CN103898068B - Express recombinant baculovirus and the application thereof of insect prothoracic gland hormone gene - Google Patents
Express recombinant baculovirus and the application thereof of insect prothoracic gland hormone gene Download PDFInfo
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Abstract
The invention discloses and a kind ofly express the recombinant baculovirus of insect prothoracic gland hormone gene and preparing the application in sterilant.Described insect thoratropic hormone is following (a) or (b) or (c): the protein that (a) is made up of the aminoacid sequence shown in sequence in sequence table 7; B protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1; C (a) or (b) is had the protein of insect thoratropic hormone function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by ().Bollworm as model insects, by insect expression in vivo prothoracic gland hormone gene, can be promoted that baculovirus is to the insecticidal power of insect, thus reach the object of Control pests to murrain by the present invention.The present invention has substantial worth for agriculture production.
Description
Technical field
The present invention relates to and a kind ofly express the recombinant baculovirus of insect prothoracic gland hormone gene and preparing the application in sterilant.
Background technology
The growing of insect, cast off a skin, participation that metamorphosis, diapause, reproduction, physiological process and the behavior reaction etc. such as polytypism all be unable to do without hormone.Moulting hormone (ecdysone) is the hormone that animal (mainly Arthropoda Insecta, Crustacea animal) can be regulated to cast off a skin secreted by prothoracic gland.The young and the adult of shrimp, crab have phenomenon of casting off a skin, and larva and the adult of Apterygota animal have phenomenon of casting off a skin, and Pterygota animal only has larva to have phenomenon of casting off a skin.Most insects larva has phenomenon of periodically casting off a skin.
Moulting hormone synthesizes using cholesterol as skeleton, but insect itself can not synthetic cholesterol, but obtains from plant directly or indirectly.Moulting hormone is stimulated by prothoracicotropic hormone and synthesizes at prothoracic gland, except prothoracic gland, in the embryo that the ovary of the positive maturation of lepidopteran, some insects of Orthoptera and lepidopteran, Orthoptera grow, also can synthesize moulting hormone class material or intermediate.Usually in the prothoracic gland of insect, α-moulting hormone (α-ecdyson) is first synthesized, resynthesis β-moulting hormone (20-hydroxyecdysone).Specific activity α-the moulting hormone of β-moulting hormone is much higher, is the major hormone regulating insect molting.
Insect hormone has become major fields of sterilant research and development, its mechanism of action is different from the past acts on neural conventional pesticides, toxicity is low, pollute less, on natural enemy and beneficial organism impact little, contribute to the development of sustainable agriculture, be conducive to that pollution-free green food is produced, be beneficial to man health.
Baculovirus (baculovirus) is the double-stranded cyclic DNA virus that a class has cyst membrane parcel, and Genome Size is 80-180kb.The constitutional features of baculovirus is virus particle is shaft-like, and outer bread is by one deck albuminous membranae, and outward appearance has certain rule, is called polyhedrosis virus (NPV).Baculovirus single-minded infection arthropods, its natural reservoir (of bird flu viruses) is mainly lepidopteran, Hymenoptera and dipteral insect, also infects some crustaceans and mite class.At occurring in nature, there is sprout virus particle (buddedvirus, BV) and embedded virus particle (occlusion-derivedvirus, ODV in baculovirus; Also known as polyhedron inclusion body, be called for short PIB or OBs) two-strain form.BV is a kind of virus particle propagated between insect cell, with the envelope protein of encoding viral, can combine, propagate in insect body or between the cell of clone with the special receptor of cell surface.ODV is baculovirus communication form in the environment, is embedded in inclusion body.Inclusion body is made up of polyhedrin protein, is polyhedron cyst membrane outside it, highly stable in the environment, plays a protective role to virus particle.After swallowing the food containing polyhedron inclusion body without the larva infected, degrade under the acid pH environment of polyhedrin in insect midgut, releasing virus particle core capsid, viruses contact the epidermic cell tissue of intestines in infected insect larva, manufacture virus particle of sprouting, bud virus particle enters in the haemocoele of host insect by sprouting through cytoplasmic membrane, cause system infections, in the baculovirus infection later stage, virus particle, by a large amount of polyhedrin bag quilt, forms polyhedron inclusion body.Baculovirus has higher pathogenic to host insect, to natural enemies security, free from environmental pollution, and especially it can form prevailing disease and long-term control insect population and not developing immunity to drugs in pest population, is obviously better than other sterilant.
Summary of the invention
The object of this invention is to provide and a kind ofly express the recombinant baculovirus of insect prothoracic gland hormone gene and preparing the application in sterilant.
The invention provides the recombinant baculovirus of expressing insect thoratropic hormone gene.
