CN1089368C - Method for producing phytase using silkworm biological reactor - Google Patents

Abstract

The present invention provides a method for using a recombinant baculovirus to express and produce a phytase in an insect body, which comprises the following steps: recombing a phytase gene and a baoulovirus vector; using the recombinant baoulovirus vector to infect an insect host; culturing the infected insect host to make the insect host express phytase; collecting body fluid containing the phytase of a domestic silkworm larva or a pupa. By using the method, the phytase can be produced in mass with low cost to be used as a feed additive.

Description

Utilize silkworm biological reactor to produce the method for phytase

50-70% phosphorus exists (Lolas M.Et a1.Food Sci.42:1094-1097,1977 with the form of phytate phosphorus (phytinic acid) in the plant; Nelson T.S.Poultry Sci.47:862-871,1967)., can not directly be utilized (Cromwell G.L.Biotechnology in the FeedIndustry, Lyons T.P.ed.Alltech Technical Publication, Nicholasville by the simple stomach animal and human, KY, 133-145), cause the waste of phosphorus source, feed cost to increase, simultaneously, phytate phosphorus can not be utilized and directly excrete by animal, also can cause the phosphorus in soil and waters to pollute.In addition, phytic acid can also with necessary trace element of animal and protein chelating, these nutritive elements can not effectively be utilized, caused reduction (the Sharma C.B.et al. of plant feed (or food) nutritive value, Phytochemistry.17:201-204,1978).The phytase (Phytase, phytase) that extensively is present in the microorganism is inositol and phosphoric acid with hydrolysis of phytic acid.Phytase is fed and is raised the existing conclusive evidence of effect, can make the utilization ratio of phosphorus in the feed improve 60%, and ight soil phosphorus excretion reduces 40%, also can reduce the anti-oxidant action of phytate phosphorus.Therefore add phytase in the feed, can reduce the inorganic phosphate add-on, significant to improving the livestock industry productivity effect and reducing phytate phosphorus to the pollution of environment.

Though phytase has good feeding effect (Ware J.H.et al., US Patent No.3297548,1967; Nelson T.S.et al., J.Nutrition 101:1289-1294,1971; Nelson T.S.et al., Poult Sci.47:1842-1848,1968), but up to the present it also is not used widely on fodder industry, its basic reason is, the expression amount of phytase is too low in natural microbial, thereby be difficult to a large amount of cheap phytase products that obtain, can not satisfy requirement (the Han Y.W. of feed industrial development, Animal Feed Sci.Technol., 24:345-350,1989). along with biotechnology, development of high-tech such as genetically engineered, people recognize by engineered means, utilize bio-reactor to efficiently express phytase gene, be expected to reach and increase substantially phytase output, the purpose that reduces production costs (Conneely O.M., Biotechnology in the Feed Industry, T.P.Lyons (Ed), AlltechTechnical Publications.Nicholasville, K Y.57-66,1992).

Several microbe-derived phytase genes have obtained separation, as yeast Saccaromycescerevisiae (Bajwa W., Nucleic Acids Res.12:7721-7739,1984), Aspergillusniger (MacRae W.D., Gene, 71:339-348,1988; Yao Bin, Journal of Agricultural Biotechnology, Vol.6, No.1,1-6,1998), A.terreus and Myceliophthora thermophila (van Loon, Patent NO.EP 0684313A2,1995), A.niger (ficuum) (van Hartimgsveldt et al., Gene, 127:87-94,1993; Ehrlich K.C., Biocem.Biophys.Res.Comm.195:53-57,1993), A.niger var.awamori (Piddington C.S.Gene, 133:55-62,1993) etc.

The research of phytase gene expression mainly concentrates on the phytase gene phyA and phyB that derives from A.niger (ficuum).Expressed the phytase gene phyA that comes from A.niger var.awamori as 1993 in A.niger ALK02268, its expression amount has improved several times than former bacterial strain, is about 329U/mL (Piddington C.S., Gene, 127:87-94,1993).The same year, the phy gene that derives from A.ficumm NRRL3135 is led back former bacterial strain, the copy number of phyA gene is increased to more than 15, thereby the expression amount that makes phytase is brought up to 7600U/mL (Van Hartingsveldt W.et al., Gene, 127:87-94,1993:Van Gorcom R.F.M.et al, Pantent No.US 5436156,1995).Moore E. etc. express phytase gene that derives from yeast Saccharomyces cerevisiae and the phyB gene that derives from A.niger 762 in A.oryzae, its result makes expression amount bring up to 840U/mL and 750U/mL (Moore E.et al. respectively, J.Ind.Microbiol, 14:396-402,1995).In addition, also once the someone has attempted Expressing Recombinant Phytase gene phyA and phyB (Nevalainen H.K.M.et al. in Trichoderma, Pantent No.Wo 94/03612,1994) and attempted transforming phytase gene and make it have better temperature resistance and in aspergillus niger, express (Van Loon A.Patent No.EP 0684313A2,1995) with engineered method.These above expression studies, in general, its expression level is also lower, but has confirmed the expression that utilizes genetic engineering means to improve phytase, the validity of this approach of turnout.

