CN105385601B - A kind of nosema locustae and its suspension culture method and application - Google Patents

Abstract

The invention discloses a kind of nosema locustae and its suspension culture method and applications.Wherein, the nosema locustae strain reached for 10 generations for the separation from Asiatic migratory locust (Locusta migratoria L.) in locust body, and in vitro culture obtains after reaching for 10 generations, and deposit number is CGMCC No.11170.Further; the invention also provides a kind of culture mediums and suspension culture method in vitro culture nosema locustae; the culture medium using insect source property, water solubility, deacetylation chitin as extracellular matrix; polyvinylpyrrolidone is as visco-supplement; it is added to protein extract simultaneously; microcarrier suspension amplification cultivation is carried out using culture medium disclosed by the invention, using bioreactor, the microsporidian number through microcarrier suspension culture is enabled to reach 60 times that starting is cultivated.The present invention provides a kind of method of external mass propgation nosema locustae, the large-scale use for nosema locustae as biological insecticides provides technical support.

Description

A kind of nosema locustae and its suspension culture method and application

Technical field

The present invention relates to one plant of nosema locustae, further relate to a kind of in vitro culture nosema locustae culture medium and Cultural method, the invention belongs to agricultural biological technical fields.

Background technique

Locust is the pest for influencing agriculture-stock production, in China pastoral area, seriously endangers grassland, in order to kill locust, all the year round Chemical pesticide, but chemical pesticide not only somewhat expensive, but also pollution environment are largely used, causes residue of pesticide in livestock products, together When kill Natural Enemies of Grasshopper, cause community function to deteriorate.It therefore can as biological insecticides by using entomopathogenic microorganism Greatly reduce the destruction to natural environment.

Microsporidian is one of locust pathogenic microorganisms, and microsporidiosis is caused by microsporidian, in the place of infection It is bred in chief cell by schizont, spore phase.Since microsporidian plays a significant role in adjusting insect population.Locust is micro- Sporozoite cause of disease introduces pest population, causes microsporidian disease popularity, facilitates the solution inscribed between locust, and be possible to longer Phase plays the effect of its natural contral.

Nosema locustae is to belong to Microspora (Microspora), Microsporidea (Microsporea), micro- spore Sub- worm mesh (Microsporida), Nosema (Nosema) single-celled protozoal animal, battalion cytozoicus life.It is typical Stage is the spore of dormant period, there is the polar filament of a sporoplasm and a helical coil in spore, after host eats spore Under the action of middle intestines alkaline digest liquid, spore inner disk around polar filament quickly turn up, pierce through the funnel and enteric epithelium of host, Sporoplasm, which injects host's midgut epithelial cells by hollow polar filament pipe, simultaneously becomes small deformation body.A part of deformable body is just thin in middle intestines Development intracellular, and most of deformable body reaches sensitivity tissue by hemolymph, generates segmenta through schizogamy after, then Into the sporogenesis phase, by double-core gleba and spore mother cell's stage, cell, which further breaks up, eventually forms ellipse Mature spore.Mitochondrial is free of in microsporidian body.

There are two types of the circulation way of nosema locustae in nature, i.e. horizontal transmission and vertical transmission.First is that due to Host has eaten by the food of spore contamination or cannibalism and has made mutually to infect between individual, second is that due to organizing generating process In, the surface that segmenta or gleba have infected ovary or spore contamination ovum makes pathogen be directly delivered to filial generation from parental generation. It is mainly shown as feeding exception after locust infection, grows irregular, mutual residual, fecundity and service life reduction.Due to cytoclasis and The result of nutrient consumption causes polypide dead.The size of illness depends on susceptible degree and range and the hair of infected host Educate the stage.

At home and abroad nosema locustae was once widely studied the safety of vertebrate, and result of study shows: locust Worm microsporidian is safe to vertebrate, while also not having infectivity to honeybee, and therefore, nosema locustae becomes commodity The microsporidian insecticide of change is promoted the use of.

In the past 20 years, it is frequently found some and typical locust granulosis cause of disease-nosema locustae throughout our country The form of (Nosema bombycis, Nb), special-shaped microsporidian of different sizes.These special-shaped microsporidians are mainly derived from Field Asiatic migratory locust (Locusta migratoria L.), and the rising of nosema locustae disease incidence and field Ya Zhou Migratory locusts microsporidian is related to the cross-infection of locust.Investigation finds that a variety of fields advantage locust also has very high micro- spore Sick Natural infection rate, wherein 1 plant of microsporidian being collected into out of field Asiatic migratory locust body all has under certain food locust Appeal.

Microsporidian conventional sorting methods mainly according to its history of life, ultra microstructure, serological relation, host range, post Main tissue specificity, spore shape size etc..The development of Protocols in Molecular Biology carries out system to locust cause of disease from molecular level Study on Evolution and taxonomic identification are widely used.The SSU rRNA of microsporidian, rRNA ITS gene order have height Conservative, thus become a kind of important auxiliary tool for carrying out molecular biology classification and the systematic growth research of microsporidian. The SSU rRNA gene of microsporidian determines micro- spore suitable for the affiliation between analysis Nosema in the level of category The classification position of sub- worm.RRNA ITS is larger in biological species and subspecies variation, point that can be used in the level of microsporidian kind Class.The present invention carries out biological characteristic research to the 1 plant of microsporidian collected out of Xinjiang field Asiatic migratory locust body, is based on SSU RRNA, rRNA ITS gene order carry out the Molecular Phylogeny analysis of microsporidian, are its taxonomic identification and tracking locust The infection genesis of microsporidiosis, and effectively the harm of control locust provides foundation.

Nosema locustae is used for biological locust elimination as biological insecticides, first has to solve the problems, such as its production, currently the only Reliable method is living body propagation, but breeds microsporidian with the locust nymph of a locust, many challenges is faced, as onerous toil is strong Degree needs the mass rearing of the nymph of a locust, is crushed, the prevention of nymph of a locust other diseases.And directly with appropriate media culture or suitable elder brother The extracorporeal culturing method of worm cell culture nosema locustae is still in the exploratory stage.

Summary of the invention

The first technical problem to be solved by the present invention be to provide it is a kind of obtained through separation, and can be suitable for external outstanding The nosema locustae strain of floating culture；

The second technical problem to be solved by the present invention, which is to provide, a kind of can be directly used for culture nosema locustae Culture medium and extracorporeal culturing method.

In order to achieve the above object, present invention employs following technological means:

Inventor is separated to 1 plant of micro- spore out of In Altay, xinjiang Burqin County field Asiatic migratory locust body Worm, research find its biological characteristics with micro- spore category (Nosema): microscopically observation polypide shape is in oval, With nosema locustae (Nosema locustae) no significant difference.Fat body cells and haemocyte of the main parasitic in locust In, all stage cores are double-core.Mature spore is oval, and the fresh microsporidian size for applying on piece is 4.5 ± 0.16 μ ms 2.2 ± 0.07 μm (n=10), it is 1.4 ± 0.07 μm of 3.3 ± 0.06 μ m (n=10) that ultra-thin section, which powers on sem observation size,.It is super Micro-structure and nosema locustae are most like.The micro- spore history of life big multistage is double-core, includes many Electron-dense bodies.Jim The spore of monokaryon is also accidentally observed in the smear of Sha's dyeing and ultra-thin section face, has monokaryotic stage in the spore development later period.Polar filament with Spore long axis oblique angle is 60-70 degree, and polar filament inclination angle and locust sporozoite are variant.Ultra microstructure shows micro- spore category of separation In the micro- spore category of locust.

It is host with the 2 age in days nymph of a locust of locust, observes this plant of microsporidian in locust (Locusta migratoria L.) children The history of life growth course of enteral in worm.As a result, it has been found that: most of stages of its history of life are double-core, and the spore development later period is presented Monokaryon is the result for have binary fission.The spore fragmentation phase (sporogony) is double-core, generates the spore of single form.Respectively A stage of development is all in the cytoplasm of host.Spore shape be oval or oval cylindricality, 200-300nm of inner wall thickness, outside 40-60nm of wall thickness.Mature spore contains 3-12 polar filament circles, and there are a large amount of vacuoles in spore rear end.Outside immature spore, there is electronics Dense matter package, side rise and fall, the cytosome (shell of the tight in front-end and back-end) with two side shells；Cytosome Slightly locate rearward positioned at double-core center or double-core.Polar filament pipeloop number 15-20,1-2 row arrange irregular alignment.This plant of microsporidian spore The internal structure of son has double-core and similar polar filament circle number, but its polar filament inclination angle 60-70 degree as the micro- spore of locust, It is smaller than locust sporozoite.The history of life of this plant of microsporidian and locust sporozoite are very close, can find in adipose tissue, raw There are uninucleate stage, the single form spore containing outer wall and inner wall in the history development starting phase living.The history of life in all stages directly and host Cytoplasm contact, is proliferated in a manner of binary fission, eventually forms double-core maturation spore.

With anti-locust sporozoite serum aggregation can occur for isolated microsporidian, illustrate there is phase with locust sporozoite Same surface antigen.

From form, from the history of life, preliminary judgement belongs to the micro- spore category of locust to isolated microsporidian.

Separate the SSU rDNA and nosema locustae N.locustae and Antonospora of micro- spore The SSU rDNA sequence of scoticae is compared, and finds there is 5% between micro- spore and nosema locustae N.locustae Difference and Antonospora scoticae have 10% difference.The phylogenetic tree that microsporidian SSU rRNA sequence is established Confirm that this plant of microsporidian belongs to Nosema category.The similitude and Genetic Distance Analysis of SSU rRNA sequence show this plant of micro- spore The affiliation of worm and nosema locustae is close.The similitude and Genetic Distance Analysis of rRNA ITS gene order show the strain The similitude of microsporidian and nosema locustae is higher, and degree of differentiation is smaller.In summary result of study infers this plant of micro- spore Worm and nosema locustae are to belong to xenogenesis microsporidian.

The feature of isolated microsporidian: (1) the cluster difference that small unit rDNA sequence and other micro- spores belong to is less than 10%, it is maximum with the difference of Microsporidium Vairimorpha ceraces (Nosema/Vairimorpha) group, reach 50%；(2) it is living Shi Zhong, single spore shape have young Spore Stages；(3) morphological feature: there is development schizont and sporont body in (i) (meront/sporont) there is electron dense object in the stage in core；(ii) spore polar filament circle 3-12；The pole that the micro- spore of locust worm belongs to Wire ring 3-12；(iii) there is a large amount of vacuole in spore rear end, and the micro- spore category vacuole of locust is few；(iv) epispore contains electron dense Object；(4) tissue specificity specially parasitizes epithelial cell；Host is Orthoptera locust, scab locust and locust.