Described insect thoratropic hormone is following (a) or (b) or (c): the protein that (a) is made up of the aminoacid sequence shown in sequence in sequence table 7; B protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1; C (a) or (b) is had the protein of insect thoratropic hormone function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by ().
Described insect thoratropic hormone gene is following 1) or 2) or 3) or 4) or 5) DNA molecular: the 1) DNA molecular shown in sequence 6 of sequence table; 2) the coding region sequence 2 that is sequence table is from the DNA molecular shown in 5 ' end the 30 to 710 Nucleotide; 3) DNA molecular shown in sequence 2 of sequence table; 4) under strict conditions with 1) or 2) or 3) DNA sequence dna that limits hybridizes and the DNA molecular of code for said proteins; 5) with 1) or 2) or 3) DNA sequence dna that limits has the DNA molecular of more than 90% homology and code for said proteins.
The preparation method of described recombinant baculovirus is as follows: recombinant baculovirus plasmid is imported insect cell and culturing cell, obtains recombinant baculovirus; Described recombinant baculovirus plasmid is the baculovirus plasmid with described insect thoratropic hormone gene.
In described recombinant baculovirus plasmid, the expression of described insect thoratropic hormone gene can be started by op166 promotor (baculovirus early promoter) and/or P10 promotor (baculovirus late promoter).Described op166 promotor is specifically as shown in the sequence 5 of sequence table.Described P10 promotor is specifically as shown in the sequence 8 of sequence table.
Described recombinant baculovirus plasmid can have expression cassette first and expression cassette second; Described expression cassette first is the expression cassette for expressing described insect thoratropic hormone gene; Described expression cassette second is the expression cassette of the encoding gene for expressing baculovirus polyhedrin (Hapolh albumen).
Shown recombinant baculovirus plasmid specifically can be the recombinant plasmid N obtained in the attB site in described expression cassette first and described expression cassette second insertion Hz8 ⊿ egtBacmid.
Described recombinant vectors specifically can be the recombinant plasmid Q obtained in the attB site comprised between Tn7L and Tn7R site in recombinant plasmid M in the DNA fragmentation insertion Hz8 ⊿ egtBacmid of described expression cassette first and described expression cassette second.Described recombinant plasmid M specifically can be and the encoding gene of described op166 promotor, described insect thoratropic hormone gene and described polyhedrin is inserted expression vector respectively (as carrier pFastBac
tMdUAL) recombinant plasmid that multiple clone site obtains.Described op166 promotor can insertion vector pFastBac
tMbetween NcoI and the SmaI restriction enzyme site of DUAL.Described insect thoratropic hormone gene can insert pFastBac
tMbetween NcoI and the KpnI restriction enzyme site of DUAL.The encoding gene of described polyhedrin can insertion vector pFastBac
tMbetween BamHI and the HindIII site of DUAL.
Described Hz8 ⊿ egtBacmid is the recombinant plasmid will obtained after steroidal uridine diphosphate glucose transferase gene of casting off a skin (also known as EcdysteroidUDP-Glucosyltransferase gene, the being called for short egt gene) deactivation in HaBacHZ8Bacmid.The recombinant plasmid that the open reading frame that described Hz8 ⊿ egtBacmid is specially the egt gene replaced in HaBacHZ8Bacmid by the egfp gene shown in the sequence 9 of sequence table obtains from the DNA fragmentation shown in 5 ' end, 116 to 1470 Nucleotide.
Described recombinant baculovirus can be the form of virus particle of sprouting or the form (polyhedron inclusion body, compared with virus particle of sprouting, has the ability of oral infection) of polyhedron inclusion body.
The virus particle of sprouting of described recombinant baculovirus specifically prepares by following method: described recombinant plasmid N or described recombinant plasmid Q is imported insect cell (as bollworm HzAm1 cell) and cultivates, collect culture supernatant, be the virus liquid of virus particle of sprouting described in containing.
The polyhedron inclusion body of described recombinant baculovirus prepares by following method: described recombinant plasmid N or described recombinant plasmid Q is imported insect cell (as bollworm HzAm1 cell) and cultivates, collecting cell precipitates, namely containing described polyhedron inclusion body in cell precipitation.The polyhedron inclusion body of described recombinant baculovirus obtains by described virus particle of sprouting is inoculated insect and collected insect bodies.The polyhedron inclusion body of described recombinant baculovirus specifically prepares by following method: by the described virus particle inoculation insect that sprouts, collect corpse after insect death and smash to pieces, add water and stir evenly rear filtered through gauze, collect filtrate and the centrifugal 10min of 300g, collect supernatant liquor and the centrifugal 30min of 3000g, collecting precipitation, is described polyhedron inclusion body.Described insect can be bollworm.
