ g/ml) dyeing 5 minutes with Bt, and collecting cell is washed in physiological saline 3 times, with rHa-lectin, rHa-CRD1 and rHa-CRD2 (every larva 5 μ g) and Bt (1 * 10
7/ larva) mixes, be expelled in insect larvae (as the bollworm) body, take out hemolymph and hemocyte after 24 hours, examine under a microscope the statistics hemocyte and engulf number of bacteria.
Method (7): rHa-lectin, rHa-CRD1 and rHa-CRD2 are added in the feed of the insect of propagating artificially, and the control microorganism is propagated the infection of insect artificially to silkworm etc.
Method (8): rHa-lectin, rHa-CRD1 and rHa-CRD2 are sprayed on the feed of the insect of propagating artificially, and the control microorganism is propagated the infection of insect artificially to silkworm etc.
Embodiment 1
L is to having the primer amplification C-type agglutinin gene mature peptide (84-1013bp) of restriction enzyme site in design, promptly removes the signal peptide part.Primer is as follows: LectEco:5 '-TACTCA
GAATTCGCGTTTACATGCGACTAC-3 ' (EcoR I); LectXho:5 '-TACTCA
CTCGAGTTATTCAACTTTGTTATT-3 ' (Xho I), the PCR reaction, condition is 94 ℃ of 30sec, 60 ℃ of 45sec, 72 ℃ of 1min, 72 ℃ of 10min.PCR fragment and pET-30a (+) carrier are cut with EcoRI and XhoI enzyme respectively, by T
4Dna ligase is connected the cDNA fragment of purifying with pET-30a (+) carrier of purifying, to connect product Transformed E .coli DH5 α, screening positive clone, extract plasmid, get the expression plasmid that 1 μ L builds, dilute 10 times, transform expression strain E.coli BL21 (DE3) competent cell, cultivate in the LB/Kan substratum, the adding final concentration is that the IPTG of 0.5mM induces the insertion expression of gene, get the bacterium liquid after inducing, the centrifugal 5min of 6000rpm collects thalline, with 3ml PBS (140mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 1.8mM KH
2PO
4, pH7.4) with 30 μ l 20%Triton X-100, abundant mixing, the broken 10min of ice-bath ultrasonic, 4 ℃ of broken good thalline, 12, the centrifugal 20min of 000rpm collects upward cleer and peaceful precipitation respectively, uses nickel affinity column purification of recombinant proteins.
Embodiment 2
Design 1 couple of primer amplification rHa-CRD1 that has restriction enzyme site, primer is as follows: CRD1Eco:5 '-TACTCA
GAATTCTGCGACTACAAATACAGTCTA-3 ' EcoRI, CDR1Xho5 '-TACTCA
CTCGAGAAAGCAGATGTAAGGTCTGGG-3 ' XhoI, the step of back is with embodiment 1.
Embodiment 3
Design 1 couple of primer amplification rHa-CRD2 that has restriction enzyme site, primer is as follows: CRD2Eco5 '-TACTCA
GAATTCTGTGGTACTCCTGATGATGGA-3 ' EcoRI, CDR2Xho5 '-TACTCA
CTCGAGCTCACAAATGAATGGAGCTGG-3 ' XhoI, the step of back is with embodiment 1.
Embodiment 4
RHa-lectin, rHa-CRD1 and rHa-CRD2 are added in the feed of the insect of propagating artificially (as silkworm), 1mg/L, the control microorganism is propagated the infection of insect artificially to silkworm etc.Test is found: this method can reduce the generation of the silkworm infectious diseases that the pathogeny microorganism causes in the environment, infects and the Bacillus thuringiensis infection as the green mold bacterium, and sickness rate reduces 60%.
Embodiment 5
On the basis of embodiment 1,2,3, mutator gene obtains expression product, has the effect of identical control infected by microbes.As: 1) with the 3rd bit base sudden change of the codon of arbitrary coded amino acid in the gene and do not change amino acid, obtain to have rHa-lectin, rHa-CRD1 and the rHa-CRD2 of same acid sequence, as with tac, aca, gtt, ttt, tgc (encode respectively Y, T, V, F, C amino acid), be mutated into tat, act, c, g, gtc, a, g, ttc, tgt (still encode respectively Y, T, V, F, C amino acid), other sequence is also like this in the gene; 2) partial sequence in the gene is lacked, acquisition has rHa-lectin, rHa-CRD1 and the rHa-CRD2 of identical effect, as with the 1-78 base deletion in the genes encoding frame or with the 473-503 base deletion or with the 926-1010 base deletion, obtain rHa-lectin with identical effect; 3) rHa-lectin, rHa-CRD1 and the rHa-CRD2 that the amino acid mutation in the dna encoding the protein is obtained to have identical effect, as SLLTK being mutated into SLLAK, other amino acid mutation in the dna encoding the protein is also like this.