It is separated to nosema locustae, after per oral inoculation infects locust, initial stage without obvious external symptom, is developed to the nymph of a locust Aged or adult stage, disease pest body colour just start to become bronzing from canescence, and serious susceptible polypide pinkiness, abdomen feels like jelly, Slightly swelling, skin is de- sometimes does not get off, wing shrinkage.Later period disease pest shows hypoevolutism, breeding time extends, the bodily form reduces, last dead It dies.Spore is only separated to after dissection out of fat-body.It can be seen that microsporidian main infection adipose tissue.Day by day continuous dissection is through connecing The locust disease pest corpse of kind, 3 ages are inoculated into 5 age in days majorities and separate spore, and 3 ages in days are inoculated into adult stage majority and also separate To spore.5 ages in days, which are seeded in the adult later period, can see spore.Most started to generate spore earlier than 7-10 days after inoculation, it 2 weeks or so can See mature spore, spore largely generates within 25 days or so.Mature spore is round, and size is irregular, and mean size is 4.5 × 2.2 μm. Show that the microsporidian can infect locust, and proliferation generates a large amount of spores in vivo at it.Originally locust proliferation host is single in the early stage Head sporulation quantity is not high, but is directed to its multiplication characteristic, when by finding out suitable cause of disease inoculum density, host insect age and harvest Between after, single head sporulation quantity can be made to be increased to 6 × 10⁹It is more than a spore.Using 30cm × 40cm × 50cm insect cage single cage 500-700 head can be supported, 20 rearging cages can raise locust 10000-140000 head, and average single head spore output is by 4 × 10⁹A spore Son is increased to 6 × 10⁹A spore.

By aforementioned present invention inventor nosema locustae isolated from Asiatic migratory locust, 10 are reached in locust body Then Dai Hou reached for 10 generations using culture medium in vitro culture, obtained nosema locustae is named as XJ-20, and classification naming is Nosema locustae (Nosema locustae), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms The heart, the preservation time is: on November 3rd, 2015, preservation address are the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Institute of microbiology, deposit number CGMCC No11170.

Whether meet locust spore with the composition that culture medium culture in vitro culture nosema locustae depends on appropriate media The history of life different developmental phases requirement of sub- worm.

After nosema locustae after separation reaches 10 generations, identification in locust body, synthesis and selection are suitable for culture Base is packed into culture bottle or bioreactor, cultivates 30-50 days, micro- spore count is made to reach certain amount, and then dilution continues to cultivate, To reach requirement concentration.

Further, the invention also provides the nosema locustaes to prepare the purposes in biological insecticides.

There is provided one kind can cultivate the culture medium and method of nosema locustae in vitro for another aspect of the present invention, Enable microsporidian the surface of culture vessel complete its history of life go forward side by side line splitting proliferation, and then reach breeding proliferation mesh 's.

Inventor is examined by the history of life each step to microsporidian and analysis, selects and adjust culture medium Composition proposes the culture medium for microsporidian in vitro culture, wherein reagent is supplemented containing certain protein extracts and viscosity, To be suitble to nosema locustae to complete the history of life and culture.

Inventor's detection and analysis different extracellular matrixes coated cell culture vessel is if appropriate for microsporidian The history of life and proliferation, as a result, it has been found that when the chitin of culture vessel pan coating insect source property, water solubility, deacetylation, enhancing Adhesiveness of micro- spore on culture vessel surface, and the division growth of micro- spore can be promoted.

Therefore, on the basis of the studies above, the invention proposes a kind of for cultivating the culture medium of nosema locustae, The culture medium includes:

1) or mixtures thereof or mixtures thereof or mixtures thereof inorganic salts, sugar, amino acid and or mixtures thereof vitamin；

2) as the insect source property of extracellular matrix, water solubility, deacetylation chitin；

3) protein extract；And

4) as the polyvinylpyrrolidone of visco-supplement, preferably PVP K-90；

Each ingredient is substantially soluble in pure water, adjusting Medium's PH Value is 6.0-7.0, preferably 6.2-6.5, after aseptic filtration To obtain the final product；

Wherein, the insect source property, water solubility, deacetylation chitin content be 0.01-10g/L culture medium, It is more preferably 0.1-10g/L culture medium, most preferably 1-10g/L culture medium；

Wherein, the protein extract includes lactoalbumin hydrolysate, yeast extract and tryptosePhosphate broth, Its content is 500-3000mg/L culture medium, and more preferably content is 1000-3000mg/L culture medium, and most preferred content is 1000-2000mg/L culture medium；

Wherein, the content of the polyvinylpyrrolidone is 100-1000mg/L culture medium, is more preferably 100- 500mg/L culture medium, most preferably 200-500mg/1L culture medium.

In the present invention, insect source property, water-soluble chitin (hereinafter referred to as water-soluble chitin), have carried out removing acetyl The unique modification changed.The desirable any insect of the chitin, preferably locust slough off the skin fallen.

According to the present invention, the water-soluble chitin has following several acquisition patterns:

For prepare as extracellular matrix the insect source property used, water solubility, deacetylation chitin, selection hydrophily Chitin, preferably interior surface area be 180m²/ g or bigger chitin.The chitin extracted from locust husking has compared with imperial palace Portion's area and highly endothermic property.Therefore locust husking, particularly oriental migratory locust husking can be used as extracellular matrix use.

The skin fallen is sloughed off with 1N salt acid soak locust, in the environment for having nitrogen, 100 DEG C are handled 20 minutes.Then salt is outwelled Acid, with 1N sodium hydroxide solution, 80 DEG C of processing remove protein in 36 hours, chitin or chitin shell are made.It is water-soluble for preparation Chitin, at room temperature with aqueous slkali dissolve chitin, high viscosity alkali chitin aqueous solution places at room temperature long-time make with Machine removes acetyl group, and when deacetylation reaches 45-55%, obtained deacetylation is dissolved in water up to chitin (i.e. chitosan).

Above-mentioned water-soluble chitin alkali or acid processing can remove acetyl group, be related to heating.Some reactions can also be used As Clemmenssen reduction reaction removes acetyl group.The present invention preferably uses a certain concentration alkali process, and guarantees only to remove acetyl Change modification, without other such as methylolation, the chemical modification of tosylation.Deacetylation is in uniform soluble alkali soluble It is carried out in liquid.It takes the above method that deacetylation is made to reach 40-60%, preferably 45-50% and chitin and water is made to have height affine Power.

The insect source water solubility chitin of acquisition passes through unique chemical modification deacetylation, the chitin containing 40-60% Matter has 40-60% chitosan structure.In other words, water solubility chitin in insect source modifies deacetylation pair by sole chemical Water has high affinity, and 40-60% is chitin, and 40-60% is chitosan.Since nosema locustae surface has negative electricity Lotus, microsporidian can be adsorbed on the enterprising line splitting of chitosan positive charge amino and duplication.

Wherein there are two types of the application methods of water-soluble chitin:

1, obtained water-soluble chitin is dissolved in water by 0.01-10g/L, pours in vitro culture vessel into, arrive coating micro- The culture face of sporozoite, naturally dry.Then the other compositions being added in culture medium of the present invention are used for microsporidian culture；At this In invention, 0.5 milliliter of chitin aqueous solution coated cell culture bottle surface area is preferably 200 square millimeters.

2, obtained water-soluble chitin is dissolved in culture medium by 0.01-10g/L, by the culture containing water-soluble chitin Base is added in vitro culture vessel for microsporidian culture.

In the present invention, microsporidian in vitro culture vessel include Tissue Culture Flask, rolling bottle, culture dish, culture dish, culture Vessel surface is coated with water-soluble chitin, and being sticked on Tissue Culture Flask by water-soluble chitin microsporidian can be grown And division.

In the present invention, it is preferred to, the protein extract further comprises myosin, cromoci, creatinine And bovine plasma albumin V；

Wherein, the content of myosin is 1-100mg/L culture medium, is more preferably 1-50mg/L culture medium, most preferably 5-15mg/L culture medium；The content of cromoci is 1-500mg/1 culture medium, is more preferably 1-100mg/L culture medium, optimal It is selected as 10-100mg/L culture medium；The content of creatinine is 1-500mg/L culture medium, is more preferably 1-200mg/L culture medium, optimal It is selected as 10-200mg/L culture medium；The content of bovine plasma albumin V is 1000-10000mg/L culture medium, is more preferably 100- 10000mg/L culture medium, most preferably 1000-10000mg/1L culture medium.

In the present invention, it is preferred to, or mixtures thereof or mixtures thereof described inorganic salts, sugar, amino acid or its mixing It is thin that or mixtures thereof object and vitamin derive from the known inorganic salts being commercially available, sugar, amino acid, the insect of vitamin content Born of the same parents' culture medium is added 2) -4 on this basis) in component.

In the present invention, it is preferred to, it include NaH in or mixtures thereof described inorganic salts₂PO₄·2H₂O, NaHCO₃, KCl, CaCl₂·2H₂O, CuCl₂·H₂O, CoCl₂·6H₂O, FeSO₄·7H₂O, MgCl₂·4H₂O, MgSO₄·7H₂O, MnCl₂·4H₂O, NaCl, NaH₂PO₄·4H₂O, (NH₄)₆(Mo₇O₂₄) 4H2O and ZnCl₂Middle one or more；

It include D-Glucose, fructose, sucrose, malic acid, alpha-KG, amber in or mixtures thereof described sugar Acid, one or more of fumaric acid and maltose；

In or mixtures thereof described amino acid include L-alpha- alanine, beta- alanine, L- R-gene, Altheine, L-Aspartic acid, L-cysteine, Pidolidone, L-Glutamine, glycine, L-Histidine, the different bright ammonia of L- Acid, L-Leu, l-cysteine disodium, L- hydroxyproline, l-Lysine hydrochloride, l-methionine, L-phenylalanine, L- dried meat ammonia Acid, DL-tryptophan, L-threonine, L-Trp, l-tyrosine, l-tyrosine disodium, in Valine and L-Histidine It is one or more kinds of；

It include biotin, D-VB5 calcium, choline chloride, folic acid, I- inositol, cigarette in or mixtures thereof described vitamin Acid, puridoxine hydrochloride, riboflavin, thiamine hydrochloride, one or more of vitamin B12 and p-aminobenzoic acid.