The present invention also protects a kind of preparation method of recombinant baculovirus, comprises the steps: recombinant baculovirus plasmid to import insect cell and culturing cell, obtains recombinant baculovirus; Described recombinant baculovirus plasmid is the baculovirus plasmid with described insect thoratropic hormone gene.
In described recombinant baculovirus plasmid, the expression of described insect thoratropic hormone gene can be started by op166 promotor (baculovirus early promoter) and/or P10 promotor (baculovirus late promoter).Described op166 promotor is specifically as shown in the sequence 5 of sequence table.Described P10 promotor is specifically as shown in the sequence 8 of sequence table.
Described recombinant baculovirus plasmid can have expression cassette first and expression cassette second; Described expression cassette first is the expression cassette for expressing described insect thoratropic hormone gene; Described expression cassette second is the expression cassette of the encoding gene for expressing baculovirus polyhedrin (Hapolh albumen).
Shown recombinant baculovirus plasmid specifically can be the recombinant plasmid N obtained in the attB site in described expression cassette first and described expression cassette second insertion Hz8 ⊿ egtBacmid.
Described recombinant vectors specifically can be the recombinant plasmid Q obtained in the attB site comprised between Tn7L and Tn7R site in recombinant plasmid M in the DNA fragmentation insertion Hz8 ⊿ egtBacmid of described expression cassette first and described expression cassette second.Described recombinant plasmid M specifically can be and the encoding gene of described op166 promotor, described insect thoratropic hormone gene and described polyhedrin is inserted expression vector respectively (as carrier pFastBac
tMdUAL) recombinant plasmid that multiple clone site obtains.Described op166 promotor can insertion vector pFastBac
tMbetween NcoI and the SmaI restriction enzyme site of DUAL.Described insect thoratropic hormone gene can insert pFastBac
tMbetween NcoI and the KpnI restriction enzyme site of DUAL.The encoding gene of described polyhedrin can insertion vector pFastBac
tMbetween BamHI and the HindIII site of DUAL.
Described recombinant baculovirus can be the form of virus particle of sprouting or the form (polyhedron inclusion body, compared with virus particle of sprouting, has the ability of oral infection) of polyhedron inclusion body.
The virus particle of sprouting of described recombinant baculovirus specifically prepares by following method: described recombinant plasmid N or described recombinant plasmid Q is imported insect cell (as bollworm HzAm1 cell) and cultivates, collect culture supernatant, be the virus liquid of virus particle of sprouting described in containing.
The polyhedron inclusion body of described recombinant baculovirus prepares by following method: described recombinant plasmid N or described recombinant plasmid Q is imported insect cell (as bollworm HzAm1 cell) and cultivates, collecting cell precipitates, namely containing described polyhedron inclusion body in cell precipitation.The polyhedron inclusion body of described recombinant baculovirus obtains by described virus particle of sprouting is inoculated insect and collected insect bodies.The polyhedron inclusion body of described recombinant baculovirus specifically prepares by following method: by the described virus particle inoculation insect that sprouts, collect corpse after insect death and smash to pieces, add water and stir evenly rear filtered through gauze, collect filtrate and the centrifugal 10min of 300g, collect supernatant liquor and the centrifugal 30min of 3000g, collecting precipitation, is described polyhedron inclusion body.Described insect can be bollworm.
Arbitrary described baculovirus polyhedrin is following (d) or (e) above: the protein that (d) is made up of the aminoacid sequence shown in sequence in sequence table 3; (e) by the aminoacid sequence of sequence 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have polyhedrin function the protein derived by sequence 3.
The encoding gene of arbitrary described baculovirus polyhedrin can be following 6 above) or 7) or 8) or 9) DNA molecular: 6) sequence 4 of sequence table is from the DNA molecular shown in 5 ' end 157-897 position Nucleotide; 7) DNA molecular shown in sequence 4 in sequence table; 8) under strict conditions with 6) or 7) DNA sequence dna that limits hybridizes and the DNA molecular of polyhedrin of encoding; 9) with 6) or 7) DNA sequence dna that limits has more than 90% homology and the DNA molecular of polyhedrin of encoding.
The present invention also protects a kind of sterilant, and its activeconstituents is at least one in following material: (I) recombinant baculovirus containing insect prothoracic gland hormone gene, recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium; (II) insect prothoracic gland hormone gene; (III) insect prothoracic gland hormone.Described insect prothoracic gland hormone can be above arbitrary described insect prothoracic gland hormone.Described insect prothoracic gland hormone gene can be above arbitrary described insect prothoracic gland hormone gene.Described recombinant baculovirus can be above arbitrary described recombinant baculovirus.Described sterilant can be bollworm sterilant.