The purpose of this invention is to provide a kind of baculovirus expression system that utilizes, especially silkworm (Bm)-silkworm baculovirus (BmNPV) expression system efficiently expresses, produces the method for phytase in the hope of reaching a large amount of, the cheap production of phytase, widespread use in feed and food.

The present invention relates to a kind of method of utilizing recombinant baculovirus (Baculovirus) at insect expression in vivo, production phytase, comprising: phytase gene and baculovirus vector are recombinated; Use the recombining virus carrier infection insect host; Cultivating infected insect host makes it carry out the phytase expression.

Baculovirus comprises BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV, TnNPV etc., insect host comprise silkworm (Bombyx mori), wild silkworm (Bombyxmandarina), Semen Ricini silkworm (Philosamia cynthia ricim), wild silkworm (Dictyoploca japanica), Philosamia cynthia (Philosamia cynthia pryeri), tussah (Antheraea pernyi), yamama (Antheraeayamamai), wild giant silkworm (Antheraea polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn mythimna separata (Spodoptera frugiperda), cabbage looper (Trichoplusiani), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), cigarette beetle (Heliothisvirescens), oriental armyworm (Pseudaletia separata), gypsymoth (Lymantria dispar) etc.

This bio-reactor of baculovirus expression system is that the eighties is set up.(Smith since nineteen eighty-three utilizes baculovirus expression system to efficiently express people's Interferon, rabbit first, Mol.Cell Biol., 3:2156-2165,1983), existing dozens of foreign gene has obtained efficiently expressing, only just there are Interferon, rabbit (Yang Guanzhen etc. in China, Acta Biochimica et Biophysica Sinica, 22:355-361,1990), arrowhead proteinase inhibitor (season equality, silkworm industry science, 21:223-227,1995), Mareks disease virus Glycoprotein B (Xiao Qingli etc., silkworm industry science, 23:104-108,1997) etc. multiple.Utilize the advantage of this system phytase generating in next life to be: 1. the expression efficiency of this expression system is high, and output can reach the level of milligram level/worm, thereby can reduce the production cost of phytase greatly and make phytase scale operation become possibility; 2. this expression system is an eukaryotic expression system, and the exogenous protein of its expression can carry out posttranslational modification, makes it similar to natural product with biological activity etc. at biochemical property, and this provides assurance for the phytase that gives expression to has normal biologic activity.3. the phytase of producing with this system need not purifying, only need with after the larva roughing of Expressing Recombinant Phytase directly as fodder additives, greatly reduce production cost.At present, baculovirus expression system, silkworm especially wherein (Bm)-silkworm baculovirus (BmNPV) expression system are one of individual expression systems of eukaryote that has most in the world business development value.

Adopt baculovirus expression system, use insect larvae and pupa as bio-reactor scale operation phytase efficiently, use in the feed of monogastric animal or food, this invention is for both at home and abroad first.The baculovirus expression system of optimizing among the present invention is silkworm (Bm)-silkworm baculovirus (BmNPV) expression system, by gene recombination, with the phytase gene into silkworm baculovirus BmNPV that recombinates, obtain recombinant silkworm baculovirus rBmNPV (phyA2), the rBmNPV (phyA2) that carries phytase gene is bred in the silkworm (larva, pupa etc.) of different developmental phases, use silkworm and produce phytase as host's bio-reactor.Equally, utilize other baculovirus expression system, also can express the production phytase in the same way as AcNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV, TnNPV etc.