In the present invention, it is preferred to, it is 10-30% (v/ that volumn concentration is further comprised in the culture medium V) animal blood serum, the animal blood serum are fetal calf serum or locust lymph.

In the present invention, it is preferred to, the culture medium contains following component:

1) insect source property, water solubility, deacetylation chitin 10g/L；

2) protein extract:

Include lactoalbumin hydrolysate 1500mg/L, yeast extract 1500mg/L, trypsasePhosphate broth 1500mg/ L, myosin 10mg/L, cromoci 50mg/L, inosine 100mg/L and bovine serum albumin(BSA) V 5000mg/L；

3) viscosity replenishers PVP K-90 250mg/L；

4) inorganic salt mixt:

Include NaH₂PO₄·2H₂O 507mg/L；NaHCO₃300mg/L；KCl 1720mg/L；CaCl₂·2H₂O 750mg/L；CuCl₂·H₂O 0.1mg/L；CoCl₂·6H₂O 0.03mg/L；FeSO₄·7H₂O 0.28mg/L；MgCl₂·4H₂O 1140mg/L；MgSO₄·7H₂O 3269mg/L；MnCl₂·4H₂O 0.01mg/L；NaCl 1425mg/L；NaH₂PO₄·4H₂O 580mg/L；(NH₄)₆(Mo₇O₂₄)·4H₂O 0.02mg/L and ZnCl₂0.02mg/L；

5) sugared mixture:

Include D-Glucose 2917mg/L；Fructose 20.9mg/L；Sucrose 11865mg/L；Malic acid 306mg/L；alpha- Ketoglutaric acid 169mg/L；Succinic acid 27.4mg/L；Fumaric acid 25.2mg/L；Maltose 500mg/L；

6) ispol:

Include L-alpha- alanine 131.5mg/L；Beta- alanine 234mg/L；L- R-gene 692mg/L；L- Asparagine 797mg/L；L-Aspartic acid 797mg/L；L-cysteine 10.5mg/L；Pidolidone 1000mg/L；L- paddy ammonia Amide 750mg/L；Glycine 371mg/L；L-Histidine 1142mg/L；L-Isoleucine 396mg/L；L-Leu 157mg/L； L-cysteine disodium 60mg/L；L- hydroxyproline 400mg/L；L-Lysine hydrochloride 610mg/L；L-methionine 521mg/L；L- Phenylalanine 562mg/L, L-PROLINE 396mg/L；DL-tryptophan 559mg/L；L-threonine 173mg/L；L-Trp 91.5mg/L, l-tyrosine 21mg/L；L-tyrosine disodium, 180mg/L；Valine, 292mg/L and L-Histidine 1142mg/L；

7) vitamin mixtures:

Include biotin 0.123mg/L；D-VB5 calcium 0.89mg/L；Choline chloride 10.85mg/L；Folic acid 0.125mg/L； I- inositol 0.285mg/L；Niacin 0.165mg/L；Puridoxine hydrochloride 0.285mg/L；Riboflavin 0.125mg/L；Thiamine hydrochloride 0.125mg/L；Vitamin B12 0.12mg/L and p-aminobenzoic acid 0.245mg/L；

8) fetal calf serum that volumn concentration is 20% or 30%；

Adjust medium pH to 6.3, after aseptic filtration to obtain the final product.

In the present invention, the lactoalbumin hydrolysate, yeast extract, phosphoric acid tryptoseMeat soup can be from Difco company Purchase obtains.Myosin comes from fetal calf serum, and cromoci comes from horse liver, can buy from Sigma company.Known inorganic salts, Sugar, amino acid, vitamin content insect cell medium can be bought from commercial company, and the above composition is added.

Studies have shown that culture medium of the invention can cultivate nosema locustae, it is particularly suitable for embryonic tissue, the fat of locust Body tissue, reproduction or ovary tissue and digestive system tissue, these are that nosema locustae completes history of life and rich in Tissue, thus the breeding micro- spore of locust can be cultivated with the liquid nutritional environment of culture medium simulation locust tissue, cell development.

It is another aspect of the present invention to provide a kind of suspension culture methods of nosema locustae comprising following steps:

(1) the described in any item culture mediums of claim 2-7 are added into bioreactor, are then added under drying regime The microcarrier that average diameter is 50-300 μm, the starting final concentration of microcarrier reach 0.1-3g/L；

(2) in the culture medium containing microcarrier obtained to step (1), nosema locustae is added, the locust is micro- Sporozoite is named as XJ-20, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.11170；

(3) control parameter pH 7.0-7.2, PO are adjusted₂It is 25%, temperature is 28-30 DEG C, maintains to carry out not less than 40rpm Stirring, changed liquid with the culture medium in fresh step (1) every 10 days；

(4) at the 50th day of culture, after microcarrier sedimentation separation, the lemon containing trypsase is added into the culture solution of retention Lemon acid sodium solution, is slightly agitated for, and confirms the case where sporozoite is detached from microcarrier every 15-30 minutes microscopically observations, uses pancreas Enzyme inhibit liquid terminate reaction to get；

Wherein, it is preferred that the volume of culture bioreactor is 3-30L, preferably 10L.

The micro- spore of separated locust is passed on by breeding with locust, and sterile working separates micro- spore, is added and this hair is housed The Tissue Culture Flask of bright culture medium, condition of culture are 28-30 DEG C of temperature, and incubation time is 50 days, during which complete division growth Microsporidian moves into the new culture medium Tissue Culture Flask subculture of the present invention that is equipped with and expands.Pass through culture and subculture, micro- spore Borer population mesh is continuously increased.Microsporidian number through microcarrier suspension culture can reach 60 times of starting culture.

Detailed description of the invention

Fig. 1 is different operating volume bioreactor culture microsporidian growth curve.

Specific embodiment

The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But examples are merely exemplary, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art answer It should be appreciated that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form repair Change or replace, but these modifications and replacement are fallen within the protection scope of the present invention.

The separation of 1 nosema locustae of embodiment and Preliminary Identification

1 materials and methods

The separation and purifying of 1.1 microsporidians

Field Asiatic migratory locust microsporidian is obtained from the separation of Altay Prefecture of Xinjiang Uygur Autonomous Regions Burqin County, is tested The 3 age in days nymphs of a locust (n=50) of room raising, mix a small amount of wheat bran as feed, by micro- spore of separation using maize leaves by 30 ± 1 DEG C of room temperature Spore suspension is made in sub- insect infection tissue homogenate, and the microsporidian of suspension, every feeding 10 are counted with blood cell counting plate⁵Or 10⁷ Spore is applied in maize leaves, and the 4-5 age nymph of a locust is fed after drying.After feeding 24 hours, fresh corn leaf is replaced.Disease pest raising exists Equipped with 4 watts of fluorescent lamps, illumination round the clock, in the transfer room that temperature is 30 ± 1 DEG C.It is developed to the aged or adult stage to the nymph of a locust, collects It dies of illness worm corpse, appropriate distilled water is added and smashs to pieces, differential centrifugation obtains precipitating spore.Spore is resuspended in distilled water, be placed in- It is saved backup in 20 DEG C of refrigerators.

The nymph of a locust is squeezed with phosphate buffer (Phosphate-buffered saline, PBS), and 2 milliliters of suspensions is taken to be put into 2 milliliters containing 25%, 50%, on the PBS gradient liquid of 75%Percoll, 2000g is centrifuged 30min, from the low collection sporozoite of pipe, PBS centrifuge washing is used repeatedly, is put into sterile distilled water, and homogeneous liquid is filtered with adsorption cotton, and 900g is centrifuged 5 minutes, repeatedly with distillation Water centrifuge washing.

The size and form of 1.2 microsporidians are observed

Nosema locustae is purchased from U.S. M&R Durango company.Locust sample is derived from the pasture of Xinjiang region in 2013, sample This number n=315, abdominal incision remove tissue and organ sample.

It is observed under phase contrast microscope.The fresh locust for removing tissue and organ is homogenized with dismembyator, detects whether to infect Or it is prepared into the microsporidian suspension of transfection.Fresh micro- spore suspension can also be prepared from the excrement of the locust of suspected infection.This Two kinds of suspensions can directly be observed under the microscope with distilled water or the mixing of Ringer ' the s solution of 1/4 ionic strength, it is also possible to first Alcohol is fixed, Jim Sha dyes or CalcoXuor White M2R is dyed or CalcoXuor and DAPI double staining observation core.

To carry out projection Electronic Speculum observation, it is solid that infected tissue 2.5% (V/V) glutaraldehyde and 0.1M can block 4 DEG C of sour (pH7.4) Fixed 1 hour, then with 1.% (W/V) OsO₄After fixation, 1% acetic acid uranium dyeing, sample is embedded in Spurr ' s resin, uses concentration Incremental acetone dehydration.For pathological observation, 0.5-1 μm of slice is placed in slide, micro- with Nikon E-600 with methylene blue staining Sem observation.Projection Electronic Speculum observation is such as carried out, ultra-thin section is dyed with lead citrate, with H -300 Hitachi and JEM 100CX Microscope is observed in 80-100kV.

The measurement of the serological relation of 1.3 microsporidians and nosema locustae

Monoclonal antibody coated colloid pearl M11 and M12 are purchased from Tokyo Yakult company.8 microlitres of colloid pearls and 8 micro- It rises spore suspension mixing to drip in slide hole, 2 control slides are containing only spore liquid and coating pearl.When 50% spore absorption pearl is aggregated into Block is then the positive.90% spore does not adsorb pearl, then is feminine gender.

It is 1 × 10 that nosema locustae, which is diluted to concentration with 9g/L NaCl solution,⁹The spore liquid of a/mL, Freund are complete The complete immune male rabbit of adjuvant injection, 1 × 10⁹A/mL spore liquid adds incomplete Freund's adjuvant each after first time is 2,3 weeks immune Booster immunization is primary, takes blood to produce Nb immune antiboidy serum after finally immune 1 week.1 is pressed with 9g/L NaCl solution when test: The dilution of 32 volume ratios.The spore liquid of 1 plant of microsporidian and anti-Nb serum are subjected to serological reaction with slide agglutination, observation is solidifying Poly- response situation, while negative control is made with normal rabbit serum.