At least one of the present invention also in following material is preparing the application in sterilant: (I) recombinant baculovirus containing insect prothoracic gland hormone gene, recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium; (II) insect prothoracic gland hormone gene; (III) insect prothoracic gland hormone.Described insect prothoracic gland hormone can be above arbitrary described insect prothoracic gland hormone.Described insect prothoracic gland hormone gene can be above arbitrary described insect prothoracic gland hormone gene.Described recombinant baculovirus can be above arbitrary described recombinant baculovirus.Described sterilant can be bollworm sterilant.
Bollworm as model insects, by by prothoracic gland hormone gene process LAN in insect body, can be promoted that baculovirus is to the insecticidal power of insect, thus reach the object of Control pests to murrain by the present invention.The present invention has substantial worth for agriculture production.
Accompanying drawing explanation
Fig. 1 is the structural representation of recombinant plasmid pFBD-HaPH-op166-HaPTTH.
Fig. 2 is the genomic structural representation of HaBac △ egt.
Fig. 3 is that the PCR of Hz8 ⊿ egtBacmid and HaBac △ egt-HaPTTHbacmid identifies electrophorogram; 1:HaBac △ egt-HaPTTHbacmid; 2:Hz8 ⊿ egtBacmid; .
Fig. 4 is the fluorescence photo cultivated in embodiment 4 after 3 days.
Fig. 5 is the electrophorogram of pcr amplification product in embodiment 6.
Fig. 6 is the median lethal time of polyhedron inclusion body in embodiment 7.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.The English full name of Bacmid is Baculovirusplasmid, and Chinese implication is baculovirus plasmid.Grace ' s substratum pulvis: purchased from Invitrogen.Intestinal bacteria DH10B: purchased from Invitrogen company, article No. 18290-015.
Carrier pFastBac
tMdUAL: reference: Bac-to-BacBaculovirusExpressionSystemVersionD6April2004.
Helper plasmid: reference: Bac-to-BacBaculovirusExpressionSystemVersionD6April2004.
This plasmid of HaBacHZ8Bacmid(has improved Baculovirus Gene group DNA, described transformation refers to: insert the attB site of inserting for transposable element in polyhedron gene, former polyhedrin gene inactive): reference: Han-zhongWang, FeiDeng, Pijlman, GorbenP, Xin-wenChen, Xiu-lianSun, Vlak, JustM, Zhi-hongHu.CloningofbiologicallyactivegenomesfromaHelico verpaarmigerasingle-nucleocapsidnucleopolyhedrovirusisol atebyusingabacterialartificialchromosome.VirusRes.2003No v, 97 (2): 57-63., Yan-HongSI, Ming-GangFANG, YiHUANG, Fang-LiangZHENG, TingLI, Zhi-HongHU, Han-ZhongWANG.ConstructionandCharacterizationofaHelicove rpaarmigeraNucleopolyhedrovirusBacterialArtificialChromo somewithDeletionofEcdysteroidUDP-GlucosyltransferaseGene .Bioscience, Biotechnology, andBiochemistryVol.71 (2007) No.10P2435-2441.
Bollworm HzAm1 cell: Zhang Youhong; Wang Hualin; Zhu Xiongwei; National muscial instrument; Chen Yan; Lv Zhong; Ma Jie; Zhang Jing; Be applicable to Screening of Media and low serum domestication [J] of bolworm cell HzAm1 growth; Biotechnology journal; 04 phase in 2006.
Bollworm (Helicoverpaarmigera): reference: ZhaoX., K.Wu, G.LiangandY.Guo.2009.ModifiedfemalecallingbehaviorinCry1 Ac-resistantHelicoverpaarmigera (Lepidoptera:Noctuidae) .PestManagementScience.65 (4): 353-357..
The thoratropic hormone of bollworm is as shown in the sequence 1 of sequence table (being made up of 226 amino-acid residues).The cDNA of the encoding gene (HaPTTH gene) of the thoratropic hormone of bollworm is as shown in the sequence 2 of sequence table, its open reading frame such as sequence 2 of sequence table forms from 5 ' end the 30 to 710 Nucleotide, 430-783 position nucleotide coding reactive site amino acid.
LB substratum (pH7.0): solvent is water, solute and concentration as follows: yeast extract paste 5g/L, peptone 10g/L, NaCl10g/L.
