CN103030692A - Recombinant serine protease inhibitor, fungus expression vector and fungus insecticide of recombinant protein - Google Patents
Recombinant serine protease inhibitor, fungus expression vector and fungus insecticide of recombinant protein Download PDFInfo
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Abstract
The invention discloses a recombinant serine protease inhibitor, a fungus expression vector and a fungus insecticide of recombinant protein. The invention relates to the recombinant serine protease inhibitor; a molecular biology technology is used to construct a recombinant nucleotide sequence; and the recombinant serine protease inhibitor comprises a signal peptide sequence of encoded coccidio beauveriabassiana chitinase and a serine protease inhibitor gene of encoded drosophila, and has a nucleotide sequence shown as SEQ ID NO. 1 and an amino acid sequence shown as SEQ ID NO. 2. The obtained recombinant serine protease inhibitor can inhibit immunoreaction of insects, increase multiplication speed of fungi in haemocoeles, and therefore improve characteristics of insecticide fungus toxicity.
Description
Technical field
The present invention relates to a kind of new serine protease and suppress son, relate in particular to the recombinant protein that the coding sequence of secretory signal peptide that contains fungi and insect serine protease suppress sub-serpin sequence.
The present invention relates to express the fungus expression vector of above-mentioned recombinant protein.
The invention still further relates to the disinsection fungal agent of expressing above-mentioned recombinant protein.
Background technology
Fungi is one of principal element of control nature the quantity of insects' population; biological pesticide take it as the main active ingredient (Feng Mingguang that has been used widely in China, America and Europe and Africa; " plant protection 21 century prospect and the 3rd the youth of the nation plant protection scientific worker scientific seminar collection of thesis ", 1998).But biological pesticide comprises that bacterium, virus and Mycophyta sterilant all exist to knock down the shortcomings such as insect overlong time and preventive effect be unstable, therefore how to improve the efficient of these biological species sterilants, and studies the research emphasis that its mechanism of action has become this field.
In fungal infection host's process, insect can utilize the system of defense of self to resist the intrusion of fungi.Insect body wall can provide physical barriers for it, and the immunity system of self then can be identified the pathogenic agent of invasion, and immunocyte is attached to the pathogenic agent surface, engulfs pathogenic agent, forms melanochrome.Immunity system also can produce a large amount of antibacterial proteins in addition, the growth of Antifungi.Investigators have furtherd investigate the immune response of insect take fruit bat as model animals.The immunity system of fruit bat comprises three reactions, and humoral immunization produces a series of antibacterial peptide by fatty body, engulfs and melanism.The antibacterial peptide that fruit bat is subject to immunity to be produced when coercing comprises Drosomycin, Metchnikowin, Defensin, Attacin, Cecropin, Drosocin and Diptericin, and wherein Drosomycin, Metchnikowin and Cecropin have restraining effect to fungi.Studies show that, fruit bat can be activated sp tzle/Toll/cactus signal pathway (Toll approach) (Schneider DS when being subject to fungi and coercing
Et al.,
Genes Dev, 1991,5:797-807), wherein sp tzle is the part of Toll, by the serine stretch protein enzyme activation.Sp tzle is combined this approach of activation with Toll, cause the degraded of I κ B-like albumen and Cactus albumen and the nuclear translocation of NF-κ B-like, Dorsal and Dif, thereby activate expression (the Nicolas E of genes involved
Et al.,
J Biol Chem, 1998,273:10463-10469).Toll in this approach, sp tzle, Tube or Pelle undergo mutation, and all can cause the expression of anti-fungus peptide Drosomycin to reduce, and insect is to extremely sensitive (the Lemaitre B of fungi infestation
Et al.,
Cell, 1996,86:973 – 83).Utilize Aspergillus fumigatus (
Aspergillus fumigatus) spore processes respectively Toll mutant fruit bat and wild-type fruit bat, the mutant fruit bat is all dead afterwards at 2-3 days, and the wild-type fruit bat only has 1/3 death after processing 6 days.Analyze demonstration, in the Toll mutant, the expression of antifungal protein Drosomycin significantly reduces (Lemaitre B
Et al.,
Cell, 1996,86:973 – 83).Above-mentioned studies show that, Toll signal pathway are the main signal approach of insect when being subject to fungal infection, prevent this approach can cause insect that the resistibility of fungal pathogens is reduced.Therefore, this approach becomes the potential target of bacterial strain genetic improvement, but does not also report accordingly at present.