Use gene recombination technology, will derive from various microorganisms (as aspergillus niger, Aspergillus fumigatus, fungies such as yeast, intestinal bacteria, genus bacillus, bacteriums such as lactobacillus), plant (corn, soybean, paddy rice etc.) phytase gene is building up to various baculovirus delivery carriers (as AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, BlueBacII (pETL), p2Bac, p2Blue, p89B310, pAc360,373, pAcAB3,4, pAcAS3, pAcC129, C4, DZ1, pAcGP67, pAcIE1, pAcJP1, pAcMLF2,7,8, pAcMP1,2, pAcRP23,25, pAcRW4, pAcsMAG, pAcUW1,21,2A, 2B, 3,31,41,42,43,51, pAcVC2,3, pAcYM1, pAcJcC5, pBacl, 2, pBlueBacIII, pBlueBacHis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhe1, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX 1.1, pSYN XIV VI+, pSYNVI+wp, pSYNXIV VI-, pVL1391,1392,1393, pVL941,945,985, pVTBac, pBM030, pUAC-5) on, make phytase gene in the polyhedron promotor, under the control of p10 promotor or other virus and Eukaryotic strong promoter, by in the body or external (in vivo/vitro) reorganization, phytase gene is incorporated on the genome of baculovirus, obtain recombinant virus.Insect larvae or the pupal cell (optimal time be 1-2 days early stage tender pupa) of various means through epidermis infection 1-5 age (optimal time was four or five ages) be eaten down or be adopted to recombinant virus can by per os, expresses the production phytase.Optimized scheme is to derive from black mold (Aspergillus niger 963 among the present invention, CGMCC0332) phytase gene phyA2 (Chinese patent, publication number: CN1184156A) be inserted on the delivery carrier B acPAK6, by reorganization in the body phytase gene is transferred on the genome of silkworm baculovirus parent plant Bm-BacPAK6 again, substitute the lacZ gene on the genome, by the white plaque select technology of indigo plant, obtain to carry the recombinant silkworm baculovirus rBmNPV (phyA2) of phyA2.With the silkworm larva or the pupa in its infected silkworm clone or percutaneous puncture-inoculation 1-5 age, can breed rBmNPV (phyA2) in a large number.When rBmNPV (phyA2) duplicated in the silkworm body, phyA2 expressed under the control of polyhedron gene (polh) promotor, produces phytase PHYA2.After infecting 3-6 days (the best is 5 days), collect and contain the silkworm larva of phytase or the body fluid of pupa (or whole homogenate), after killing infectious cause of disease, itself and carrier (as wheat bran, the dregs of rice, stone flour etc.) are mixed and stirred, just obtain phytase preparation after oven dry, the pulverizing, can add in the monogastric animal feed as fodder additives.In acidic gastric juice, phytase hydrolysis phytic acid is degraded to inositol and phosphoric acid with phytate phosphorus.Behind the phytase process separation that the present invention produces, the purifying, also can add in the food, promote the absorption of nutritive ingredient in the food, improve Nutritive value of food.

The phytase gene phyA2 that clones from black mold had once realized high expression level (Yao Bin etc., Chinese patent, publication number: CN1184156A) in Yeast system.We further advance silkworm baculovirus with this gene recombination, obtain recombinant virus rBmNPV (phyA2), and behind rBmNPV (phyA2) infected silkworm larva and the pupal cell, phytase gene obtains high expression level, and every milliliter of body fluid can produce the phytase more than 1 milligram.Article one, the enzyme activity of silkworm (or pupa) product is about 500,000 units (U).

The production cost of phytase is produced phytase (Yao Bin etc., Chinese patent, publication number: CN1184156A) suitable with utilizing Yeast system in this invention.Therefore the leftover bits and pieces silkworm chrysalis of silk mill is the protein source of feed always, adds with the phytase of originally researching and producing, and has also added albumen to feed simultaneously, has increased feed nutritive value, and need not investment and found the factory, the no three wastes, and electric power and the consumption of water resources equal energy source are few.Except that the mixed feed that can be used for suitability for industrialized production, also can therefore tally with the national condition directly for the peasant is used in various simple stomach livestock and poultry animals (as pig, the chicken etc.) feed, the utmost point has application and popularization value.Because silkworm China Ministry of Health approval so this kind phytase behind partial purification, can be used as foodstuff additive, improves Nutritive value of food for food medicine dual-purpose insect.In general, this invention can reduce the production cost of phytase significantly, can produce good economy, ecology and social benefit.