The structure of embodiment 1, expression vector
1, composition sequence table double chain DNA molecule shown in sequence 4 (polyhedron gene of bollworm, also known as Hapolh gene, the sequence 4 that its open reading frame is sequence table is from shown in 5 ' end 157-897 position Nucleotide; The Hapolh albumen shown in sequence 3 of Hapolh gene coded sequence table) and be inserted into carrier pFastBac
tMbetween BamHI and the HindIII site of DUAL, obtain recombinant plasmid pFBD-HaPH.
2, composition sequence table the double chain DNA molecule (baculovirus early promoter, also known as op166 promotor) shown in sequence 5 and between NcoI and the SmaI restriction enzyme site being inserted into recombinant plasmid pFBD-HaPH, obtain recombinant plasmid pFBD-HaPH-op166.
3, composition sequence table the double chain DNA molecule shown in sequence 2 and it can be used as template, with PHN and PHK composition primer pair carry out pcr amplification, obtain pcr amplification product.
PHN:5’-CATG
CCATGG ATGATAACTCGGCCGTTAC-3’；
PHK:5’-CC
GGTACCTTATTTCTCAGTAGCGTA-3’。
In PHN, the restriction endonuclease recognition sequence of underscore mark restriction enzyme NcoI, the encoding gene of square frame mark signal peptide.In PHK, the restriction endonuclease recognition sequence of underscore mark restriction enzyme KpnI.
4, use the pcr amplification product of restriction enzyme NcoI and KpnI double digestion step 3, reclaim digestion products.
5, with restriction enzyme NcoI and KpnI double digestion recombinant plasmid pFBD-HaPH-op166, the carrier framework of about 6474bp is reclaimed.
6, the digestion products of step 4 is connected with the carrier framework of step 5, obtains recombinant plasmid pFBD-HaPH-op166-HaPTTH.According to sequencing result, structrual description carries out to recombinant plasmid as follows: between NcoI and the KpnI restriction enzyme site of recombinant plasmid pFBD-HaPH-op166, insert the double chain DNA molecule shown in sequence 6 of sequence table (in sequence 6, be the encoding gene of signal peptide from 5 ' end the 1 to 63 Nucleotide, the 64 to 744 Nucleotide is HaPTTH gene; Protein shown in sequence 6 encoding sequence 7).
The preparation of embodiment 2, Hz8 ⊿ egtBacmid
Egfp gene shown in the sequence 9 of sequence table is replaced the open reading frame of the egt gene in HaBacHZ8Bacmid from the DNA fragmentation shown in 5 ' end, 116 to 1470 Nucleotide, obtain Hz8 ⊿ egtBacmid.
The acquisition of embodiment 3, HaBac △ egt-HaPTTHbacmid
Mechanism description: in intestinal bacteria, Helper plasmid promotes the generation of swivel base, the DNA fragmentation comprising HaPTTH gene and Hapolh gene (inserting the polyhedrin gene inactive of the Hz8 ⊿ egtBacmid caused in order to cover exogenous sequences) between Tn7L and the Tn7R site of recombinant plasmid pFBD-HaPH-op166-HaPTTH inserts the attB site of Hz8 ⊿ egtBacmid by transposition, obtain recombinant baculovirus plasmid.
1, Helper plasmid is imported the competent cell of intestinal bacteria DH10B, obtain recombinant bacterium I.
2, by the recombinant bacterium I that Hz8 ⊿ egtBacmid steps for importing 1 obtains, recombinant bacterium II is obtained.
3, the recombinant bacterium II that the recombinant plasmid pFBD-HaPH-op166-HaPTTH step of converting 2 embodiment 1 obtained obtains, then LB culture medium flat plate is coated (containing 50 μ g/mL kantlex, 7 μ g/mL gentamicins, 10 μ g/mL tsiklomitsins, 100 μ g/mLX-gal and 40 μ g/mLIPTG), cultivate 24-48 hour for 37 DEG C, check the blue hickie on flat board, white colony is rule again at a fresh LB culture medium flat plate (containing 50 μ g/mL kantlex, 7 μ g/mL gentamicins, 10 μ g/mL tsiklomitsins, 100 μ g/mLX-gal and 40 μ g/mLIPTG), cultivate 24-48 hour for 37 DEG C, extracting waste bacterium colony, be the bacterium colony containing recombinant baculovirus plasmid (also known as HaBac △ egt-HaPTTHbacmid).
The PCR of Hz8 ⊿ egtBacmid and HaBac △ egt-HaPTTHbacmid identifies that electrophorogram is shown in Fig. 3, adopt the primer pair that the primer of the primer of corresponding Hz8 ⊿ egtBacmid (5 '-CCCAGTCACGACGTTGTAAAACG-3 ') and the described HaPTTH gene of correspondence (5 '-ATCCTGATGAGTTGTCTGCT-3 ') forms, HaBac △ egt-HaPTTHbacmid shows the object fragment of about 1.9kb, and Hz8 ⊿ egtBacmid does not show described object fragment.