Summary of the invention
For the prior art above shortcomings, one object of the present invention is to provide a kind of recombinant serine protease to suppress son, serine protease inhibition that it contains the coding sequence of secretory signal peptide that derives from fungi and derives from insect, have the characteristic that suppresses the Insect immunity reaction and improve the disinsection fungal virulence, infect inefficient problem to solve insect pathogenic fungus.
Another object of the present invention is to provide the expression vector that contains above-mentioned nucleotide sequence.
A further object of the present invention is to provide the disinsection fungal agent that produces albumen of the present invention.
Realize above-mentioned purpose, the present invention adopts following technical scheme: a kind of recombinant serine protease suppresses son, it is characterized in that, adopts Protocols in Molecular Biology to make up a recombinant nucleotide sequence, its contain the coding beauveria bassiana (
Beauveria bassiana) signal peptide sequence of chitinase and the serine protease of coding fruit bat suppress subbase because of, it has nucleotide sequence and the aminoacid sequence shown in SEQ ID NO. 2 shown in SEQ ID NO. 1.
Described recombinant serine protease suppresses the construction process of son, comprises the steps:
1) fruit bat serpin gene cloning:
In the serpin of fruit bat gene (NM_080112), an intron is arranged, utilize the method for PCR lap to remove intron; With P1/P2, P3/P4 is primer respectively; Primer sequence is as follows:
P1:5 '-
GAATTCATGGCGAGCAAAGTCTCGATCCTTCTCCTGC-3 ', the underscore sequence is the EcoRI site;
P2: 5’-GGGGTACGAACTTGGCATAGGAAGCTGCCGAGGCC-3’；
P3: 5’-GGCCTCGGCAGCTTCCTATGCCAAGTTCGTACCCC-3’；
P4:5 '-
CCCGGGTTAGACGCTCATGGGCGTGGGAT-3 ', the underscore sequence is the SmaI site;
Drosophila gene group DNA is masterplate, with N end and the C end (take intron as cut-point) of Phusion archaeal dna polymerase (NEB) amplification serpin gene; Reaction system: 5 μ L, 5 * Phusion Taq polymerase buffer, 2 μ L, 2.5 mM dNTP, each 1 μ L(10 μ m of upstream and downstream primer), 0.4 U Phusion Taq DNA polymerase, cumulative volume 25 μ L;
PCR reaction parameter: 98
oC (2 min); 30 circulations: 98
oC (20 s), 56
oC (30sec), 72 (1min), 72
oC (5 min); Reclaim respectively N end (1222bp) and C end (139bp) fragment, carry out assembling without primer;
Reaction system: 5 μ L, 5 * Phusion Taq polymerase buffer, 2 μ L, 2.5 mM dNTP, N end fragment (50ng), C end fragment (50ng), 0.4 U Phusion Taq DNA polymerase, cumulative volume 25 μ L;
Take above-mentioned assembling product as masterplate, the serpin cDNA sequence take P1 and P4 as the primer amplification total length, the clone advances pEASY-Blunt carrier (full formula gold product); The carrier called after pEASY-serpin that checks order correct;
2) structure of fusion gene:
Design primer P5-P7, the fusion gene of structure Bbsp:serpin;
Primer sequence is as follows:
P5:5 '-
TCTAGAGAGCTCATCGCTTGGCAACG-3 ', the underscore sequence is the XbaI site;
P6:5 '-AAAAAA
GCGGCCGCATGGCTCCTTTTCTTCAAACC-3 ', the underscore sequence is the NotI site;
P7:5 '-GG
ACTAGTGCCGGCTCGCGGCGCCAAGGG-3 ', the underscore sequence is the SpeI site;
With beauveria bassiana (
Beauveria Bassiana) genome is masterplate, with the signal peptide of P6 and P7 amplification beauveria bassiana chitinase gene Bbchit1, the clone advances pGEM-T carrier (Promega), and sequence verification obtains carrier pGEM-Bbsp; Take pEASY-serpin as masterplate, with the maturation protein zone of P4 and P5 amplification serpin gene, the clone advances the pGEM-T carrier, gets pGEM-serpin;
NotI and SpeI double digestion pGEM-Bbsp reclaim Bbsp fragment (90bp); XbaI and SmaI double digestion pGEM-serpin get the ripe regional fragment (1362bp) of serpin.
