CN101348776B - Transgenic lactobacillus secreting acidic cellulase, preparation and use thereof - Google Patents

Abstract

The invention relates to a transgenic engineering lactobacillus for secreting acid cellulase, a preparation method thereof and application of the same. A cellulase gene comes from fungi and is converted into a recipient strain and then expressed; and the lactobacillus has the function of secreting the acid cellulose as verified. The transgenic engineering lactobacillus not only can be used for processing coarse fodders such as silage and so on but also can be directly fed to animals to be permanently planted and multiplied in alimentary canals of the animals and then continuously secret the acid cellulase and decompose cellulose in the alimentary canals, thereby saving the direct economic cost and the labor service cost caused by addition of the cellulase into the fodders, and avoiding waste caused by the fact that redundant enzyme is only digested in the form of proteins. The transgenic engineering lactobacillus has great significance in animal nutrition, particularly ruminant nutrition and especially animal nutrition under the conditions of collective breeding and high-proportion concentrated feeding stuff.

Description

Transgenic lactobacillus of secreting acidic cellulase and its production and application

Technical field

The present invention relates to the genetically engineered field, specifically, the present invention relates to transgenic engineering milk-acid bacteria of secreting acidic cellulase and its production and application.

Background technology

1, the proposition of problem

Along with the increase of the size of population, the more grain of human needs, this demand is more serious because of the frequent natural disaster that the non-farmlandization in increasing soil, desertification, degeneration and climate change cause; The raising of living standards of the people, quality and quantity demand to livestock product such as meat, egg, milk, skins is increasing sharply, the livestock industry of development grain-saving type is to solve people and animals to strive one of effective way of grain phenomenon, especially the solution ruminating animal the low problem of roughage utilization rate is just become a real problem.Though Mierocrystalline cellulose is renewable organic resource of enriching, nature exist widely in also can degraded cellulose microorganism, comprising (Tomme etc. such as bacterium, fungi and actinomycetes, 1995), but people depend on discovery and the understanding of people to cellulase more to cellulosic effective utilization.The research and development of cellulase produce far-reaching influence to the development that comprises food, feed, the many industries of weaving with utilization; in recent years; molecular biology and genetically engineered researchdevelopment will be accelerated the process of cellulase broader applications in industrial and agricultural production, and this solves difficult problems such as energy dilemma, food shortage, environmental pollution and all has realistic meaning to the mankind.

Utilize cellulase to improve the content that cellulosic utilization ratio mainly contains following two aspects: the interpolation of external source cellulase and the regulation and control of endogenous cellulase.Research in the past few decades concentrates on mostly: seed selection, the suitability for industrialized production of cellulase, the promotion and application of the plain enzyme superior strain of eccrine fiber, the i.e. interpolation of external source cellulase.As time goes on the development of scientific-technical progress, the external source of digestive ferment is added and has as if been reached a metastable platform phase, so the research of digestive ferment is progressed into the regulation and control stage of studying endogenous cellulase.

Why ruminating animal can effectively utilize roughage mainly is the effect that relies on rumen microorganism.Rumen microorganism is the basic reason that causes ruminating animal and non-ruminant animal digest supersession characteristic to the fermentation of feed.But roughage is to finish (Flint and Forsberg by a series of decomposition of cellulose and the acting in conjunction of non-decomposition of cellulose microorganism in the microbiological deterioration process of cud, 1995), come down to by the complicated enzymatic reaction that multiple microorganism is participated in, multiple digestive ferment participates in, so all factors that influence rumen microorganism quantity, kind and enzyme activity all will influence the utilization of ruminating animal to roughage.As everyone knows, enzymatic reaction is except mainly being subjected to concentration of substrate and enzyme activity influence, also be subjected to the influence of temperature and pH value, because the interior temperature of the body of ruminating animals such as ox, sheep is substantially constant again, so the ruminant tumor gastric microorganism utilizes the major influence factors of cellulosic enzymatic reaction to be the pH value.The ability of ruminant tumor gastric cellulose-decomposing bacteria digestion plant cell wall is the function that the ruminal environment at place takes place for a bacterial species, herbage kind and digestion, has become research focus as the pH value that influences fiber digestion basic environmental factors.

Studies show that the pH of cud is subjected to the influence of feed type and composition.Concentrated feed easily ferments, and the VFA that unit weight produces is more than roughage, and the density of concentrated feed is big and particle is little simultaneously, makes to search for food and to ruminate time of consumption few, so saliva production is less relatively, makes ruminal pH value reduction (Zhou Jianmin etc., 1999).Reports such as Hao Zhengli, when feeding low-quality forage, the rumen fluid ph value is higher; Roughage causes the pH value to reduce through pulverizing or granulating; When feeding the higher feed of concentrated feed ratio, pH is often lower, and food back lowering speed is fast.