The acquisition of embodiment 4, recombinant baculovirus (virus particle of sprouting)
One, the acquisition of recombinant baculovirus
1, inoculation 5 × 10 in the capsule (Grace ' the s substratum containing 10%FBS is housed) of 35mm
5individual bollworm HzAm1 cell, 27 ° of C overnight incubation, inhale and abandon supernatant liquor, add 1mlGrace ' s substratum room temperature and place 1 hour in Biohazard Safety Equipment.
2, use Grace ' s substratum to be diluted to 100 μ l respectively 5 μ gHaBac △ egt-HaPTTHbacmid and 10 μ lLipofectin, then both are mixed, every 7min mixing once, after 45min, add 400 μ lGrace ' s substratum, mixing.
3, get the capsule of completing steps 1, inhale and abandon supernatant liquor and add the mixed solution that step 2 obtains, 27 ° of C cultivate the every 15min of 30min(and shake up capsule once); Then add 400 μ lGrace ' s substratum, mixing, 27 ° of C cultivate 6 hours; Then add Grace ' the s substratum of 1mL containing 20%FBS, mixing, collects supernatant liquor after 27 ° of C cultivate 6 days, is the f1 disease venom of recombinant baculovirus (also known as HaBac △ egtHaPTTH).27 ° of C cultivate in the process of 6 days, observe the generation situation of green fluorescence every day under fluorescence inverted microscope, cultivate the fluorescence photo after 3 days and see Fig. 4 (can observe green fluorescence, represent and infect successfully).
4, at 25cm
2batch cultur bottle in press MOI=0.1 dosage f1 disease venom infect bollworm HzAm1 cell, cultivate 96 h before harvest supernatant liquors, be s-generation virus liquid for 27 DEG C.
5, at 25cm
2batch cultur bottle in press MOI=0.1 dosage s-generation virus liquid infect bollworm HzAm1 cell, cultivate 96 h before harvest supernatant liquors, be third generation virus liquid for 27 DEG C.
Two, the acquisition of control baculovirus (virus particle of sprouting)
Replace HaBac △ egt-HaPTTHbacmid carry out step one with Hz8 △ egtBacmid, obtain f1 disease venom, s-generation virus liquid and the third generation virus liquid of baculovirus (also known as HaBac △ egt).The genomic structural representation of HaBac △ egt is shown in Fig. 2.
Three, the acquisition of control baculovirus (virus particle of sprouting)
Recombinant plasmid pFBD-HaPH-op166-HaPTTH is replaced to carry out embodiment 3 recombinant plasmid pFBD-HaPH-op166, the DNA fragmentation comprising Hapolh gene between Tn7L and the Tn7R site of carrier pFBD-HaPH inserts the polyhedron gene site (polyhedrinlocus) of Hz8 ⊿ egtBacmid by transposition, obtain recombinant baculovirus plasmid (also known as HaBac △ egt-Abacmid).
Replace HaBac △ egt-HaPTTHbacmid carry out step one with HaBac △ egt-Abacmid, obtain f1 disease venom, s-generation virus liquid and the third generation virus liquid of baculovirus (also known as HaBac △ egt-A).
The acquisition of embodiment 5, polyhedron inclusion body
One, the acquisition of HaBac △ egtHaPTTH polyhedron inclusion body
1, bollworm is supported to 3 age Mos, put to numb with cold before injection on ice, the third generation virus liquid of HaBac △ egtHaPTTH is got with the microsyringe of sterilization, second from the bottom and be injected in hemolymph between the 3rd at bollworm abdominal foot, every bollworm injects 5 μ l, after 5 days, bollworm liquefaction is dead, collects worm corpse.
2, add distilled water after being smashed to pieces by the worm corpse of step 1 and stir evenly, collect filtrate by 3 layers of filtered through gauze, centrifugal for filtrate 300g 10min is collected supernatant liquor, supernatant liquor is carried out the centrifugal 30min of 3000g, collecting precipitation (HaBac △ egtHaPTTH polyhedron inclusion body).
Two, the acquisition of HaBac △ egt polyhedron inclusion body
Replace the third generation virus liquid of HaBac △ egtHaPTTH with the third generation virus liquid of HaBac △ egt, other is with step one, obtains HaBac △ egt polyhedron inclusion body.
Three, the acquisition of HaBac △ egt-A polyhedron inclusion body
Replace the third generation virus liquid of HaBac △ egtHaPTTH with the third generation virus liquid of HaBac △ egt-A, other is with step one, obtains HaBac △ egt-A polyhedron inclusion body.