Description of drawings: Fig. 1. construction procedures Fig. 2 of reorganization delivery carrier pBacPAK8 phyA2. the Southern blotting of recombinant silkworm baculovirus rBmNPV (phyA2) analyzes

1. the rBmNPV that cuts without enzyme (phyA2); 2. silkworm baculovirus parent plant Bm-BacPAK6 (gal ⁺); 3. the rBmNPV that cuts through the SmaI enzyme (phyA2); 4. the rBmNPV that cuts through the EcoRI enzyme (phyA2); 5. the rBmNPV that cuts through the BamHI enzyme (phyA2); Fig. 3. the proteins gel electrophoresis of the phytase of in silkworm larva and silkworm chrysalis, expressing

1. expression standard molecular weight; 2. silkworm recombinant virus infection silkworm chrysalis; 3. silkworm recombinant virus infection silkworm larva; 4. silkworm baculovirus parent plant infected silkworm larva; 5. without the silkworm larva that infects; Fig. 4. the accumulation of phytase and infection back time relation Fig. 5 behind the infecting silkworm with recombinant baculovirus silkworm larva. the dynamics of the phytase of expressing behind the pH value performance chart 7. infecting silkworm with recombinant baculovirus silkworm larvas of the phytase of expressing behind the temperature characteristics figure 6. infecting silkworm with recombinant baculovirus silkworm larvas of the phytase of expressing behind the infecting silkworm with recombinant baculovirus silkworm larva

Three. experiment:

1. bacterial strain, virus strain and carrier coli strain E.coli TG1 and DH _5aAvailable from Promega company; Carry the escherichia coli vector pYY-01 (Chinese patent of phytase gene, publication number: CN1184156A) preserved, turn round plasmid pBacPAK8 available from Clontech company, bombyx mori cell strain BmN, silkworm nuclear-polyhedrosis virus parent plant Bm-BacPAK6 (gal by Chinese Academy of Agricultural Sciences's feed ⁺) be so kind as to give by Susumu doctor Maeda of California, USA university.Cultivated silkworm breed variety is JY1, is preserved by the Ministry of Agriculture of Inst. of Silkworm, Chinese Academy of Agricultural Sciences silkworm biotechnology emphasis open laboratory.

2. enzyme and test kit restriction enzyme, ligase enzyme are Boehringer company product.

3. biochemical reagents IPTG, X-Gal, SDS and sodium phytate are Sigma company product.Ammonium persulphate, TEMED, acrylamide and methylene diacrylamide are Promega company product.Lipofectin, low melting point agarose (LMP), Nick Translation test kit, T ₄Dna ligase, RNA enzyme, Proteinase K, bombyx mori cell substratum TC-100, foetal calf serum and other reagent are purchased the company in GIBCOBRL;

Substratum intestinal bacteria substratum be LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).Bombyx mori cell strain BmN nutrient solution is TC-100.

Experiment one

Carry the preparation of DNA of the carrier pYY-01 of phytase gene

The intestinal bacteria streak inoculation that will contain the pYY-01 plasmid is on the LB solid medium that contains 100g/ml penbritin (Amp), cultivated 16 hours for 37 ℃, the single bacterium colony of picking is transferred and is contained in the LB substratum of 100g/ml Amp in 3ml, 37 ℃ of shaking culture 16 hours (200 rev/mins), draw 1ml the activatory intestinal bacteria be added to 100ml and contain in the LB substratum of 100ug/mlAmp, 37 ℃ are continued shaking culture 16 hours (200 rev/mins), ice bath 15 minutes, it is centrifugal that (5000rpm * 5min) collects thalline, thalline is suspended in 3ml solution I [50mM glucose, 10mM EDTA, 25mM Tris-HCl (pH 8.0), the 5mg N,O-Diacetylmuramidase] in, placed on ice 20 minutes, add 6ml solution II (0.2N NaOH, 0.1%SDS), slowly reversing makes mixing for several times, placed on ice 10 minutes, add 4.5ml solution III [3M NaAc (pH4.8)] again, careful mixing, placed on ice 30 minutes, centrifugal (12000rpm * 20min), collecting supernatant liquor changes in another pipe, 95% ethanol that adds 2.5 times of volumes, place after 2 hours for-20 ℃, centrifugal (12000rpm * 15min), the collecting precipitation plasmid DNA, lyophilize postprecipitation 1ml TE[10mMTris-HCl, 1mM EDTA (pH 8.0)] dissolving, add 10 1RNA enzymes (10mg/ml), placed 15～30 minutes for 37 ℃, last Sepharose 2B column purification plasmid DNA, collect first peak, use phenol, each extracting of chloroform is once got supernatant liquor, the 3M NaAc (pH 6.5) that adds 1/10 volume, and add the dehydrated alcohol of 2 times of volumes, place more than 2 hours centrifugal (the precipitation plasmid DNA of 12000rpm * 15min) for-20 ℃, wash precipitation once with 75% ethanol, vacuum-drying, DNA is dissolved among the 200 l TE, and-20 ℃ of preservations are standby.