Further, a kind of fungus expression vector of recombinant protein, with above-mentioned Bbsp(90bp) and serpin(1362bp) jointly make up pUC-Pgpda-TtrpC, get the pUC-Pgpda-Bbsp:serpin-TtrpC carrier, it has the expression original paper of fusion rotein Bbsp:serpin, a Fungal community composition promotor Pgpda is contained in the upstream, and a terminator TtrpC is contained in the downstream.
Further again, the present invention also provides a kind of disinsection fungal agent of recombinant protein, with above-mentioned carrier pUC-Pgpda-Bbsp:serpin-TtrpC XbaI single endonuclease digestion, get the expression original paper Pgpda-Bbsp:serpin-TtrpC fragment (4.4kb) of fusion rotein Bbsp:serpin, make up fungus expression vector pK2-BAR:GFP, get the pK2-BAR:GFP-Pgpda-Bbsp:serpin-TtrpC plasmid and change agrobacterium tumefaciens lba4404 over to; Utilize the Agrobacterium-mediated transformation method to obtain the disinsection fungal transformant of constructive expression Bbsp:serpin.
Compared to existing technology, the present invention has following beneficial effect:
Among the present invention, serine protease suppresses the adjusting that sub-serpin has participated in sp tzle/Toll/cactus cascade reaction.Serpin can suppress the to degrade activity of sp tzle serine protease makes sp tzle keep complete, thereby prevents this signal pathway.Studies show that, in the wild-type fruit bat, when pathogen infection is arranged, sp tzle is become activity form by protease cracking, but suppress subbase because of serpin(Spn43AC at serine protease) mutant in, sp tzle is in active state always, anti-fungus peptide Drosomycin constructive expression (Levashina EA
Et al.,
Science, 1999,285:1917 – 1919).Therefore, the immune response of serpin energy negative regulation insect produces the ability that serpin then can improve fungi antagonism host immune system, thereby improves the insecticidal effect of fungi in fungal infection host's process.
The present invention is by expressed fusion protein Bbsp:serpin in disinsection fungal, fungi is secreted in infecting host's process produce serpin, suppress host's Toll signal path, thereby suppress the immune response of insect, strengthen the rate of propagation of fungi, shorten the time that fungi is knocked down insect.Also do not carry out at present the report of strain improvement for host immune response, so the control that the success that obtains of the present invention both can be insect provides supper toxic strain, also can provide a new thinking for strain improvement.
The recombinant serine protease that the present invention obtains suppresses son, can suppress the immune response of insect, strengthens the rate of propagation of fungi in haemocoele, thereby improves the characteristic of disinsection fungal virulence, improves the insecticidal effect of fungi.
Provided by the invention turn recombinant serine protease suppress subbase because of beauveria bassiana transform bacterial strain; Virulent Analysis is the result prove, the virulence of transformant contrasts and is significantly improved; Transformant rate of propagation in the insect haemocoele increases significantly than wild type strain.
Description of drawings
Fig. 1 is 70 aminoacid sequences of Serpin N end; Wherein arrow is depicted as the signal peptidase cleavage site of supposition.
Fig. 2 is the plasmid map of fusion gene Bbsp:serpin of the present invention.