The medial lethal dose of embodiment 6, polyhedron inclusion body
HaBac △ egtHaPTTH polyhedron inclusion body embodiment 5 prepared respectively, HaBac △ egt polyhedron inclusion body and HaBac △ egt-A polyhedron inclusion body carry out the detection of following medial lethal dose:
1, select uniform two age Mo cotton bollworm larvae, be placed in 96 aseptic orifice plates, hungryly in 28 DEG C of constant temperature illumination boxs cultivate 16 hours, allow their decortications enter in next age.
2, the sterilized water dilution being used by polyhedron inclusion body the sucrose containing 4g/100mL and 1mg/mL to eat light blue is 1 × 10
6individual polyhedron inclusion body/mL, 3 × 10
5individual polyhedron inclusion body/mL, 1 × 10
5individual polyhedron inclusion body/mL, 3 × 10
4individual polyhedron inclusion body/mL or 1 × 10
4the suspension (with blood counting chamber counting polyhedron inclusion body) of individual polyhedron inclusion body/mL.
3, the cotton bollworm larvae of completing steps 1 is divided into five groups, often organize 48 larvas, feed with the suspension that step 2 obtains respectively, timing from feeding, the larva that in 10min, middle intestines become blueness proceeds in 24 orifice plates containing fresh artificial diet, observes 2 every day and adds up mortality ratio until all for trying dead larvae or pupating.Carry out repeating experiment for three times.Dense dosage (the LC of median lethal of polygonal inclusion body is calculated with Probit regression analysis
50) and standard error, compare two-strain LC
50between the significance of difference.
The toxic limit medium dose of HaBac △ egtHaPTTH polyhedron inclusion body is 2.43 × 10
4individual polyhedron inclusion body/ml, standard deviation is 0.39 × 10
4individual polyhedron inclusion body/ml, 95% fiducial interval is 1.75-3.29 × 10
4individual polyhedron inclusion body/ml.The toxic limit medium dose of HaBac △ egt polyhedron inclusion body is 8.12 × 10
4individual polyhedron inclusion body/ml, standard deviation is 1.16 × 10
4individual polyhedron inclusion body/ml, 95% fiducial interval is 6.16-10.7 × 10
4individual polyhedron inclusion body/ml.The toxic limit medium dose of two kinds of polyhedron inclusion bodys has significant difference (z=7.27, p<0.05).The toxic limit medium dose of HaBac △ egt-A polyhedron inclusion body is consistent with the toxic limit medium dose of HaBac △ egt polyhedron inclusion body.
4, the bollworm of 48 hours after step 3 of learning from else's experience process, extracts total serum IgE and reverse transcription is cDNA, take cDNA as template, adopts ACTIN gene to be reference gene, by the expression amount of semiquantitive PCR qualification HaPTTH gene.
Primer for the identification of ACTIN gene is as follows:
Upstream primer: 5 '-CCTGGTATTGCTGACCGTATGC-3 ';
Downstream primer: 5 '-CTGTTGGAAGGTGGAGAGGGAA-3 '.
Primer for the identification of HaPTTH gene is as follows:
Upstream primer: 5 '-TGCCGAAGGTGATGGCAATG-3 ';
Downstream primer: 5 '-CCTTTTCCGGGTCCTCGTTTCG-3 '.
The electrophorogram of pcr amplification product is shown in Fig. 5.Compared with the bollworm of HaBac △ egt polyhedron inclusion body of feeding, in the Helicoverpa armigera of HaBac △ egtHaPTTH polyhedron inclusion body of feeding, the expression amount of HaPTTH gene significantly increases.
Result shows, process LAN HaPTTH gene can impel the toxic limit medium dose of baculovirus to decline, and greatly improves baculovirus to the lethal efficiency of bollworm.
The median lethal time of embodiment 7, polyhedron inclusion body
HaBac △ egtHaPTTH polyhedron inclusion body embodiment 4 prepared respectively, HaBac △ egt polyhedron inclusion body and HaBac △ egt-A polyhedron inclusion body carry out the detection of following median lethal time:
1, select uniform two age Mo cotton bollworm larvae, be placed in 96 aseptic orifice plates, hungryly in 28 DEG C of constant temperature illumination boxs cultivate 16 hours, allow their decortications enter in next age.
2, the sterilized water dilution being used by polyhedron inclusion body the sucrose containing 4g/100mL and 1mg/mL to eat light blue is suspension.
3, get 96 larvas of completing steps 1, the suspension obtained by step 2 carries out feeding, and (dosage of feeding is 100 times of LC
50), timing from feeding, the larva that in 10min, middle intestines become blueness proceeds in 24 orifice plates containing fresh artificial diet, observes 4 times (being respectively 7:00,13:00,18:00 and 23:00) every day and adds up mortality ratio until all for trying dead larvae or pupating.Carry out repeating experiment for three times.