Embodiment 5
On the basis of embodiment 1,2,3, use other expression vector instead, as pGEX serial carrier, Yeast expression carrier such as pPIC serial carrier and insect baculovirus carrier, obtain the lectin product of identical function.
Embodiment 6
On the basis of embodiment 1,2,3, use other expression system instead, as yeast expression system, insect cell expression system, obtain the lectin product of identical function.
Embodiment 7 administering modes
RHa-lectin (every larva 15 μ g) is mixed with Bt (30/larva), be expelled in insect larvae (as the bollworm) body, reduce and infect mortality ratio.Test is found: compare with the control group of injecting normal saline, the statistics mortality ratio is reduced to 23% from 97% after 48 hours.Injection rHa-CRD2 (every larva 15 μ g) mixes with Bt (30/larva), and with the control group comparison of injecting normal saline, the statistics mortality ratio is reduced to 38.2% from 97% after 48 hours.
Method (5): rHa-lectin, rHa-CRD1 and rHa-CRD2 (every larva 3 μ g) are mixed with Bt (30/larva), be expelled in insect larvae (as the bollworm) body, take out hemolymph after 24 hours, with being coated on LB flat board (1% peptone after the physiological saline dilution, 0.5% yeast extract, 1% NaCl, 1.5% agar, pH7.0), counting statistics bacterium number.Test is found: compare with the control group of injecting normal saline, bacterial count is from 3.87 * 10 in the hemolymph
5Individual/μ l is reduced to 2.77～0.93 * 10
5Individual/μ l.
With 70% Ethanol Treatment 10 minutes, collecting cell added Sumitomo Acridine Orange RK conc (1 μ g/ml) dyeing 5 minutes with Bt, and collecting cell is washed in physiological saline 3 times, with rHa-lectin, rHa-CRD1 and rHa-CRD2 (every larva 5 μ g) and Bt (1 * 10
7/ larva) mixes, be expelled in insect larvae (as the bollworm) body, take out hemolymph and hemocyte after 24 hours, examine under a microscope the statistics hemocyte and engulf number of bacteria.Test is found: compare with the control group of injecting normal saline, the quantity of engulfing the hemocyte of bacterium is increased to 54.41% from 19.02%.
RHa-lectin, rHa-CRD1 and rHa-CRD2 are added on the feed of the insect of propagating artificially, and the control microorganism is propagated the infection of insect artificially to silkworm etc.Test is found: this method makes the generation of insect infection disease be reduced to 30% from 90%.
Claims (10)
1. the bio-pharmaceutical of two carbohydrate recognition structure territory Ha-CRD1 of a dna recombinant expression bollworm C-type lectin and this gene and Ha-CRD2, it is characterized in that, with two the carbohydrate recognition structure territory Ha-CRD1 and the Ha-CRD2 of bollworm C-type agglutinin gene and this gene, recombinant expressed in intestinal bacteria.
2. two carbohydrate recognition structure territory Ha-CRD1 of a kind of dna recombinant expression bollworm C-type lectin as claimed in claim 1 and this gene and the bio-pharmaceutical of Ha-CRD2, it is characterized in that, described bollworm C-type agglutinin gene is Ha-Lect, in intestinal bacteria recombinant expressed bollworm C-type lectin full-length gene, functional domain and on the basis of said gene, modify or gene that base mutation produces, obtain recombinant protein lectin or lectin functional domain.
3. the method for two carbohydrate recognition structure territory Ha-CRD1 of a dna recombinant expression bollworm C-type lectin and this gene and Ha-CRD2 comprises the steps:
[1] designs the primer of band restriction enzyme site respectively at the gene order of the Ha-Lect, the Ha-CRD1 that insert expression vector and Ha-CRD2, with PCR method increase respectively Ha-Lect and Ha-CRD1 and Ha-CRD2;
[2] amplification and extraction expression vector-Ha-Lect, expression vector-Ha-CRD1 and expression vector-Ha-CRD2 plasmid in intestinal bacteria;
[3] expression vector-Ha-Lect, expression vector-Ha-CRD1 and expression vector-Ha-CRD2 plasmid are changed over to respectively in the host, use the inductor abduction delivering;
[4] use Ni
2+Post or other filler purification of Recombinant rHa-Lect, rHa-CRD1 and rHa-CRD2.