Fig. 3 is the fungus expression vector collection of illustrative plates of Bbsp:serpin of the present invention.
Fig. 4 is the beauveria bassiana transformant PCR checking of Bbsp:serpin of the present invention.
Fig. 5 is wild type strain and the propagation situation of transgenosis bacterial strain in the insect haemocoele.
Embodiment
Below in conjunction with specific embodiments and the drawings the present invention is described in further detail.
A kind of recombinant serine protease suppresses son, contains a fusion rotein, and the coding sequence of secretory signal peptide that described fusion rotein origin comes from the mykose enzyme forms with sub fusion of serine protease inhibition that derives from insect.Described fusion rotein origin come from beauveria bassiana (
Beauveria bassiana) chitinase signal peptide (hereinafter to be referred as Bbsp) with derive from fruit bat (
Drosophila) serine protease suppress son (hereinafter to be referred as serpin) and merge and form.
Further, the code book invention recombinant serine protease nucleotide sequence that suppresses son contains the signal peptide that coding derives from the chitinase gene of fungi and derives from the gene that the insect serine protease suppresses son with coding.Adopt Protocols in Molecular Biology to make up a recombinant nucleotide sequence, its contain the coding beauveria bassiana (
Beauveria bassiana) signal peptide sequence of chitinase and the serine protease of coding fruit bat suppress subbase because of, it has nucleotide sequence and the aminoacid sequence shown in SEQ ID NO. 2 shown in SEQ ID NO. 1.
In addition, the present invention also provides a kind of disinsection fungal to transform bacterial strain, and the plasmid vector that makes up with the present invention transforms disinsection fungal and the disinsection fungal that obtains to express recombinant protein of the present invention transforms bacterial strain, wherein disinsection fungal can be beauveria bassiana (
Beauveria bassiana), Metarhizium anisopliae (
Metarhizium anisopliae), yellowish green green muscardine fungus (
Metarhizium flavoviride), muscardine (
Beauveria brongniartii), paecilomyces fumosoroseus (
Paecilomyces fumosoroseus), Tang Pusen by the hair spore (
Hirsutella thompsonii) or powder Paecilomyces varioti (Paecilomyces farinosus) etc.Also make up beauveria bassiana and transformed bacterial strain.
In the following implementation, used experiment material is commercially available purchase product if no special instructions.
One, serine protease suppresses clone and the fungus expression vector structure of sub-serpin.
[embodiment 1]:
1. fruit bat serpin gene cloning:
In the serpin of fruit bat gene (NM_080112), an intron is arranged, utilize the method for PCR lap to remove intron.With P1/P2, P3/P4 is primer respectively.Primer sequence in the vector construction process is as follows:
P1:5 '-
GAATTCATGGCGAGCAAAGTCTCGATCCTTCTCCTGC-3 ' (the underscore sequence is the EcoRI site);
P2: 5’-GGGGTACGAACTTGGCATAGGAAGCTGCCGAGGCC-3’；
P3: 5’-GGCCTCGGCAGCTTCCTATGCCAAGTTCGTACCCC-3’；
P4:5 '-
CCCGGGTTAGACGCTCATGGGCGTGGGAT-3 ' (the underscore sequence is the SmaI site),
P5:5 '-
TCTAGAGAGCTCATCGCTTGGCAACG-3 ' (the underscore sequence is the XbaI site);
P6:5 '-AAAAAA
GCGGCCGCATGGCTCCTTTTCTTCAAACC-3 ' (the underscore sequence is the NotI site);
P7:5 '-GG
ACTAGTGCCGGCTCGCGGCGCCAAGGG-3 ' (the underscore sequence is the SpeI site);
P8：5’-TCCAATTGCTTCCGATCTGG-3’。
Drosophila gene group DNA is masterplate, with N end and the C end (take intron as cut-point) of Phusion archaeal dna polymerase (NEB) amplification serpin gene.Reaction system: 5 μ L, 5 * Phusion Taq polymerase buffer, 2 μ L, 2.5 mM dNTP, each 1 μ L(10 μ m of upstream and downstream primer), 0.4 U Phusion Taq DNA polymerase, cumulative volume 25 μ L.PCR reaction parameter: 98
oC (2 min); 30 circulations: 98
oC (20 s), 56
oC (30sec), 72 (1min); 72
oC (5 min).Reclaim respectively N end (1222bp) and C end (139bp) fragment, carry out assembling without primer.Reaction system: 5 μ L, 5 * Phusion Taq polymerase buffer, 2 μ L, 2.5 mM dNTP, N end fragment (50ng), C end fragment (50ng), 0.4 U Phusion Taq DNA polymerase, cumulative volume 25 μ L.Take above-mentioned assembling product as masterplate, the serpin cDNA sequence take P1 and P4 as the primer amplification total length, the clone advances pEASY-Blunt carrier (full formula King Company product).The carrier called after pEASY-serpin that checks order correct.