What the rumen ph dynamic change was taken food has the greatest impact.Animal searches for food behind the feed, carbohydrate ferments in cud and produces voltaile fatty acid, the speed that voltaile fatty acid produces is fast, quantity is many, and the speed that the cud epithelium is slower than generation to the absorption and the outflow of voltaile fatty acid, so the concentration of voltaile fatty acid raises gradually in the cud, cause the pH value to descend, the acid that is produced of will fermenting theoretically can make the pH value of rumen fluid reduce to 2.5～3.0.And the decline of the volatile fat acid concentration that produces along with carbohydrate fermentation, the cud epithelium is accelerated relatively to the absorption and the effusive speed of voltaile fatty acid, the density loss of voltaile fatty acid in the cud, the saliva of ruminating animal contains abundant sodium bicarbonate and phosphoric acid salt in addition, saliva constantly flows into cud and makes voltaile fatty acid obtain neutralization to cause pH value rising, so generally its pH value but maintains 5.5～6.5.The salivation of ruminating animal is by searching for food, chew and ruminate the process decision.Feed structure is coarse more, the roughage ratio is high more in the feed, and the time that animal searches for food, chews and ruminates is just long more, and the saliva of generation is just many more, and just strong more to the buffering of cud, pH value is rising (Zhao Guangyong, 1999) just.

Have only a pH scope that is rather narrow to be suitable for the digestion of cud fiber.Discoveries such as Hoover, the optimum pH value of Mierocrystalline cellulose and dry-matter digestion is 6.5, no matter 5.5 still is 7.5 digestibilities that can both reduce fiber significantly.PH value is lower than 6.2 to the feed that Zhou Shao and Japanese plum acute hearing are just reported high fine fodder level between the 2.5～8.0h of back feeding, and can influence normal activity and fodder cellulose and the proteinic utilization of rumen microorganism at this moment.When ruminal pH value was lower than 6.0, the growth of cellulose-decomposing bacteria and methanogen and Metabolic activity reduced; PH≤5.5 o'clock protozoon disappearance (Zhang Qingcai, 1998).Stewart (1977) finds to add the barley ruminal pH value and Mierocrystalline cellulose decomposes the cause that decline thereupon mainly is the quantity minimizing of cellulose-decomposing bacterium, when pH value 6.9, every milliliter of rumen content has 106 filter paper degradation bacteria, drops to 103 when the pH value is 6.0.

Discontinuous ruminating animal feed posterior tuberosity stomach inner pH value of feeding presents periodical change, the back ruminal pH value of promptly searching for food descends, and reaches low value (Zhen Yuguo, horse are peaceful, 1998) usually in 1.5～6.0 hours in the back of feeding, in (Reddy and Joji etc., 1985 of ging up to some extent again in back 8 hours of searching for food; Reddy etc., 1987,1993).Zhou Shao and Japanese plum acute hearing (2003) find that also the pH value descends gradually after feeding, reduce to minimumly about 4 hours, after this go up gradually, and pH value minimum value significant difference (p＜0.05) between different fine fodder level.

Evidence shows: the fiber hydrolysis is not subjected to the restriction of fiber hydrolysis microbe population and vigor, and it is relevant with the obtainable fiber number of microorganism with environment PH of living in, as long as maintain time sufficiently long 6 or more in breeding cycle pH value, they just can be to be equal to or greater than speed that fiber the passes through cud also hydrocellulose of growing; And when low pH value cellulase-producing thin/fungi growth can be suppressed, and then Mierocrystalline cellulose decomposes and also be suppressed, so the existence that ruminal pH value keeps relative stability to micropopulation in the cud plays an important role.Cud acidity increases to the stable maximum that threatens of the cud ecosystem, because the pH value has very big influence to microbe species and quantity.Cellulose-decomposing bacteria is responsive especially for the variation of pH value, its optimum pH value is 6.2～7.0, rskov reports such as (1990), and rumen ph is lower than 6.2 o'clock cellulose-decomposing bacterias with its growth of severe inhibition, and then the digestibility decline (Feng Yanglian, 2004 that cause Mierocrystalline cellulose or hemicellulose; Zhou Jianmin etc., 1999; Zhao Guangyong, 1999; Hope big county etc., 1991).

So just have contradiction: ruminant tumor gastric pH value is done the high cycle variation in low earlier back with feed, its pH value Schwellenwert will be lower than 6.2, and when the pH of rumen content value or rumen fluid ph value are lower than 6.2, with the microbial growth speed of serious restricted fermentation structural carbon hydrate and the total number of microorganism (Feng Yanglian, 2004).Rumen microorganism is lower than in the pH value under 6.2 the situation still can the secreting acidic cellulase, thereby fiber is hydrolyzed and improves the digestibility of fiber in whole breeding cycle and just become the problem of pendulum in face of ours.By molecular biotechnology the rumen bacteria of anti-low pH value being introduced the fiber hydrolysis function may be the effective way that strengthens fiber hydrolysis ability under the acidic conditions, and especially fiber digestion should have more future under the acid cud condition of the high fine fodder feed generation of the high stocking density of improvement intensification.