The history of life of 1.4 microsporidians observes and infection locust

The susceptible nymph of a locust of inoculation 15-20 days is taken, dissection takes out fritter fat-body, is applied on cover plate, is fixed with 95% methanol, Jim Sha dyeing, om observation.The spore of purification is diluted to 1 × 10⁸Spore/mL, drop are consolidated on cover plate with 25% glutaraldehyde It is fixed, ethanol series dehydration, metal spraying, the film making of H600 (Hitachi, Tokyo, Japan) scanning electron microscopic observation.Take susceptible later period rouge Fat tissue is starved acid pair with glutaraldehyde one and is fixed, epoxy resin embedding, L K B ultrasound slice machine-cut piece, one citric acid of acetic acid uranium The double dyeing of lead, the film making of AE1EM-801 transmission electron microscope observing.

Spore liquid is diluted to l x 10⁷Spore/milliliter feeds the 2-5 age in days nymph of a locust, every group processing 30, counts day by day It dies of illness borer population, then the worm corpse that will die of illness is smashed to pieces respectively, hydraulic pressure piece processed, one by one microscopy, having found spore, person is susceptible worm corpse.

Spore measures fixed: extracting spore liquid to every worm corpse of dying of illness, is counted after purification with blood counting chamber.

It is observed with Giemsa staining method.From 5 microsporidians of field Asiatic migratory locust and the development of nosema locustae The history of life.It is inoculated with 1 plant of field Asiatic migratory locust microsporidian with 2 age of the day Asiatic migrotory locust nymph of a locust, after different time takes middle intestines after infection Portion's tissue smear fixes 3min with 95% methanol, and Giemsa dyes 10min, and oil under the microscope, is taken pictures.

The Molecular Phylogeny of 1.6 field Asiatic migratory locust microsporidians is analyzed

1.6.1 prepared by sporozoite DNA

Containing 5 × 10⁶Spore suspension 900g is centrifuged 2min, and precipitating is resuspended in 5 μ l distilled water and 200 μ l of STE liquid In (100mM NaCl, 100mM Tris-HCl, pH 8.0,1mM EDTA), be vortexed concussion, and 800g is centrifuged 1min.Precipitating is used 150mg bead (Sigma G-8772,425-600 μ m diameters) and 150 μ l STE liquid, which are vortexed, to be shaken 30 seconds, and 2 microlitres of addition is dense 10 milligrams/ml Proteinase K Solution is spent, 55 DEG C are incubated for 2 hours, are put into 95 DEG C of incubation 5min, are put into centrifuge tube 17,500g centrifugation 2min, 10 times of volume supernatants are added 1 times of volume 3M sodium acetate, ethyl alcohol/phenol chloroform, ethanol precipitation DNA, are floated again with ethyl alcohol It washes, is dissolved in TE buffer (10mM Tris-HCl, pH 8.0,1mM EDTA).

1.6.2 sporozoite PCR amplification SSU rRNA

Primer:

(1) KAI-01=5 '-GAATTCAAGCTTGTAGTAGAGACCCAAATATC-3 ' and KAI-02=5 '- GAGCTCGCATGCACTGTTCAGATATGGTCCTTATCG-3’

(2) VN001F=5 '-CTGCAGGTACCACCAGGTTGATTCTGCCTGAC-3 ' and VN001R=5 ' GAGCTCGCATGCGGTTTACCTTGTTACGACTT-3 ',

KAI-01 and KAI-02 is sporozoite little subunit rRNA primer (small subunit rRNA, SSUrRNA). VN001F and VN001R is SSU rRNA conserved sequence primer.100 microlitres of PCR reaction volume, contain respective primer 5pm, 0.2mM is each DNTP, 2.5 unit Taq DNA polymerases, 10ng DNA profiling, PCR buffer (10mM Tris-HCl, pH 8.4,50mM KCl、1.5mM Mg Cl₂), mineral oil closing is instilled, in thermal cycler (Astec, Program Temp Control System PC-700 40 circular responses, reaction condition: 94 DEG C of 1min, 55 DEG C of 2min, 72 DEG C of 30s) are carried out.

3- μ l PCR product is mixed with 5 μ l TE, 2 μ the l 40% sucrose liquid containing 0.5% bromophenol blue, and 0.7% agarose is added Gel electrophoresis, DNA marker (being purchased from treasured biology Takara Shuzo Co company) while electrophoresis.Running gel ethidium bromide staining.

With primer I LSUF (5'-TGGGTTTAGACCGTCGTGAG-3') and S33R (5'- ATATAGCGTCTACGTCAGGCAG-3' it) is used for the amplification of microsporidian rRNAITS sequence, reaction system to be 25 μ L.React item Part are as follows: 94 DEG C initial denaturation 8 minutes, 94 DEG C 1 minute, 56 DEG C of minutes, 72 DEG C 1 minute, recycle 30 times；72 DEG C extend 10 minutes. 1.5% agarose gel electrophoresis detects PCR product.By 1 plant of field Asiatic migratory locust microsporidian SSU rRNA sequence and rRNA The PCR product of ITS sequence send Dalian treasured biotechnology Services Co., Ltd to be sequenced.

1.6.3 sporozoite SSU rRNA is sequenced

Micro- spore of -20 DEG C of storages contains 10⁸Spore/ml melts and moves into the micro- from pipe of 1.5-ml, is added

150 μ LTAE (40mM Tris-acetate, 2mM EDTA) are resuspended, and are added 150mg bead (0.5mm diameter), 50s is shaken in bead oscillator (being purchased from U.S. Biospec company), 3min is heated, takes 1-5 μ L as the template of standard PCR. 16S rRNA gene V1f and 1492r primer amplification.Amplification condition: 94 DEG C of denaturation 3min, 35 circulations: 94 DEG C of 45s, 45 DEG C 30s, 72 DEG C of 90s.72 DEG C extend 5 minutes.Separation PCR product is sequenced.

V1f(18f)(5'-CAC CAG GTT GAT TCT GCC TGAC-3')；1492r(5'-GGTTAC CTT GTTACG ACT T-3')；350f(5'-CCA AGG A(T/C)GGCAGCAGGCGCG AAA-3')；350r(5'- TTTCGCGCCTGCTGCC(G/A)TCCTTG-3')；530f(5'-GTGCCAGC(C/A)GCCGCGG-3')；530r(5'- CCGCGGC(T/G)G CTGGCAC-3')；1047r(5'-AACGGCCATGCACCAC-3')；1061f(5'-GGTGGT GCATGG CCG-3 ') or more PCR product splices on DNA sequence analysis software, obtain SSU rDNA whole genome sequence.

1.6.4 Phylogenetic Analysis

To separate N.locustae (AY305324) that microsporidian SSU rRNA gene order and gene pool log in, N.whitei(AY305323、)Antonospora scoticae(AF024655)、Vairimorpha necatrix (Y00266) sequence is compared, and constructs various Wild insects with the ortho position connected (Neighbor-Joining) of 5 software of MEGA The phylogenetic tree of microsporidian SSU rRNA.It uses SSU rRNA gene order as outer group, carries out 1000 repetitions, then distinguish By the SSU rRNA gene order for separating microsporidian and rRNA ITS gene order and the homeologous sequence downloaded from NCBI Column carry out similitude and degree of differentiation analysis.

2 results

Fat-body is primary infection site, can also be found in female, male gonad, haemocyte, can also be seen in cavum pericardiale Observe microsporidian.In the microsporidian history of life, there are many conditions with Host cellular response, from the schizont phase to spore shape At microvesicle, host's endoplasmic reticulum cisterna, mitochondria and helminth are close.In the schizont phase, it is seen that host's micro-pipe group and helminth matter Film is connected.When entering for the second schizont phase, there is " tubular structure " in host cell matter.In the schizont phase, helminth (spore) Massive duplication cause host cell volume reduce and core atrophy.Cause from the conversion of schizont phase to spore phase, sporogenesis Adipose tissue carries out formula damage, and last fat-body is replaced by a large amount of spores.

2.1 separation microsporidian forms, growth course and ultra microstructure

In Xinjiang region, 2011-2014 has collected 592 locust nymphs of a locust, and prevalence rate in 2011 is 80.6% (n= 151)；Prevalence rate 70% (n=40) in 2012；Prevalence rate 53.8% (n=300) in 2013；Prevalence rate 58.8% in 2014 (n=101)；Microsporidian is largely present in the excrement of infection locust.

These microsporidian spores and nosema locustae refractivity having the same, but Spore shapes are partially long, in length Oval, with nosema locustae no significant difference.Separate microsporidian each stage of the history of life of enteral in locust Form is essentially identical, and development almost synchronizes: visible spherical double-core schizont after infection 20h, schizont is in a manner of binary fission later Proliferation, 48-72h are division animated period, and schizont splits into spore bud mother cell, and spore bud mother cell binary fission generates 2 pairs Core spore bud eventually forms the mature spore of 2 double-cores.The growth cycle of this plant of Asiatic migratory locust microsporidian is 96h or so, than The growth cycle (84h) of nosema locustae is slightly long, and development models meet the feature of Nosema category.

In infection locust larva smear, mainly circular dicaryotic phase spore.Oval, Fresh spores size 4.5 ± 0.16 2.2 ± 0.07 μm of μ m (average ± SE, n=10), methanol is fixed and Jim Sha dyes 4.3 ± 0.20 μ of spore size M × 2.00 ± 05 μm (n=10).Smear sloughs polar filament, 200 μm of maximum length for sloughing pole pipe.DAPI dyeing illustrates spore The arrangement of son and archespore body center.In the spore that the painting on piece of Jim Sha dyeing is mostly double-core period, there are the 4 pairs of cores not divided Spore accidentally has the biggish nucleus of density, 2 cores or the spore of 4 core phases.The difference of spore mother cell and spore is greatly Small, spore mother cell is longer.

Electronic Speculum observation separation spore, spore are round or oval cell in the puberty, and diameter is 4.4 ± 0.18 μm of (n= 13) (thickness 7-9nm) usually, is wrapped up by single cytoplasma membrane in budding spore, as the spore in other periods, With the upright contact of host cell.Schizont (meront) cytoplasm contains many ribosomes and several rough surfaced endoplasmic reticulum (RER) cisternas, seemingly Extend from perinuclear space.Intermitotic double-core has uniform caryoplasm, and has nucleopore on nuclear membrane.The schizont puberty (Merogony) be separation the spore history of life in mainly proliferation or breeding period.Before spore cell division, schizont is elongated, in core Occurs microtublue organizing center (MTOC) (coniform spot on film.Two core spore cell chromosome numbers are no more than 8.Since nuclear fission is later than Cytokinesis, so common the spore for having double-core can be elongated.