2. the structure of fusion gene:
Design primer P5-P7, the fusion gene of structure Bbsp:serpin.With beauveria bassiana (
Beauveria Bassiana) genome is masterplate, with P6 and P7 amplification beauveria bassiana chitinase Bbchit1(Genebank accession number: signal peptide AY145440), the clone advances the pGEM-T carrier, and sequence verification obtains carrier pGEM-Bbsp; Take pEASY-serpin as masterplate, with the maturation protein zone of P4 and P5 amplification serpin gene (removed 23 at N end self signal peptide, Fig. 1 is seen in the analysis of signal peptide), the clone advances the pGEM-T carrier, gets pGEM-serpin; NotI and SpeI double digestion pGEM-Bbsp reclaim Bbsp fragment (90bp); XbaI and SmaI double digestion pGEM-serpin get the ripe regional fragment (1362bp) of serpin.Bbsp and the common structure of serpin are advanced pUC-Pgpda-TtrpC, get the pUC-Pgpda-Bbsp:serpin-TtrpC carrier, as shown in Figure 2.XbaI single endonuclease digestion pUC-Pgpda-Bbsp:serpin-TtrpC carrier, reclaim Pgpda-Bbsp:serpin-TtrpC fragment (4.4kb), make up the pK2-BAR:GFP carrier, get the pK2-BAR:GFP-Pgpda-Bbsp:serpin-TtrpC plasmid and change agrobacterium tumefaciens lba4404 over to, its plasmid map is seen Fig. 3.
3. genetic of fungi transforms and identifies:
Utilize the Agrobacterium-mediated transformation method with above-mentioned expression original paper transform goal beauveria bassiana wild-type Bb0062 bacterial strain (beauveria bassiana (
Beauveria bassiana) Bb0062 separate self-infection cabbage caterpillar (
Pieris rape), being stored in Agricultural University Of Southwest's biotechnology center, also can obtain from known approach, for example CCTCC AF93297 or separate from other approach obtains).With primer P8 and P4 checking beauveria bassiana transformant, reaction system: 12.5 μ L, 2 * Taq mixture, each 1 μ L(10 μ m of upstream and downstream primer), the 50ng genomic dna adds water and mends to 25 μ L.PCR reaction parameter: 94
oC (5 min); 32 circulations: 94
oC (30 s), 56
oC (30sec), 72 (2min); 72
oC (5 min), expection clip size 1.9kb.1.2% agarose electrophoresis detects, and the result as shown in Figure 4.
M, the λ/Eco911 15 of MBI company; C, the positive control take the pK2-BAR:GFP-Pgpda-Bbsp:serpin-TtrpC plasmid as masterplate; WT is take beauveria bassiana wild type strain DNA as masterplate; 58～67, different Bbsp:serpin transformants.Result's demonstration, 58,63,67 transformants and positive control have the band of formed objects, and showing all has changing over to of goal gene.