Experiment two

The preparation of delivery plasmid pBacPAK8 DNA

The intestinal bacteria that contain delivery plasmid pBacPAK8 are extracted plasmid DNA after the overnight incubation in the LB substratum, method is with experiment one.

Experiment three

The breeding amplification of silkworm nuclear-polyhedrosis virus parent plant and the preparation of viral DNA

Press TC-100 description of product preparation 1 * TC-100, transfer to pH6.22 with 2N NaOH, the nutrient solution after the filtration sterilization is added 10% foetal calf serum, as nutrient solution, cultivates bombyx mori cell BmN for 27 ± 1 ℃.(number of cells is about 10 to the bombyx mori cell of the about 50ml of cultivation in the T-100 nutrient solution ⁷-10 ⁸), infect the cell of logarithmic phase with silkworm nuclear-polyhedrosis virus parent plant Bm-BacPAK6, infection multiplicity is 1,27 ± 1 ℃ of cultivations, collect virus infection liquid after 3-4 days, centrifugal (3000rpm * 20min), remove precipitation, centrifugal (the 25000rpm * 60min) of supernatant liquor, remove supernatant, (contain Tris 12.1g among the 1000ml with 1ml viral DNA extract, EDTA 33.6g, KCl 14.1g, pH7.5) suspension virus particle precipitation, be transferred in the centrifuge tube of 1.5ml, add Proteinase K to final concentration be 50g/ml, 50 ℃ are incubated 2 hours, add again 35% Sarkorsel to final concentration be 1%, continue 50 ℃ of insulations 2 hours, use isopyknic phenol respectively, phenol: chloroform (1: 1), chloroform is extracting successively, in upper water phase transition to a new pipe, add the 3M NaAc of 1/10 volume, add 2 times of volume dehydrated alcohols, put 2 hours for-20 ℃, the centrifugal 10min of 5000rpm, the precipitation viral DNA, precipitation is washed once lyophilize with 75% ethanol, be dissolved among the 50 l TE, it is standby to place 4 ℃ of preservations.

Experiment four

The linearizing of the DNA of virus parent plant Bm-BacPAK6

Get 20 l viral DNAs and (be about 1～2g), 2 l restriction enzyme CvnI, 3 l10 * enzyme cutting buffering liquid, mend to volume 30 l with sterilized water, 37 ℃ are incubated 3-4 hour, and electrophoretic examinations is placed 65 ℃ * 10min with the endonuclease reaction mixture after showing the enzyme effect of cutting fully, make the enzyme complete deactivation, standby in 4 ℃ of preservations then.

Experiment five

The 1. preparation of the dna fragmentation of phytase gene phyA2 of structure (Fig. 1) of reorganization delivery carrier pBacPAK8 phyA2

Get standby plasmid pYY-01 DNA 30-50g, the damping fluid 10l of 10 * restriction endonuclease EcoRI, the EcoRI of 100unit, aseptic bi-distilled water is mended to cumulative volume 100 l, 37 ℃ were reacted 2 hours, and on the low melting point sepharose of application of sample to 1%, the 50V electrophoresis is 60 minutes under 4 ℃ of conditions, under ultraviolet lamp, cut the gel that contains the phyA2 dna fragmentation, the 3M NaCl that adds 1/10 volume, 68 ℃ of heating dissolving in 10 minutes gel, 37 ℃ of following phenol extractings, centrifugal (12000rpm * 5min), supernatant liquor is used phenol again, chloroform: primary isoamyl alcohol (24: 1) extracting successively once adds the 3M NaAc (pH6.5) of 1/10 volume, adds the dehydrated alcohol of 2 times of volumes again, place more than 2 hours for-20 ℃, it is centrifugal that (12000rpm * 15min), precipitation is washed once vacuum-drying with 75% ethanol, add 25 l TE dissolving ,-20 ℃ of preservations are standby.2. the enzyme of delivery plasmid pBacPAK8 is cut

Get delivery plasmid DNA 2g, damping fluid 2 l of 10 * restriction endonuclease EcoR I, the EcoR I of 4U, aseptic bi-distilled water mend to cumulative volume 20 l, and 37 ℃ were reacted 1 hour, get on the sepharose of 5 l application of samples to 1%, voltage 100V electrophoresis 20 minutes, ultraviolet lamp are checked the endonuclease reaction result down, after enzyme cuts entirely, 65 ℃ of water-baths were preserved enzyme deactivation standby in 10 minutes.3.phyA2 gene is connected with delivery plasmid pBacPAK8's