Survival analysis (SPSS software, Life-table or Kaplan-Meier method) is carried out to the infected test dead larvae time, and calculates ST
50value, analytical results is shown in Fig. 6.The median lethal time of HaBac △ egtHaPTTH polyhedron inclusion body is 68 hours (mean values of three tests).The median lethal time of HaBac △ egt polyhedron inclusion body is 119 hours (mean values of three tests).The median lethal time of HaBac △ egt-A polyhedron inclusion body is consistent with the median lethal time of HaBac △ egt polyhedron inclusion body.
Claims (8)
1. express the recombinant baculovirus of insect thoratropic hormone gene;
The preparation method of described recombinant baculovirus is as follows: recombinant baculovirus plasmid is imported insect cell and culturing cell, obtains recombinant baculovirus; Described recombinant baculovirus plasmid is the baculovirus plasmid with described insect thoratropic hormone gene;
Described recombinant baculovirus plasmid contains expression cassette first and expression cassette second; Described expression cassette first is the expression cassette for expressing described insect thoratropic hormone gene; Described expression cassette second is the expression cassette of the encoding gene for expressing baculovirus polyhedrin;
Described recombinant baculovirus plasmid is that the attB site by described expression cassette first and described expression cassette second being inserted in Hz8 ⊿ egtBacmid obtains;
Described Hz8 ⊿ egtBacmid is the recombinant plasmid will obtained after the steroidal uridine diphosphate glucose transferase gene deactivation of casting off a skin in HaBacHZ8Bacmid;
Described insect thoratropic hormone is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 7;
B protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
The protein of described baculovirus polyhedrin for being made up of the aminoacid sequence shown in sequence in sequence table 3.
2. recombinant baculovirus as claimed in claim 1, is characterized in that: described insect thoratropic hormone gene is following 1) or 2) or 3) DNA molecular:
1) DNA molecular shown in sequence 6 of sequence table;
2) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end the 30 to 710 Nucleotide;
3) DNA molecular shown in sequence 2 of sequence table.
3. recombinant baculovirus as claimed in claim 1 or 2, is characterized in that: in described recombinant baculovirus plasmid, is started the expression of described insect thoratropic hormone gene by op166 promotor and/or P10 promotor.
4. prepare a method for recombinant baculovirus, comprise the steps: recombinant baculovirus plasmid to import insect cell and culturing cell, obtain recombinant baculovirus; Described recombinant baculovirus plasmid is the baculovirus plasmid with insect thoratropic hormone gene;
Containing expression cassette first and expression cassette second in described recombinant baculovirus plasmid; Described expression cassette first is the expression cassette for expressing described insect thoratropic hormone gene; Described expression cassette second is the expression cassette of the encoding gene for expressing baculovirus polyhedrin;
Described recombinant baculovirus plasmid is that the attB site by described expression cassette first and described expression cassette second being inserted in Hz8 ⊿ egtBacmid obtains;
Described Hz8 ⊿ egtBacmid is the recombinant plasmid will obtained after the steroidal uridine diphosphate glucose transferase gene deactivation of casting off a skin in HaBacHZ8Bacmid;
Described insect thoratropic hormone is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 7;
B protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
The protein of described baculovirus polyhedrin for being made up of the aminoacid sequence shown in sequence in sequence table 3.
5. method as claimed in claim 4, is characterized in that: described insect thoratropic hormone gene is following 1) or 2) or 3) DNA molecular:
1) DNA molecular shown in sequence 6 of sequence table;
2) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end the 30 to 710 Nucleotide;
3) DNA molecular shown in sequence 2 of sequence table.
6. the method as described in claim 4 or 5, is characterized in that: in described recombinant baculovirus plasmid, is started the expression of described insect thoratropic hormone gene by op166 promotor and/or P10 promotor.
7. a sterilant, its activeconstituents is the recombinant baculovirus of the arbitrary described expression insect thoratropic hormone gene of claim 1-3.
8. the recombinant baculovirus of the arbitrary described expression insect thoratropic hormone gene of claim 1-3 is preparing the application in sterilant.
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| CN102382803A (en) * | 2011-11-08 | 2012-03-21 | 中国科学院武汉病毒研究所 | Construction method of novel gene engineering recombinant virus for expressing gp64 gene |
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| CN102382803A (en) * | 2011-11-08 | 2012-03-21 | 中国科学院武汉病毒研究所 | Construction method of novel gene engineering recombinant virus for expressing gp64 gene |
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