Two, the toxicity test of transformant:
[embodiment 2]:
0.05% aseptic Tween-80 collects the spore of wild-type beauveria bassiana and Bbsp:serpin transformant, vortex, and four layers of lens wiping paper filter, and are centrifugal, regulate spore final concentration 1 * 10
8～5 * 10
6Individual/mL.The spore suspension for preparing is adopted the method inoculation greater wax moth of topical inoculation, take 0.05% Tween-80 as blank.The result shows: compare with wild type strain, 96h after inoculation, the toxic limit medium dose of serpin transformant have reduced by 3.5 times, and namely under this condition, transformant only needs can reach the insecticidal effect identical with wild-type less than the concentration of wild type strain 1/3; 5 * 10
6Under the concentration, median lethal time has shortened 14% under the spore concentration of individual/mL.This result shows, can significantly improve the insecticidal effect of bacterial strain by overexpression serpin in disinsection fungal.
Three, the propagation of fungi in the insect haemocoele is observed:
[embodiment 3]:
NaCl with 0.85% prepares the spore suspension (1 * 10 of wild type strain and transgenosis bacterial strain
7Individual/ml), take greater wax moth as the examination worm, every worm is injected the spore suspension of 4 μ L, places 26 ℃ of raisings.Get the quantity that hemolymph is observed hyphal body every 12h.The result compares with wild type strain as shown in Figure 5, and the rate of propagation of transgenosis bacterial strain (serpin bacterial strain) in insect hemolymph is significantly improved.Insect is silkworm, and A inoculates 0.85% NaCl; B, inoculation 1 * 10
7Individual/ml beauveria bassiana wild type strain; C, inoculation 1 * 10
7Individual/ml Bbsp:serpin turns Strain of Beauveria bassiana.Be illustrated as the observations of the rear 36h of inoculation.
Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make according to the present invention various changes and distortion, and only otherwise break away from spirit of the present invention, these changes and distortion all should belong to the scope of claims.
＜110〉Southwestern University
＜120〉recombinant serine protease suppresses son, fungus expression vector and disinsection fungal agent thereof
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＜223〉SEQ ID NO. 1 nucleotide sequence
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atggctcctt ttcttcaaac cagcctcgcg ctccttccat tgttggcttc cactatggtc 60
agcgcctcgc ccttggcgcc gcgagccggc actagagagc tcatcgcttg gcaacggcaa 120
caacaacagc agcaacagca gcaactgcag ctacagcagc aactgctgct gcagcagcaa 180
caacaccaac gtaacccaag accggagctg ggcctccgtt ccctgcccgg aaacccgtgg 240
acccagaaca atcaggaagc cataagcgat gtggtggcgg tggacctaac caaacgtgag 300
ccggtcactc cgccacccaa tcgcccgccg cccgtcttca gctacatgga ccgcttcagc 360
tccgagctct tcaaggagat cattaagtcg caaagtcagc agaacgtggt gttctcgccc 420
ttctccgtcc acgcgctgct ggccctgatc tacggggcct cggacggaaa aacgttccgg 480
gaactgcaga aggccggaga gttcagcaag aacgccatgg ccgtggccca ggacttcgag 540
agcgtgatca agtacaagaa gcatttggag ggcgcagatc tgaccctggc aaccaaggtc 600
tactacaacc gcgagctggg tggcgtcaac cacagctacg atgagtacgc aaagttctat 660
ttcagcgccg gcacggaggc tgtcgacatg cagaacgcca aagacacggc agccaagatc 720
aacgcctggg tgatggacac gacgcggaac aagatccggg acctggtcac accgaccgac 780
gttgacccac agacgcaggc gctccttgtg aatgcagtct acttccaggg tcgttgggag 840
cacgaattcg ccaccatgga cacatcaccc tacgactttc agcacaccaa cggcagaatt 900
tccaaggtgg ccatgatgtt caacgacgat gtgtacggcc tggccgagct gcctgagctg 960
ggcgccaccg ccttggaact ggcctacaag gacagcgcca ccagcatgct aatcctgctg 1020
cccaatgaga ccaccggact gggcaaaatg ctgcagcagc tgtcccggcc ggagttcgat 1080