It is mainly vacuole, the membranous system containing electron-dense substance and folding in cytoplasm in sporozoite.In sporinite Inside there is simple plasma membrane and has the early stage spore of surface coat.Coloring matter of the core containing single density has electronics close in division Spend spiral plaque.Spore mother cell is rounded to contain a large amount of endoplasmic reticulum vacuoles.Spore mother cell has 2 kinds of rows with the spore of pole pipe Column.There is 3-12 polar filament circle on some irregular clusters, shown on section " fibril " of 20 length, some spore mother cells are containing single One 16 polar filament circles.Rare in the 4 core spores of division stage, dicaryotic phase spore is in the great majority.Cytoplasm divides immediately after disclosing core separation It splits.Quantum splitting body spore it is extracellular be spherical shape, in oblong intracellular, it is indicated that division zygoblast plasma membrane plasticity.Quantum splitting Body spore cytoplasm inhomogenous can dye, sometimes dyeing weight, and dyeing is light sometimes.With in the limpid region of cytoplasm, it is shown as compression The linear mystery of red dot or chromosome assembling.

There are 2 nucleus in the middle part of spore, two sides are endoplasmic reticulum.Pole spore after spore rear end has one, spore front end pole There are the multilayer membrane structure of referred to as polar body in silk two sides.The spore for much discharging polar filament can be observed in just dead polypide internal control Son.Polar filament is routed up from spore one end, and length is 120-130 microns, and there are a fritter sporoplasm in many polar filament front ends.

The internal structure of the microsporidian of transmission electron microscope observation separation has edge as nosema locustae The double-core of y direction arrangement, conidial cell wall constitutes by 3 layers, is followed successively by outer wall, inner wall and plasmalemma from outside to inside: outer wall compared with It is thin, color depth；Inner wall is thicker, accounts for about the 3/4 of conidial cell wall, and coloring is shallow；Plasma membrane layer is thin, color depth.The polar filament circle of each microsporidian Several and polar filament inclination angle is greater than 30 degree.Polar filament circle number is between 3-12 circle, polar filament inclination angle but its polar filament inclination angle 60-70 degree, With locust sporozoite without significant difference.

Through gradating, schizont becomes the second schizont.This period is monokaryon, can carry out same spermatium fusion.This A period could not observe coniform spot and mitosis whether occurs, and spore cell size is identical with schizont, but and fragmentation Body is had any different, and difference is: (i) has diameter 50-100nm electron dense granules in caryoplasm, originally goes out in dicaryospore cell Now, all there is particle in monokaryon and dicaryospore after.(ii) a large amount of smooth surfaced endoplasmic reticulum cisternas in spore cell matter, vacuole, thin Lamella accumulation.Iii) there are the accumulation of blister sample film lamella, diameter 30-60nm near endoplasmic reticulum cisterna.

Close to core, compact arranged small micro-pipe group forms 1-2 orthodrome structure.Small micro-pipe is usually by smooth surfaced endoplasmic reticulum packet It wraps up in.This period all schizonts undergo cell division, and the cell and sporonin not exclusively divided contains 2-4 monokaryon, rather than double Times core.Nuclear membrane boundary is unclear, also without conical spot, also without micro-pipe and chromosome.Once in a while it can be observed that internally positioned ribosomes With endoplasmic reticulum cisterna.The plasma membrane thickness about 10-12nm of transition phase has less regular Eversible structure, originally observes plasma membrane appearance Face electron dense is irregular " patch ", and the rear electron dense substances film that formed wraps up parasitic animal and plant cell, this is meaned turns to the spore phase Change.Spore mother cell's (Sporonts) size is 4.73 ± 0.17 × 3.56 ± 0.19 μm (n=12), and film is electron dense object Matter, thickness about 30nm (outer wall), shape, which is elongated, to be extended, and there is the rough surfaced endoplasmic reticulum (RER) of accumulation at two end of cell.The amphiploid ultra micro in the period Structure is similar with schizont (Meront).Spore mother cell carries out binary fission.

Cell after binary fission is gradually transformed into immature spore.Small micro-pipe group (CSTs, clusters of small Tubules the major part of cell) is occupied, micro-pipe net junket is finally divided into and forms especially micro- spore across golgiosome.Across Gao Er The polar tube protein of spore phase is mature on matrix.Immature 2.12 ± 0.08 μm of 5.74 ± 0.14 μ m of spore size (n=5), into one Step stretches and polarization.Polarization is the vesicles that polar filament top occurs and cell backend electronics densification polar filament object forms, and is immature spore The feature of sub-district not other period spores.It is a large amount of vacuole, tubular reticulum junket, micro- in addition to polar filament precursor in immature spore cytoplasm Pipe and the endoplasmic reticulum of extension surround the double-core of central location.Originally, immature sporocyst plasma membrane and electron dense object (outer wall) package. Immature epispore is multilayered structure, at least three layers in this phase.Prematurity spore feature: reducing suddenly, outer wall and plasma membrane it Between increase additional layer, there are many tubular structures on outer wall, have Electron-dense body and prematurity pole pipe with reproducibility osmium dyeing rear end Spiral.The additional layer of mature spore is converted to the terminus spore of electron lucent.

Spore is fixed, is projected under Electronic Speculum, and spore size is 1.43 ± 0.07 μm of 3.3 ± 0.06 μ m (n=10).Electronics is saturating About 0.3-0.5 μm of bright terminus spore thickness.0.05-0.2 μm of is reduced in spore tip end spore thickness.Outer wall 3-40nm is thick, Contain an electron lucent layer in two electron density layers.There is the plasma membrane of 10-12nm thickness under outer wall.Sporonin body release, in empty spore Still contain cytoplasma membrane in film.Multilayered structure is presented in polar filament, and irregular alignment is enclosed at 1-2 layers containing 15-18.Polar filament length is spore 2/5, polar filament and spore long axis oblique angle are 60-70 degree.Pole plastid front end chamber zone contains compact arranged film；Rear end is containing loose The film of arrangement and rear end is without vacuole.The cytosome of release is circle, is 3 × 6 μm in the size for applying on piece.Have 2 in cytosome Independent core, cytoplasm electron lucent simultaneously contain circular film.

The serological reaction of 2.2 field Asiatic migratory locust microsporidians

It is negative with anti-SES-NU monoclonal antibody reactive, and M11 and M12 monoclonal antibody reaction negative, the sporozoite less than 10% Pearl is coated in conjunction with NB monoclonal antibody.The mostly anti-spore reacting positive separated with 18 days nymphs of a locust of infection of locust sporozoite.

Aggregation can be occurred with locust sporozoite antiserum by separating microsporidian, illustrate that they and nosema locustae have Similar face antigen, it was demonstrated that the affiliation of this plant of field Asiatic migratory locust microsporidian and nosema locustae is close.

The Molecular Phylogeny of 2.3 microsporidians is analyzed

A 1250bp segment is obtained using primer KAI-01 and KAI-02 primer amplification separation microsporidian.Separate spore SSU rDNA sequence is 1337bp, N.locustae, N.whitei SSU rDNA sequence 1335bp and 1337bp, separates spore SSU rDNA sequence and N.locustae homology are 95-97%.The G/C content of three sequences is 65%, similar Antonospora scoticae (62%), but than N.bombycis high (33%).Phylogenetic analysis separation spore, N.locustae, N.whitei are in together in chadogram, and relationship is still unclear.Parsimony discloses separation spore closest to locust Worm sporozoite (N.locustae).ML display separation spore and N.whitei are most close, as adjacency analysis structure.All knots Fruit illustrates to separate spore, N.locustae, N.whitei form with new sister's cluster Antonospora scoticae, and contains The micro- spore cluster sequence of the locust of N.bombycis is uncorrelated.

Construct this plant of microsporidian SSU rRNA's with ortho position phase connection (Neighbor-Joining) of 5 software of MEGA Phylogenetic tree.In chadogram, this plant of field Asiatic migratory locust microsporidian is respectively positioned in the monoid of Nosema category, and explanation is Nosema belongs to microsporidian, accordingly names this plant of field Asiatic migratory locust microsporidian for Nosema sp-XJ-10, selection 20 Microsporidian SSU rRNA gene order carries out similitude and Genetic Distance Analysis, finds Nosema sp-XJ-10 and Nosema Belong to type sepecies nosema locustae (N.locustae, AY305324) have very high similitude, similarity 95%, with The equal very little of the genetic distance of N.locustae.Therefore illustrate that this field Asiatic migratory locust microsporidian belongs to Nosema category, and And it is close with the affiliation of nosema locustae (N.locustae).Choose 15 microsporidian rRNA ITS gene orders into Row similitude and Genetic Distance Analysis find that the similarity of this field Asiatic migratory locust microsporidian and nosema locustae is low In 75%, degree of differentiation is also larger, illustrates that this Asiatic migratory locust microsporidian is with nosema locustae (N.locustae) Nosema belongs to not of the same race, is that Nosema belongs to microsporidian not of the same race, with Antonospora scoticae and The genetic distance of N.bombycis respectively reaches 21.3 and 11.5.Confirmation is microsporidian not of the same race.

Embodiment 2. separates breeding in nosema locustae body

1. material and method

1.1 materials: Nosema sp-XJ-10 is proliferated through Asiatic migratory locust.Xinjiang Uygur Autonomous Regions A Le picks up from locust system Test worm is done in safe area locust egg, indoor passage raising.

1.2 test method

1.2.1 a large amount of proliferation of the micro- robe worm of locust

1) relationship of host's lethal time and cause of disease stage of development

Microsporidian spore liquid is diluted to 1 × 10⁸Spore/mL takes 10 microlitres of drops on diameter 10mm wheat seeding blade, cool The locust 4 age nymph of a locust of this room passage raising is fed with after dry, per first, about 10 altogether.For 24 hours afterwards by wheat seeding blade eat everything up The nymph of a locust is used as test worm, is placed in big plastic barrel and raises, and 30 ± 1, humidity 70%, illumination round the clock, using wheat seeding plus wheat bran as feed, often Dead borer population is recorded in its observation, collects worm corpse of dying of illness within the 10th day after inoculation, and check in worm corpse of dying of illness under phase contrast microscope Pathogen developmental state.