Get phyA2 gene fragment 6 l of preparation in the step 1, delivery plasmid pBacPAK8 DNA 1 l of preparation in the step 2,5 * T ₄DNA connects damping fluid 2 l, and T4 ligase enzyme 1 l, aseptic bi-distilled water mend to cumulative volume 10 l, and 12～14 ℃ of ligations are spent the night.4. the preparation of competent escherichia coli cell

Toothpick with sterilization is selected e. coli tg1 list bacterium colony, be inoculated in the LB substratum of 3ml, 37 ℃ of shaking culture are spent the night, getting 100 l overnight culture is inoculated in the 3ml LB substratum, 37 ℃ of shaking culture 2～2.5 hours, make the O.D value about 0.6, centrifugal (5000rpm * 5min) collect thalline is suspended in thalline 800 l 100mM CaCl of precooling ₂In, ice bath is 30 minutes then, and it is centrifugal that (5000rpm * 5min) abandon supernatant liquor adds 200 l100mM CaCl again ₂, impact tube wall gently, precipitation is suspended and mix, put on ice and be used in 4～24 hours transforming.5. colibacillary genetic transformation

Get connection mixture 5 l that prepare in the step 3, be added in the 200 l competent cells, mixing gently, ice bath 30 minutes, 37 ℃ of thermal shocks 5 minutes placed rapidly 1～2 minute on ice, add and be incubated to 37 ℃ LB substratum 500 l, cultivated 1 hour for 37 ℃, get 100-150 l and coat on the LB solid medium flat board that contains 50g/mlAmp, be inverted overnight incubation for 37 ℃.6. carry the evaluation of positive colony of reorganization delivery plasmid

The single bacterium colony of picking from the LB flat board that transforms, prepare plasmid DNA by the method among the embodiment two, use the phyA2 gene that Bam HI and EcoR I double digestion insert down, the plasmid that occurs the DNA band of about 1.3Kb behind the electrophoresis is reorganization delivery plasmid, carries positive clone's of clone's of reorganization delivery plasmid.The DNA of the reorganization delivery carrier pBacPAK8 phyA2 of preparation is standby in-20 ℃ of preservations.

Experiment six

The structure of the structure 1. recombinant baculovirus rBmNPV (phyA2) of recombinant silkworm baculovirus

The reorganization delivery plasmid pBacPAK8phyA2 DNA of the linearizing silkworm baculovirus parent plant of 1 g Bm-BacPAK6 DNA and 2g mixes, the Lipofectin that adds 30 l again supplies 60 l with aseptic bi-distilled water, mixes gently, left standstill 15 minutes, and be added to and contain 1 * 10 ⁶The 25cm of BmN cell ²Carry out cotransfection in the glass bottle, remove the 1 * TC-100 nutrient solution that contains foetal calf serum in the bottle then, wash 3 times with not containing foetal calf serum 1 * TC-100 nutrient solution, the back adds 1 * TC-100 nutrient solution that 3ml does not contain foetal calf serum, this mixture is dropwise added in another culturing bottle, 27 ℃ of cultivations were added 3ml after 4 hours is not had 1 * TC-100 nutrient solution of foetal calf serum and the foetal calf serum of 600 l, cultivated 4～5 days for 27 ℃, collect the screening purifying that supernatant liquor is used for recombinant silkworm baculovirus rBmNPV (phyA2); 2. recombinant silkworm baculovirus rBmNPV (phyA2) screens and purifying

Carry the LacZ gene on the silkworm nuclear-polyhedrosis virus parent plant, this expression of gene product can decompose substrate X-gal and make the formed plaque of the cell that is infected be blue look after infecting insect cell, exogenous origin gene integrator will replace this gene after viral genome, thereby plaque is white in color.Utilize this mark can screen recombinant virus.Inoculate an amount of cell (being advisable) can pave plate bottom area 80% in 60 or the 35mm plate in, treat cell attachment after, inhale and to remove nutrient solution, the transfection supernatant liquor that previous step is obtained in rapid is by 10 ^-3, 10 ^-4, 10 ^-5Different extent of dilution dilute, drawing 1ml joins in the attached cell, it is evenly distributed, 27 ℃ were infected 1 hour, suction removes to infect liquid, and 2% low melting point sepharose is dissolved, and is as cold as about 40 ℃, 2 * TC-100 (containing 20% foetal calf serum) with 40 ℃ of preheatings mixes then, add X-gal again and make its final concentration reach 150g, this mixture is added drop-wise to (the 60mm plate adds 4ml approximately, and the 35mm plate adds 1ml approximately) in the plate, after waiting to solidify, be inverted for 27 ℃ and cultivated 4～7 days, microscopic examination, the plaque that will have white feature is picked out, repeat above step, through the pure recombinant silkworm baculovirus rBmNPV (phyA2) of purifying acquisition of 2-3 wheel.3. the amplification of recombinant virus rBmNPV (phyA2) in bombyx mori cell