ctcaaccgcg ttgcccaccg cctgcgccgc cagtcggtcg ctgtgcgcct gcccaagttc 1140
caattcgagt tcgagcagga catgaccgag ccgctaaaga acctgggagt ccaccagatg 1200
ttcacgccca actcgcaggt gaccaagttg atggatcagc cggtgcgcgt gagcaagatc 1260
ctgcagaagg cctacatcaa tgtgggcgag gcgggcacag aggcctcggc agcttcctat 1320
gccaagttcg tacccctttc gctgcctccc aagcccacgg agttcgtcgc caaccggcca 1380
ttcgtcttcg ccgtccgcac cccctcctca gttctgttca taggtcacgt ggagtatccc 1440
acgcccatga gcgtctaa 1458
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tpmsv 485
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Claims (4)
1. a recombinant serine protease suppresses son, it is characterized in that, adopts Protocols in Molecular Biology to make up a recombinant nucleotide sequence, its contain the coding beauveria bassiana (
Beauveria bassiana) signal peptide sequence of chitinase and the serine protease of coding fruit bat suppress subbase because of, it has nucleotide sequence and the aminoacid sequence shown in SEQ ID NO. 2 shown in SEQ ID NO. 1.
2. described recombinant serine protease suppresses sub construction process according to claim 1, it is characterized in that, comprises the steps:
1) fruit bat serpin gene cloning:
In the serpin of fruit bat gene (NM_080112), an intron is arranged, utilize the method for PCR lap to remove intron; With P1/P2, P3/P4 is primer respectively; Primer sequence is as follows:
P1:5 '-
GAATTCATGGCGAGCAAAGTCTCGATCCTTCTCCTGC-3 ', the underscore sequence is the EcoRI site;
P2: 5’-GGGGTACGAACTTGGCATAGGAAGCTGCCGAGGCC-3’；
P3: 5’-GGCCTCGGCAGCTTCCTATGCCAAGTTCGTACCCC-3’；
P4:5 '-
CCCGGGTTAGACGCTCATGGGCGTGGGAT-3 ' (the underscore sequence is the SmaI site),
Drosophila gene group DNA is masterplate, with N end and the C end of Phusion archaeal dna polymerase (NEB) amplification serpin gene; Reaction system: 5 μ L, 5 * Phusion Taq polymerase buffer, 2 μ L, 2.5 mM dNTP, each 1 μ L(10 μ m of upstream and downstream primer), 0.4 U Phusion Taq DNA polymerase, cumulative volume 25 μ L;
PCR reaction parameter: 98
oC (2 min); 30 circulations: 98
oC (20 s), 56
oC (30sec), 72 (1min), 72
oC (5 min); Reclaim respectively N end (1222bp) and C end (139bp) fragment, carry out assembling without primer;
Reaction system: 5 μ L, 5 * Phusion Taq polymerase buffer, 2 μ L, 2.5 mM dNTP, N end fragment (50ng), C end fragment (50ng), 0.4 U Phusion Taq DNA polymerase, cumulative volume 25 μ L;
Take above-mentioned assembling product as masterplate, the serpin cDNA sequence take P1 and P4 as the primer amplification total length, the clone advances pEASY-Blunt carrier (full formula gold product); The carrier called after pEASY-serpin that checks order correct;
2) structure of fusion gene:
Design primer P5-P7, the fusion gene of structure Bbsp:serpin;
Primer sequence is as follows:
P5:5 '-
TCTAGAGAGCTCATCGCTTGGCAACG-3 ', the underscore sequence is the XbaI site;
P6:5 '-AAAAAA
GCGGCCGCATGGCTCCTTTTCTTCAAACC-3 ', the underscore sequence is the NotI site;
P7:5 '-GG
ACTAGTGCCGGCTCGCGGCGCCAAGGG-3 ', the underscore sequence is the SpeI site;
With beauveria bassiana (
Beauveria Bassiana) genome is masterplate, with the signal peptide of P6 and P7 amplification beauveria bassiana chitinase gene Bbchit1, the clone advances pGEM-T carrier (Promega), and sequence verification obtains carrier pGEM-Bbsp; Take pEASY-serpin as masterplate, with the maturation protein zone of P4 and P5 amplification serpin gene, the clone advances the pGEM-T carrier, gets pGEM-serpin;
NotI and SpeI double digestion pGEM-Bbsp reclaim Bbsp fragment (90bp); XbaI and SmaI double digestion pGEM-serpin get the ripe regional fragment (1362bp) of serpin.