2) it is inoculated with the relationship of cause of disease concentration and lethal time and spore output

1×10⁵Spore/mL, 1 × 10⁶Spore/mL, 1 × 10⁷Spore/mL, 1 × 10⁸Spore/mL microsporidian liquid difference 10 microlitres of drops are drawn on wheat seeding blade with micro suction dispenser, and single head is fed with the 4 age nymphs of a locust after airing, and the blade person of eating up is tried Worm is placed in single head in small plastic barrel and raises.Rearing conditions are with 1), and every processing is inoculated with 20 and is divided into 3 groups, and observation is recorded and died of illness daily Worm, single head are weighed, and survey spore output with blood counting chamber.

3) relationship in host insect age and lethal time and spore output

2,3,4, the 5 age in days nymphs of a locust are chosen, single head is fed with l x10⁶Spore/mL, method with 2), die of illness by daily observation record Worm, single head are weighed, and spore output is surveyed.

4) relationship of harvest time and spore output

Using l x 10⁶Spore/ml is sprayed on wheat blade, and the 4-5 age in days nymph of a locust is fed with after airing, renews fresh feeding after 24 hours Material feeding, in rectangle wire gauze insect cage (30cm × 30cm × 50cm), 200-300 head/cage, every batch of 4000, constant temperature 30 ± 1 degree Celsius, humidity 70%, illumination round the clock.Worm corpse of dying of illness is collected within 10-40 days, in -20 DEG C of freezer storages.

Extraction of spores: freezing disease pest corpse is pulverized with meat grinder, and differential centrifugation is after the filtering of nylon sand cloth up to thick purification spore Son counts the purifying infection nymph of a locust phosphate buffer (Phosphate- for surveying spore output microsporidian through blood counting chamber Buffered saline, PBS) it squeezes, take 2 milliliters of suspensions to be put into 2 milliliters of PBS ladders containing 25%, 50%, 75%Percoll It spends on liquid, 2000g is centrifuged 30min, from the low collection sporozoite of pipe, uses PBS centrifuge washing repeatedly, is put into sterile distilled water, homogeneous Liquid is filtered with adsorption cotton, and 900g is centrifuged 5 minutes, uses distilled water centrifuge washing repeatedly, and -20C is saved.

2 results

Asiatic migratory locust is infected with microsporidian, initial stage catches an illness feature without obvious outside, when the nymph of a locust enters the aged or adult stage Afterwards, susceptible serious polypide abdomen feels like jelly, swelling, is in bronzing；Skin is de- not to be got off or dies of the skin taken off to slough off interior, wing shrinkage, Puberty is spun out, last dead.Strong worm fat-body yellow transparent, piece volt, diease occurrence fat-body then become opaque, are creamy white, It is filled with a large amount of spores.

The 2 age nymphs of a locust are inoculated with microsporidian, before most of individuals died of for 5 ages.After the inoculation of the 3 age nymphs of a locust, then there are a small number of individuals can Develop adult.4-5, it is most of after the inoculation of the age nymph of a locust to develop to adult, but become adult in spite of illness more.

The 4 age nymphs of a locust are fed with the microsporidian of various concentration, occurred mature spore in pin main body through 10-15 days.25-30 Its visible a large amount of spore generates.Single head locust can produce 4x l0⁹Spore.Show nosema locustae Nosema sp-XJ-10 It can be proliferated in locust body, generate a large amount of spores.

Multiplication characteristic of 2.1 microsporidians in group breeding locust

2.1.1 the relationship of host's lethal time and cause of disease stage of development

The 4 age in days nymphs of a locust are inoculated with l x l0⁶It is all dead after spore/head 28 days.Start within the 10th day after inoculation to receive the worm that dies of illness, altogether 79 are received, wherein accounting for the 48.1% of total dead worm corpse dead 38 before 16 days, microscopy cause of disease is in schizogamy period, Spore is few or immature.The 18-22 days dead worm corpse 15, account for 19.0%, all maturation spores, schizont and not at Ripe spore is few.It can be seen that it is few to generate robe amount in schizogamy period for cause of disease many places in Early death worm corpse, and disease pest is dead Time delays, and the cause of disease generated in vivo is then in the spore maturity period, and it is more to generate spore amount.

2.1.2 it is inoculated with the relationship of cause of disease concentration and lethal time and spore output

It is inoculated with 1x 10⁶-1×10⁸Spore/head, the trend that average single head sporulation quantity increases with the reduction of inoculation cause of disease concentration. It is lower to be inoculated with cause of disease concentration, it is 12.6-34.5 days from the median lethal time that the median lethal time is also longer, inoculum density and cause Dead time is negatively correlated (r=0.9121).Lethal time is drawn longer, and polypide weight also increases, and 0.54-2.01 grams of average weight/ Head, the two are positively correlated (r=0.9021), and within the scope of certain lethal time (11.1 1 21.2 days), polypide weight is produced with spore Amount is positively correlated (r=0.9657).Therefore inoculum density influences host's lethal time, thus influences to produce the amount of embracing, the present embodiment knot Fruit shows that inoculum density is l x l0⁶A spore/ml can obtain higher sporulation quantity.2-4 days different worms are inoculated with identical spore liquid In age, average single head, which produces full measure, to be had the tendency that increasing with worm age and increase.Worm age is smaller more sensitive to microsporidian, thus average Lethal time shortening, weight loss, spore output are also low.Instar on the 5th is due to reducing micro- spore sensibility, although median lethal Time is elongated, and weight increased, but sporulation quantity declines instead, therefore, selects instar inoculation on the 4th to be easy to get higher spore and produces Amount.

2.1.3 the relationship of harvest time and spore output

A large amount of proliferation nosema locustaes are when to collect worm corpse of dying of illness be to obtain higher one key factor of spore output.Inspection Look into two batches different times harvest die of illness worm corpse and measure its produce embrace amount.After inoculation 10-20 days harvest disease pest corpse sporulation quantity compared with It is low.Higher (4-7 × 10 of sporulation quantity of 20 days or more harvest disease pest corpse⁹Robe/head), amount highest (5- is embraced in the production of 30-40 days harvests 7×10⁹Spore/head).

2.2 Field information effects

On the grassland of Inner Mongol, continuous 4 years progress small areas of 2011-2015 and large area field trial, as the result is shown not It is different with spore dosage locust elimination effect, it is higher than 0.65 × 10⁹Full son/mu corrects Revision insect recluced rate up to 60% or so.

3. nosema locustae in-vitro culture medium of embodiment, extracellular matrix prepare and the exoadaptation of nosema locustae Culture

Pure water is added in 3.1 self-control nosema locustae culture medium MX, volume 1L, following each substance.

Inorganic salt mixt: NaH₂PO₄·2H₂O 507mg/L；NaHCO₃300mg/L；KCl 1720mg/L；CaCl₂· 2H₂O 750mg/L；CuCl₂·H₂O 0.1mg/L；CoCl₂·6H₂O 0.03mg/L；FeSO₄·7H₂O 0.28mg/L； MgCl₂·4H₂O 1140mg/L；MgSO₄·7H₂O 3269mg/L；MnCl₂·4H₂O 0.01mg/L；NaCl 1425mg/L； NaH₂PO₄·4H₂O 580mg/L；(NH₄)₆(Mo₇O₂₄) 4H2O 0.02mg/L and ZnCl2 0.02mg/L.

Sugared mixture: D-Glucose 2917mg/L；Fructose 20.9mg/L；Sucrose 11865mg/L；Malic acid 306mg/L； Alpha-KG 169mg/L；Succinic acid 27.4mg/L；Fumaric acid 25.2mg/L；Maltose 500mg/L.

Ispol: L-alpha- alanine 131.5mg/L；Beta- alanine 234mg/L；L- R-gene 692mg/L；Altheine 797mg/L；L-Aspartic acid 797mg/L；L-cysteine 10.5mg/L；Pidolidone 1000mg/L；L-Glutamine 750mg/L；Glycine 371mg/L；L-Histidine 1142mg/L；L-Isoleucine 396mg/L； L-Leu 157mg/L；L-cysteine disodium 60mg/L；L- hydroxyproline 400mg/L；L-Lysine hydrochloride 610mg/L；L- first Methyllanthionine 521mg/L；L-phenylalanine 562mg/L, L-PROLINE 396mg/L；DL-tryptophan 559mg/L；L-threonine 173mg/L；L-Trp 91.5mg/L, l-tyrosine 21mg/L；L-tyrosine disodium, 180mg/L；Valine, 292mg/L And L-Histidine 1142mg/L.

Vitamin mixtures: biotin 0.123mg/L；D-VB5 calcium 0.89mg/L；Choline chloride 10.85mg/L；Folic acid 0.125mg/L；I- inositol 0.285mg/L；Niacin 0.165mg/L；Puridoxine hydrochloride 0.285mg/L；Riboflavin 0.125mg/L； Thiamine hydrochloride 0.125mg/L；Vitamin B12 0.12mg/L and p-aminobenzoic acid 0.245mg/L.

Protein extract combination: lactoalbumin hydrolysate 1500mg/L, TC-yeastolate 1500mg/L, trypsasePhosphorus Hydrochlorate meat soup 1500mg/L, myosin 10mg/L, cromoci 50mg/L, inosine 100mg/L and bovine serum albumin(BSA) V 5000mg/L；

Visco-supplement PVP K-90,250mg/L.

10%, 20% and 30% fetal calf serum is further added, culture medium MX10, MX20 and MX30 culture medium is made. PH to 6.3 is adjusted with potassium hydroxide, standby make is stored after aseptic filtration.

3.2 insect source property, water solubility, deacetylation chitin preparation

3.2.1 water-soluble chitin is prepared from the husking of locust pupa

(i) preparation process

The container for filling 300mL1N hydrochloric acid is added in the skin that 5 grams of locust pupas are taken off, under filled with nitrogen environment.100 DEG C of heating 20 Minute, vacuum drying neutral with warm water and distilled water flushing.Dry locust pupa back off skin immerse 300 milliliters of 1N sodium hydroxides it is molten Liquid, the protein that 80 DEG C of heating, stirring remove in husking for 36 hours obtain 0.9 gram of chitin.

(ii) water compatibility is assessed

In saturated solution, relative humidity analyzes chitin after absorbing moisture by each chitin of BET equation indirect determination Hydrophily.There are a large amount of internal areas and height heat-absorbing due to preparing chitin, chitin has high to water affine Property.