4. two carbohydrate recognition structure territory Ha-CRD1 of dna recombinant expression bollworm C-type lectin as claimed in claim 3 and this gene and the method for Ha-CRD2 is characterized in that inductor described in the step [3] is IPTG, methyl alcohol or other compound.
5. two carbohydrate recognition structure territory Ha-CRD1 of dna recombinant expression bollworm C-type lectin as claimed in claim 3 and this gene and the method for Ha-CRD2, it is characterized in that expression vector is selected from coli expression carrier, Yeast expression carrier or insect baculovirus carrier described in the step [1].Preferably, coli expression carrier is selected from pET serial carrier or pGEX serial carrier, and Yeast expression carrier is selected from the pPIC serial carrier.
6. two carbohydrate recognition structure territory Ha-CRD1 of dna recombinant expression bollworm C-type lectin as claimed in claim 3 and this gene and the method for Ha-CRD2, it is characterized in that Ha-Lect, Ha-CRD1 and Ha-CRD2 described in the step [1] modify on Ha-Lect gene and the basis thereof or gene that base mutation produces.
7. two carbohydrate recognition structure territory Ha-CRD1 of dna recombinant expression bollworm C-type lectin as claimed in claim 3 and this gene and the method for Ha-CRD2 is characterized in that the escherichia coli expression bacterial strain is DH5 α described in the step [2].
8. two carbohydrate recognition structure territory Ha-CRD1 of dna recombinant expression bollworm C-type lectin as claimed in claim 3 and this gene and the method for Ha-CRD2, it is characterized in that host described in the step [3] is that intestinal bacteria, yeast or other expression are biological; Described intestinal bacteria, preferred BL21 (DE3); Described yeast, preferred pichia spp (Pichiapastoris); Described other expressed biological, preferred insect cell.
9. the application of two carbohydrate recognition structure territory Ha-CRD1 of dna recombinant expression bollworm C-type lectin as claimed in claim 1 and this gene and the bio-pharmaceutical of Ha-CRD2 is characterized in that, is used to prevent and treat domestic insect infection venereal disease evil.Described domestic insect is selected from silkworm, bollworm, beet armyworm.
10. the application of two carbohydrate recognition structure territory Ha-CRD1 of dna recombinant expression bollworm C-type lectin as claimed in claim 9 and this gene and the bio-pharmaceutical of Ha-CRD2, it is characterized in that, rHa-lectin, rHa-CRD1 and rHa-CRD2 and pathogeny biology are expelled in the insect larvae body jointly, and described insect is selected from bollworm, silkworm, beet armyworm; Described pathogeny biology is selected from Bacillus thuringiensis Bacillus thuringiensis, klebsiella, Candida albicans, green muscardine fungus or muscardine;
Or add in the feed of the insect of propagating artificially that sterilization is cooled to 37 ℃ rHa-lectin, rHa-CRD1 and rHa-CRD2 to 1mg/L; The described insect of propagating artificially is selected from bollworm, silkworm, beet armyworm; Described microorganism is selected from Bacillus thuringiensis, green muscardine fungus or muscardine.
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| CN101864425A (en) * | 2010-05-17 | 2010-10-20 | 山东大学 | Silkworm C-type agglutinin recombination expression method and application thereof |
| CN105504038A (en) * | 2016-02-04 | 2016-04-20 | 中国水产科学研究院黄海水产研究所 | Turbot Lily-type lectin(SmLTL) recombinant protein, and preparation and application methods thereof |
| CN110241102A (en) * | 2019-06-21 | 2019-09-17 | 济宁学院 | A kind of method of enzymic degradation 2,6- dihydroxy-benzoic acid |
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2008
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| CN101864425A (en) * | 2010-05-17 | 2010-10-20 | 山东大学 | Silkworm C-type agglutinin recombination expression method and application thereof |
| CN105504038A (en) * | 2016-02-04 | 2016-04-20 | 中国水产科学研究院黄海水产研究所 | Turbot Lily-type lectin(SmLTL) recombinant protein, and preparation and application methods thereof |
| CN105504038B (en) * | 2016-02-04 | 2018-10-09 | 中国水产科学研究院黄海水产研究所 | Turbot SmLTL recombinant proteins and its preparation and application method |
| CN110241102A (en) * | 2019-06-21 | 2019-09-17 | 济宁学院 | A kind of method of enzymic degradation 2,6- dihydroxy-benzoic acid |
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