With the BmN cell of recombinant silkworm baculovirus rBmNPV (phyA2) infection normal growth, cultivate and collect supernatant liquor after 4 days, promptly contain a large amount of recombinant virus rBmNPV (phyA2) in the supernatant liquor.

Experiment seven

In the integration of checking phytase gene in recombinant virus genomes on the molecular level

Get 5 μ g recombinant virus genomes DNA, separate with EcoRI, BamHI, SmaI holoenzyme respectively, enzymolysis product carries out 0.8% agarose gel electrophoresis, the DNA in the gel is transferred to carry out Southren blotting analysis on the nylon membrane.Hybridize the phytase gene segment that be about 1.0Kb of used probe for downcutting from recombinant plasmid pYY-1 with BamHI.The probe enzyme is cut rear electrophoresis and is reclaimed, with random primer labelling Kit label probe.Result (Fig. 2) shows, recombinant virus dna occurs 1,2,1 specific hybrid band respectively after with EcoRI, BamHI, SmaI enzymolysis, and this proof phytase gene correctly is incorporated in the viral genome.

Experiment eight

Use silkworm and produce phytase as the host

Mulberry leaf are raised silkworm to five ages, and the silkworm surface with 75% ethanol disinfection, is drawn recombinant virus respectively with microsyringe, inject 10 by every ⁶Pfu virus quantity (about 5 l supernatant liquors) is at uromere the 2nd and the 3rd intersegmental membrane place percutaneous puncture-inoculation recombinant virus reciprocal, 24-25 ℃ routinely mulberry leaf raise silkworm, and freshen food paraxin every other day, raise after 4-5 days, from the silkworm body, prepare phytase.Get 1-2 days the tender pupa in back of pupating, the sterilization of 75% ethanol epidermis is drawn recombinant virus respectively with microsyringe, by every injection 10 ⁶Pfu virus quantity (about 5 l supernatant liquors) for preventing infectation of bacteria, adds the gentamicin of 10-20U/ pupa at abdomen intersegmental membrane place percutaneous puncture-inoculation recombinant virus in recombinant virus liquid, cultivated 4-5 days for 24-25 ℃, therefrom the phytase of preparation expression.

The concrete grammar of phytase preparation is as follows: collect the body fluid of larva and pupa, or directly with silkworm larva body and pupal cell homogenate, (10000rpm * 10min), remove precipitation and lipid layer promptly contains the phytase of expression in the supernatant liquor through centrifugal then.Supernatant liquor is with after the carrier wheat bran mixes by 1: 1 (V/W), and phytase preparation is made in 50 ℃ of air seasonings.

Silkworm larva body and pupal cell infected recombinant virus after 4-5 days, and the phytase expression amount reaches maximum value, and at this moment the expression amount in larva and the pupa is respectively up to 1.427mg/ml hemolymph and 1.902mg/ml hemolymph.The phytase of expressing can confirm by proteins gel electrophoresis, electrophoresis result (Fig. 3) shows, behind recombinant silkworm baculovirus infection silkworm larva or silkworm chrysalis, a specific phytase band appears at the 65KD place, and this proof phytase gene has obtained efficiently expressing.

Experiment nine

Use silkworm as the enzyme assay of the phytase of host's production and the Physiology and biochemistry property analysis of enzyme

Phytase in metainfective larva and the pupa hemolymph is carried out determination of activity, measuring method is: the enzyme diluent of 0.2mL adds the sodium phytate of 0.8mL 1.25mmol/L, 37 ℃ of insulation 30min, add 1mL 10%TCA and stop enzyme reaction alive, add 2mL ferrous sulfate-ammonium molybdate colour developing liquid then, 700nm measures content of inorganic phosphorus.Contrast makes enzyme-deactivating for add 1mTCA earlier in the enzyme diluent of 0.2mL, adds the substrate insulation with volume again.A unit of enzyme activity (U) is defined as: under certain condition, it is a unit of enzyme activity that per minute discharges the required enzyme amount of 1 μ mol inorganic phosphorus.The result shows, the expression amount of phytase began to express behind recombinant virus infection in 48 hours, peaked during by 120 hours (Fig. 4), and at this moment the expression amount of phytase reaches 4.667 * 10 respectively in larva and pupa ⁵U/ml hemolymph and 5.985 * 10 ⁵The U/ml hemolymph.This expression amount is high 3000 times than primary phytase gene source bacterial strain Aspergillus niger963.