(iii) chitin deacetylation

The above method prepares 3 grams of chitin 40% sodium hydroxide solutions of addition, 25 DEG C place 70 hours it is uniform to be formed Solution system.There are about 74% chitin deacetylations.It is detected with infrared spectrometer, deacetylation degree is 45-48%, in room temperature Under be dissolved in water.Under saturated pressure, deacetylation chitin adsorbs 6.3 times that water exceeds former above-mentioned non-acetylation chitin, There is high-affinity to water.The micro- spore cell epimatrix of outer locust can be done.

The in vitro culture of 3.3 microsporidians

(1) after the microsporidian Nosema sp-XJ-10 that embodiment 2 is bred takes out, 37 DEG C of water-baths 30 seconds to 1 point are put into Clock, with sterile Carlson's solution (penicillin 100000U/100mL, 0.05%gentamicin, 0.05% Antiformin it) washs three times；

(2) it is transferred in 15 milliliters of centrifuge tubes, 1000-1500rpm is centrifuged 10-20 minutes；

(3) supernatant is removed, balanced salt solution is added in precipitating, such as Emhorn liquid or Hank s ' Hank s ' liquid, 1000- 1500rpm is centrifuged 10-15 minutes, and precipitating samples counting containing a large amount of micro- spore.

(4) what is prepared from locust pupa removes acetyl, water-soluble chitin solution containing 10g/L, is applied to culture bottle wall surface, room temperature It spontaneously dries, is washed with sterile saline secondary.

(5) culture medium MX30 moves into the culture bottle of pan coating chitin, is added final concentration of 1 × 10⁵-1×10⁶/ mL's Micro- spore carries out static gas wave refrigerator, and 28-30 DEG C of cultivation temperature, CO2 concentration is 3-5%.

(6) after cultivating 10 days, liquid, the fresh training with half volume in the 20th day are changed with the fresh culture MX30 of 1 half volumes It supports base MX20 and changes liquid, change liquid with the fresh culture MX10 of half volume within the 30th day, microsporidian raised growth is bred after 30 days, Until 40 days, microsporidian number reached maximum.At 30-40 days, microsporidian was floated from culture surface, can harvest liquid centrifugation The first generation microsporidian of in vitro culture is obtained, 10% fetal calf serum culture medium is adapted to.It can with this 10% fetal calf serum culture medium Carry out nosema locustae culture and breeding.

Subculture in vitro separately culture is carried out to nosema locustae Nosema sp-XJ-10 using 10% fetal calf serum culture medium, The 10th generation nosema locustae that subculture in vitro separately culture is obtained is named as XJ-20, and classification naming is nosema locustae (Nosema locustae) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation time Be: on November 3rd, 2015, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microbe researches Institute, deposit number CGMCC No11170.

The external shaking flask culture of 4. nosema locustae of embodiment

1. material and method

1.1 microsporidian strains: what embodiment 3 obtained reached for 10 generations in locust body, reached for 10 generations in vitro, is named as The nosema locustae of Nosema sp-XJ-20 (abbreviation XJ-20), deposit number CGMCC No11170.Culture medium preparation is as implemented Example 3.

1.2 carry out microsporidian vitro propagation method using shaking flask

(1) the nosema locustae culture medium containing 10% fetal calf serum, the water solubility chitin of acetylation containing 10g/L is prepared MX10；(2) Nosema sp-XJ-20, inoculum density preferably 1 × 10 are inoculated with⁵-1×10⁶Spore/mL；(3) condition of culture: culture 28-30 DEG C of temperature, CO₂Concentration is 3-5%, incubation time 30-50 days, shaking flask revolving speed 60rpm/min.Every 10 days, measurement OD600 value and micro- spore count.It is respectively 0.5L, 1.5L using rolling bottle volume.

The nosema locustae culture medium without containing acetylation water solubility chitin is prepared simultaneously as control, remaining ingredient It is identical as the culture medium in above-mentioned steps (1).

The observation of 1.3 Electronic Speculum

10,20,30,40,50 days in culture are separately sampled, are incubated for 15 at 37 DEG C with 2.5% glutaraldehyde (PBS preparation) Minute, 4 DEG C are stored 24 hours, are embedded on 1 millimeter long 2% agarose square and are sliced, in 100mM phosphoric acid solution (pH7.4) It after being fixed with 1% osmium tetroxide, places 1 hour on ice, slice is washed with distilled water, small with the double uranium processing 1 of 1% acetic acid at 4 DEG C When, ethyl alcohol and embedding are dehydrated in the epoxy, are dyed with 1% uranium acetate, lead citrate solution, Philips CM10 electricity 60kV, 30 microns of aperture observations on mirror.

2. result

2.1 nosema locustae shaking flask cultures

Contain in culture medium and without removing acetyl water solubility chitin culture microsporidian situation such as table 1.Culture 10 days, locust The density of microsporidian is 1.3 × 10⁶Spore/mL, culture 20 days are 1.4 × 10⁶Spore/mL, culture 30 days are 1.8 × 10⁶Spore Son/mL, culture 40 days are 2.7 × 10⁶Spore/mL, culture 50 days are 3.6 × 10⁶Spore/mL..Acetyl water is removed compared to being free of The culture medium of dissolubility chitin increases.And 10-20 days after being inoculated with nymph of a locust feeding breeding microsporidian, the disease pest of harvest Corpse sporulation quantity is lower.The sporulation quantity of 20 days or more harvest disease pest corpse is higher, reaches 4-7 × 10⁹Robe/head harvests for 30-40 days Sporulation quantity highest, reaches 5-7 × 10⁹Spore/head.It can be seen that compared to the method for the method bred in vivo, it can using in vitro culture Disposably obtain greater amount of nosema locustae.

The culture medium (removing acetyl water solubility chitin containing 10g/L) of 1. invention of table and without removing acetyl water solubility chitin The density (× 10 of culture medium spinner culture microsporidian⁶Spore/mL)

Embodiment 5 continuously cultivates amplification nosema locustae in single 2L bioreactor

5.1 used materials

Bioreactor: 2.4 liters of working volumes can handle bioreactor, be purchased from Millipore Corp. of the U.S., equipped with pH, PO2, temp probe and agitating paddle.

3 ball of microcarrier Cytodex that average diameter is 50-300 μm under drying regime is purchased from U.S. GE company, uses phosphorus It after acid buffer PBS (pH7.4) aquation 24 hours, is washed three times with PBS, after the sterilizing of PBS mesohigh, microsporidian training is added Nutrient solution makes microballoon stick surface area up to 4400cm²/g。

Microsporidian: nosema locustae XJ-20, deposit number CGMCC No11170.

Culture medium preparation such as embodiment 3.

5.2 originate microcarrier activity and microsporidian protection agent assessments operation sequence during secondary culture

When microsporidian first time secondary culture, it is micro- that very low concentration is assessed using microcarrier concentration 0.1g/L and 0.3g/L Carrier is to microsporidian growth effect.

Microsporidian is added in the 2L culture medium MX10 (chitin containing deacetylation is shown in embodiment 3) of the microcarrier containing 0.6g XJ-20 makes its final concentration reach 5.0 × 10⁶Spore/mL imports the first bioreactor (Bio1), makes to stick area after mixing Reach 25000 spores/cm², originate microcarrier concentration 0.3g/L.Adjust control parameter pH7.0-7.2, PO₂It is 25%, temperature is 28-30℃.Microsporidian XJ-20 is added in microcarrier containing 0.2g, 2L culture medium MX10 (chitin containing deacetylation), makes its end Concentration reaches 5.0 × 10⁶Spore/mL imports the second bioreactor (Bio 2) after mixing, make to stick area and reach 25000 spores Son/cm², originate microcarrier concentration 0.1g/L.Adjust control parameter pH 7.0-7.2, PO₂It is 25%, temperature is 28-30 DEG C.

In culture 50 days, microsporidian carries out enzymatic treatment by following procedure: after microcarrier sedimentation separation, bioreactor was about There is the retention of 300mL culture solution, 0.025M sodium citrate solution (PBS, the not calcium ions of 300mL trypsase containing 600mg are added And magnesium ion), it is slightly agitated for, actually detaches microcarrier every 15-30 minutes microscopically observation confirmation sporozoites, use 300mL Pancreatin inhibits liquid (inhibitor containing 1mg/mL is purchased from Sigma Co., USA) to terminate reaction.After microsporidian cell count, remove Sporozoite suspension is separately added into 2.4g microcarrier (Bio1) or 0.6g microcarrier (Bio2) in 2L bioreactor, adjusts micro- Spore count, which makes to stick area, reaches 25000/cm²Carry out second of secondary culture.During culture, liquid is changed through 2 times, changes liquid for the first time Time is the 4-6 hour being added after naked microcarrier, and changing liquid for the second time is the 20th day.The micro- spore of count evaluation after the 50th day harvest The growing state of worm.The protection of microsporidian is made using same Programmable detection deacetylation chitin and polyvinylpyrrolidone With.

5.3 appraisal procedures that cow's serum acts on during secondary culture

Program for example above-mentioned 5.2, detection culture medium are 3 culture medium MX10 of embodiment, microcarrier initial concentration 0.3g/L, difference In the 5th and the 8th day progress trypsin treatment.Microcarrier is washed three times with 600mL washing lotion (PBS) except serum deprivation before enzymatic treatment.

5.4 result

For the effect for assessing microsporidian growth parameter(s), duplicate sample is acquired from bioreactor suspension in certain time, is used Cell counter detects the microsporidian number of microsporidian concentration, the output of accumulation.

5.4.1 " starting microcarrier concentration " parameter

In continuous microsporidian culture, " starting microcarrier concentration " is that 0.1g/L is minimum, but when microsporidian number is double Between it is most fast.The microsporidian number of accumulation is all higher in test.When first time secondary culture, when microcarrier concentration is 0.1g/L, Microsporidian growth is had no adverse effect.In the bioreactor of higher microcarrier concentration, microsporidian growth has active Self-replacation trend.The results are shown in Table 2:

2. microsporidian of table continuously three times culture in originate microcarrier various concentration effect

5.4.2 " microsporidian protects reagent " parameter

PVP and water-soluble acetylation chitin can increase microsporidian to microcarrier in microsporidian is continuously cultivated Adhesive attraction.