3. the fungus expression vector of a recombinant protein, it is characterized in that, with the described Bbsp(90bp of claim 2) and serpin(1362bp) jointly make up pUC-Pgpda-TtrpC, get the pUC-Pgpda-Bbsp:serpin-TtrpC carrier, it has the expression original paper of fusion rotein Bbsp:serpin, a Fungal community composition promotor Pgpda is contained in the upstream, and a terminator TtrpC is contained in the downstream.
4. the disinsection fungal agent of a recombinant protein, it is characterized in that, with carrier pUC-Pgpda-Bbsp:serpin-TtrpC XbaI single endonuclease digestion claimed in claim 3, get the expression original paper Pgpda-Bbsp:serpin-TtrpC fragment (4.4kb) of fusion rotein Bbsp:serpin, make up fungus expression vector pK2-BAR:GFP, get the pK2-BAR:GFP-Pgpda-Bbsp:serpin-TtrpC plasmid and change agrobacterium tumefaciens lba4404 over to; Utilize the Agrobacterium-mediated transformation method to obtain the disinsection fungal transformant of constructive expression Bbsp:serpin.
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Cited By (4)
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| CN103484386A (en) * | 2013-09-09 | 2014-01-01 | 华中农业大学 | Recombinant paecilomyces lilacinus strain PNVT8 and application thereof |
| CN104357413A (en) * | 2014-11-26 | 2015-02-18 | 西南大学 | Recombinant glucose-fructose oxidoreductase and fungal expression vector as well as fungal insecticide thereof |
| CN104726351A (en) * | 2015-04-14 | 2015-06-24 | 南方医科大学 | Bivalent recombination beauveria bassiana capable of killing mosquito larvae and preparation method of bivalent recombining beauveria bassiana |
| CN107418903A (en) * | 2017-06-27 | 2017-12-01 | 山东大学 | A kind of FOS synthesis engineered strain for expressing glucose oxidase and its construction method and application |
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| CN103484386A (en) * | 2013-09-09 | 2014-01-01 | 华中农业大学 | Recombinant paecilomyces lilacinus strain PNVT8 and application thereof |
| CN104357413A (en) * | 2014-11-26 | 2015-02-18 | 西南大学 | Recombinant glucose-fructose oxidoreductase and fungal expression vector as well as fungal insecticide thereof |
| CN104357413B (en) * | 2014-11-26 | 2017-07-07 | 西南大学 | One kind restructuring dextrose fructose oxidoreducing enzyme and its fungus expression vector and fungus insecticide |
| CN104726351A (en) * | 2015-04-14 | 2015-06-24 | 南方医科大学 | Bivalent recombination beauveria bassiana capable of killing mosquito larvae and preparation method of bivalent recombining beauveria bassiana |
| CN104726351B (en) * | 2015-04-14 | 2018-06-01 | 南方医科大学 | A kind of bivalent Recombinant Strain of Beauveria bassiana for killing mosquito larvae and preparation method thereof |
| CN107418903A (en) * | 2017-06-27 | 2017-12-01 | 山东大学 | A kind of FOS synthesis engineered strain for expressing glucose oxidase and its construction method and application |
| CN107418903B (en) * | 2017-06-27 | 2020-11-20 | 山东大学 | Fructooligosaccharide synthetic engineering strain for expressing glucose oxidase and construction method and application thereof |
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