After getting the liquid of haemolymph that contains phytase and suitably diluting, under different pH condition, (use the HCl-Gly damping fluid below the pH3.5; PH3.5-5.5 HAc-NaAc damping fluid; The above Tris-HCl damping fluid of using of pH5.5) measures enzymic activity for 37 ℃, the optimum pH of finding it is 2.5 and 5.7 (Fig. 5), this and primary derive from the phytase (Chinese patent of A.niger, publication number: CN1184156A) compare, first optimal pH 5.7 does not change, but second optimal pH moves on to 2.5 by original 1.8, and this may cause with the albumen higher structure that phytase forms in different expression systems is different.The result (Fig. 6) who under pH5.7, different temperature the activity of the phytase of expressing is measured shows that its optimum temperuture is 55 ℃, and this is identical with the primary phytase.Measure the enzyme kinetics character of the phytase of expressing under the condition of 37 ℃, pH5.7 and different sodium phytate concentration of substrate, result (Fig. 7) shows that the Km value of the phytase of expression is 0.7695nM.

Claims (8)

1. a method of utilizing recombinant baculovirus at insect expression in vivo, production phytase comprises: phytase gene and baculovirus vector are recombinated; Use the recombining virus carrier infection insect host; Cultivating infected insect host makes it carry out the phytase expression; Collection contains the insect body of phytase.

2. in accordance with the method for claim 1, it is characterized in that described baculovirus is BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, S1MNPV, SeMNPV or TnNPV.

3. in accordance with the method for claim 1, it is characterized in that described insect host is silkworm, wild silkworm, Semen Ricini silkworm, wild silkworm, Philosamia cynthia, tussah, yamama, wild giant silkworm, autographa california, tea geometrid, cabbage army worm, prodenia litura, autumn mythimna separata, cabbage looper, armyworm, bollworm, U.S. bollworm, oriental tobacco budworm, cigarette beetle, oriental armyworm or gypsymoth.

4. in accordance with the method for claim 1, it is characterized in that described insect is a silkworm, described baculovirus is a silkworm baculovirus.

5. in accordance with the method for claim 1, it is characterized in that described baculovirus delivery carrier is AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, BlueBacII (pETL), p2Bac, p2Blue, p89B310, pAc360,373, pAcAB3,4, pAcAS3, pAcC129, C4, DZ1, pAcGP67, pAcIE1, pAcJP1, pAcMLF2,7,8, pAcMP1,2, pAcRP23,25, pAcRW4, pAcsMAG, pAcUW1,21,2A, 2B, 3,31,41,42,43,51, pAcVC2,3, pAcYM1, pAcJcC5, pBac1,2, pBlueBacIII, pBlueBacHis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhel, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX 1.1, pSYN XIV VI+, pSYNVI+wp, pSYNXIV VI-, pVL1391,1392,1393, pVL941,945,985, pVTBac, pBM030, or pUAC-5.

6. in accordance with the method for claim 1, it is characterized in that described recombinant virus is through port food or sees through epidermis and infect the 1-5 insect larvae or the pupal cell in age.

7. in accordance with the method for claim 6, it is characterized in that described pupal cell is 1-2 days an early stage tender pupa.

8. in accordance with the method for claim 1, it is characterized in that described phytase is by the phytase gene phyA2 of black mold coding; Described delivery carrier is BacPAK6; Described reorganization is meant to be transferred to phytase gene on the genome of silkworm baculovirus parent plant Bm-BacPAK6, substitute the lacZ gene on the genome, by blue white plaque select technology, obtain to carry the recombinant silkworm baculovirus rBmNPV (phyA2) of phyA2 then; Described infection be will obtain recombinant silkworm baculovirus infection bombyx mori cell system or the silkworm larva or the pupa in percutaneous puncture-inoculation 1-5 age; After infecting 3-6 days, collect and contain the silkworm larva of phytase or the body fluid of pupa.

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