5.4.3 " serum " parameter

Serum has in microsporidian is continuously cultivated and increases microsporidian to the adhesive attraction of microcarrier and proliferation work With.

The bioreactor culture nosema locustae of embodiment 6.

6.1 material therefor

Bioreactor: working volume 2L, 5L, 10L bioreactor (German Sartorius product), equipped with pH, pO2, Temp probe and agitating paddle, remaining material is as embodiment 5.Autoclave sterilization is spare after calibration.

3 ball of microcarrier Cytodex that average diameter is 50-300 μm under drying regime is purchased from U.S. GE company, uses phosphorus It after acid buffer PBS (pH7.4) aquation 24 hours, is washed three times with PBS, after the sterilizing of PBS mesohigh, microsporidian training is added Nutrient solution makes microcarrier stick surface area up to 4400cm²/g。

Culture medium is the culture medium MX10 containing 10% calf serum, the water solubility chitin of deacetylation containing 10g/L, according to reality Apply the preparation of 3 method of example.

Nosema locustae: nosema locustae XJ-20, deposit number CGMCC No11170.

6.2 operation sequences:

6.2.1 10L bioreactor culture nosema locustae is used

4 liters of culture medium MX10 and 4g microcarriers containing 10% calf serum, the chitin of water solubility containing deacetylation Cytodex3 be added bioreactor, counted after microsporidian freeze thawing, adjust number after culture medium is added, make its final concentration of 1 ×10⁵Spore/mL, culture solution, which is added, makes microcarrier initial concentration be 0.5g/L after microsporidian kind is added.Adjust control ginseng Number pH 7.0-7.2, PO₂It is 25%, temperature is 28 DEG C, and revolving speed 40rpm makes microcarrier suspension in culture medium.Maintenance is not less than 40rpm is stirred.It was changed with fresh containing 10% calf serum, deacetylation water solubility chitin culture medium MX10 every 10 days Liquid.It 70th day, is digested by following procedure: stopping stirring, stops adjusting control PO², pH, keep temperature regulation.Microcarrier After sedimentation, there are about 500 milliliters of culture solutions in tank, and the 0.025M sodium citrate solution of 800 milliliters of trypsase containing 600mg is added (PBS, not calcium ions and magnesium ion), suitably stirring liquid, every 15-30 minute microscopically observation confirmation sporozoite reality It is detached from microcarrier, inhibits liquid (inhibitor containing 1mg/mL is purchased from Sigma Co., USA) to terminate reaction with 800mL pancreatin.Culture period Between every 5 days, measure micro- spore density.

6.2.2 10L bioreactor culture nosema locustae is used

4 liters of culture medium MX10 and 7g microcarriers containing 10% calf serum, the chitin of water solubility containing deacetylation Cytodex3 be added bioreactor, counted after microsporidian freeze thawing, adjust number after culture medium is added, make its final concentration of 1 ×10⁵Spore/mL, culture solution, which is added, makes microballoon concentration 1.4g/L.Adjust control parameter pH7.0-7.2, PO₂It is 25%, temperature It is 28 DEG C, revolving speed 40rpm makes microcarrier suspension in culture medium.Maintenance is stirred not less than 40rpm.Every 10 days with fresh Change liquid containing 10% calf serum, deacetylation water solubility chitin culture medium MX10.70th day, enzyme was carried out by following procedure Solution: stop stirring, stop adjusting control PO², pH, keep temperature regulation.After microcarrier sedimentation, there are about 800 milliliters of culture solutions to exist In tank, the 0.025M sodium citrate solution (PBS, not calcium ions and magnesium ion) of 1000 milliliters of trypsase containing 600mg is added, Appropriate stirring liquid actually detaches microcarrier every 15-30 minutes microscopically observation confirmation sporozoites, is pressed down with 1000mL pancreatin Liquid (inhibitor containing 1mg/mL is purchased from Sigma Co., USA) processed terminates reaction.Every 5 days during culture, it is close to measure micro- spore Degree.

6.2.3 2L bioreactor culture nosema locustae is used

0.5 liter of culture medium MX10 and 0.6g microcarrier containing 10% calf serum, the chitin of water solubility containing deacetylation Cytodex3 be added bioreactor, counted after microsporidian freeze thawing, adjust number after culture medium is added, make its final concentration of 1 ×10⁵Spore/mL, culture solution, which is added, makes microcarrier concentration 0.3g/L after microsporidian kind is added.Adjust control parameter pH 7.0-7.2, PO₂It is 25%, temperature is 28 DEG C, and maintenance is stirred not less than 40rpm.Every 10 days with fresh small containing 10% Cow's serum, deacetylation water solubility chitin culture medium MX10 change liquid.It 70th day, is digested by following procedure: stopping stirring, Stop adjusting control PO², pH, keep temperature regulation.After microcarrier sedimentation, there are about 400 milliliters of culture solutions in tank, is added 500 The 0.025M sodium citrate solution (PBS, not calcium ions and magnesium ion) of milliliter trypsase containing 600mg, suitably stirring liquid, Microcarrier is actually detached every 15-30 minutes microscopically observation confirmation sporozoites, inhibits liquid (to contain 1mg/mL with 500mL pancreatin Inhibitor is purchased from Sigma Co., USA) terminate reaction.Every 5 days during culture, micro- spore density is measured.

6.3 result

It is grown for detection microsporidian, microcarrier suspension duplicate sample is periodically taken in incubation, with cell counter system meter Number, takes duplicate sample average value, sees Fig. 1.

Claims (7)

1. a kind of nosema locustae (Nosema locustae), is named as XJ-20, it is characterised in that the micro- spore of the locust Sub- worm is the separation from Asiatic migratory locust (Locusta migratoria L.), reaches for 10 generations in locust body, in vitro culture reaches It is obtained after 10 generations, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation time is: 2015 November 3, deposit number are CGMCC No.11170.

2. nosema locustae described in claim 1 is preparing the application in biological insecticides.

3. a kind of for cultivating the culture medium of nosema locustae described in claim 1, it is characterised in that the culture medium Contain following component:

1) insect source property, water solubility, deacetylation chitin 10g/L；

2) protein extract:

Include lactoalbumin hydrolysate 1500mg/L, yeast extract 1500mg/L, trypsasePhosphate broth 1500mg/L, tire Globulin 10mg/L, cromoci 50mg/L, inosine 100mg/L and bovine serum albumin(BSA) V 5000mg/L；

3) viscosity replenishers PVP K-90 250mg/L；

4) inorganic salt mixt:

Include NaH₂PO₄·2H₂O 507mg/L；NaHCO₃300mg/L；KCl 1720mg/L；CaCl₂·2H₂O 750mg/L； CuCl₂·H₂O 0.1mg/L；CoCl₂·6H₂O 0.03mg/L；FeSO₄·7H₂O 0.28mg/L；MgCl₂·4H₂O 1140mg/ L；MgSO₄·7H₂O 3269mg/L；MnCl₂·4H₂O 0.01mg/L；NaCl 1425mg/L；NaH₂PO₄·4H₂O 580mg/ L；(NH₄)₆(Mo₇O₂₄)·4H₂O 0.02mg/L and ZnCl₂0.02mg/L；

5) sugared mixture:

Include D-Glucose 2917mg/L；Fructose 20.9mg/L；Sucrose 11865mg/L；Malic acid 306mg/L；Alpha- ketone penta Diacid 169mg/L；Succinic acid 27.4mg/L；Fumaric acid 25.2mg/L；Maltose 500mg/L；

6) ispol:

Include L-alpha- alanine 131.5mg/L；Beta- alanine 234mg/L；L- R-gene 692mg/L；L- asparagus fern Amide 797mg/L；L-Aspartic acid 797mg/L；L-cysteine 10.5mg/L；Pidolidone 1000mg/L；L-Glutamine 750mg/L；Glycine 371mg/L；L-Histidine 1142mg/L；L-Isoleucine 396mg/L；L-Leu 157mg/L；L- Guang Propylhomoserin disodium 60mg/L；L- hydroxyproline 400mg/L；L-Lysine hydrochloride 610mg/L；L-methionine 521mg/L；L- phenylpropyl alcohol Propylhomoserin 562mg/L, L-PROLINE 396mg/L；DL-tryptophan 559mg/L；L-threonine 173mg/L；L-Trp 91.5mg/ L, l-tyrosine 21mg/L；L-tyrosine disodium 180mg/L；Valine 292mg/L and L-Histidine 1142mg/L；

7) vitamin mixtures:

Include biotin 0.123mg/L；D-VB5 calcium 0.89mg/L；Choline chloride 10.85mg/L；Folic acid 0.125mg/L；I- flesh Alcohol 0.285mg/L；Niacin 0.165mg/L；Puridoxine hydrochloride 0.285mg/L；Riboflavin 0.125mg/L；Thiamine hydrochloride 0.125mg/L；Vitamin B12 0.12mg/L and p-aminobenzoic acid 0.245mg/L；

8) fetal calf serum that volumn concentration is 20% or 30%；

Each ingredient is substantially soluble in pure water, adjusting medium pH to 6.3, after aseptic filtration to obtain the final product.

4. purposes of the culture medium as claimed in claim 3 in culture nosema locustae, the nosema locustae, name For XJ-20, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.11170。

5. a kind of suspension culture method of nosema locustae, it is characterised in that the following steps are included:

(1) culture medium as claimed in claim 3 is added into bioreactor, average diameter under drying regime, which is then added, is The starting final concentration of 50-300 μm of microcarrier, microcarrier reaches 0.1-3g/L；

(2) in the culture medium containing microcarrier obtained to step (1), nosema locustae, the micro- spore of the locust is added Worm is named as XJ-20, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.11170；

(3) control parameter pH 7.0-7.2, PO are adjusted₂It is 25%, temperature is 28-30 DEG C, and maintenance is stirred not less than 40rpm, Liquid was changed with the culture medium in fresh step (1) every 10 days；

(4) at the 50th day of culture, after microcarrier sedimentation separation, the citric acid containing trypsase is added into the culture solution of retention Sodium solution is slightly agitated for, and is confirmed the case where sporozoite is detached from microcarrier every 15-30 minutes microscopically observations, is used tryptose Enzyme inhibit liquid terminate reaction to get.

6. suspension culture method as claimed in claim 5, which is characterized in that the volume of culture bioreactor is 3-30L.

7. suspension culture method as claimed in claim 6, which is characterized in that the volume of culture bioreactor is 10L.