2, the problem of the application of molecular biotechnology and existence, solution route

Improve and introducing exogenous metabolism function to the animal cud thereby transplant microorganism, have very tempting prospect by gene manipulation techniques.Though closely decades, this respect had many great Study on Value that have, and had only the only a few genetically modified microorganism to carry out verification experimental verification on animal.The most successful example of reorganization rumen microorganism is exactly to reduce the toxicity that contains the fluoroacetate plant.From native source catarrhalis (Moraxella), obtain to contain gene, be incorporated into Butyrivibrio fibrisolvens afterwards and belong among OB156 and the AR14 the fluoroacetate dehalogenation.The bacterium of transforming can reduce the toxicity of gifblaar poison and retain in sheep rumen 5 months and do not lose any gene (Gregg etc., 1994).Xue etc. (1997) are transferred to the plasmid of Neocallimastix Paericiorum xylanase gene among the Butyrivibrio fibrisolvens H17C, though this transformation bacterium has the xylan of reinforcement capacity of decomposition, but it can not field planting in cud (Krause etc., 2001b).Though attempt to be incorporated into not success the Butyrivibrio fibrisolvens the cellulose enzyme gene of Neocallimastix patriciarum and Ruminococcus albus (CelA and CelD) and from the 0-acetyl esterase gene that Neocallimastixpatriciarum obtains with same strategy, but the genetically modified microorganism of degraded fluoroacetate shows on this method and technology it is feasible (McSweeney etc., 1999).

Although the cellulose degradation enzymic activity of cud fungi is higher than rumen bacteria, its quantity be at most 1,000,000 of rumen bacteria/, so the still rumen bacteria that in rumen microorganism, cellulose degradation is played a major role.Over 20 years of past, people have done a large amount of effort and have understood the zymetology of cellulose digestion and carry out genetically engineered rumen bacteria and strengthen its ability to cellulose hydrolysis, its reason be rumen microorganism group fiber hydrolysis capabilities limits cellulosic digestion, but the exactness of this inference awaits further confirmation.A large amount of evidences show, the cellulose hydrolysis dynamics relevant with content of cellulose and available surface-area thereof is very important, in other words, the fiber hydrolysis is not subjected to the restriction of fiber hydrolysis microbe population and vigor, and relevant with environment PH of living in the obtainable fiber number of microorganism (Feng Yanglian, 2004).Known low pH value by stop the fiber hydrolysis thin/fungal growth suppresses cud fiber digestion (the minimum pH value that thin/fungal growth requires is 5.9) (Russel and Dombrowski, 1980).By genetic engineering the rumen bacteria that tolerates low pH value being introduced the fiber hydrolysis function may be approach (Russell and the Wilson that strengthens fiber hydrolysis ability under the acidic conditions, 1996), improve especially that fiber digestion has more future under the acid cud condition that high fine fodder feed produces.

The degree of improving the fiber hydrolysis not only depends on the fiber hydrolysis ability of former cud cellulose-decomposing bacteria and genetically engineered bacterium, also depends on the dynamics of ruminal pH value in the breeding cycle.For example, how long the pH value is positioned within the level that allows fiber hydrolysis bacteria growing, and the hydrolysis bacterium of attachment fiber suppresses engineered bacterium near fiber.Because fiber hydrolysis bacterial classification substantially exceeds fiber and digests needed cell concn, so as long as pH value maintains time sufficiently long 6.0 or more in breeding cycle, they just can be grown to be equal to or greater than the speed that fiber passes through cud.Though these through genetically engineered microorganism can not as the pH value greater than 6.0 condition under rapid digestion fiber the dominant fiber hydrolytic bacteria, but it can be grown under lower pH value condition by degrading close feed granules, thereby make it be lower than under 6.0 the condition still hydrocellulose (Feng Yanglian, 2004) effectively in the pH value.

Milk-acid bacteria wide industrial purposes and important ecological significance have caused the very big interest of people, lactobacillus is as the main member of normal microflora in the humans and animals body, extensively be distributed in digestive tube, urogenital tract, respiratory tract and the body surface of humans and animals, it has most important important effect (Gu Ruixia, Xie Jizhi, 1995 to body health; Jia Shifang, Guo Xinghua, 1996; Li Pinglan etc., 2000), give birth to function (Meng Tao, Guo Xinghua, 1993 so have many benefits; Jia Shifang etc., 1996; Jin Shilin, 1998; Yang Jiebin etc., 1996; Yang Rude etc., 1998; Zhang Hong, 1999).In recent years, carrier receptor system (Leer, 1996 of a series of different purposes in lactobacillus, have been set up; Sanders, 1997; Pouwels, 1993).For the security that milk-acid bacteria is used in food and medicine, the research of food grade expression system then is the forward position and the focus in this field.The general strategy of lactobacillus food grade expression system construction is: the host is a food-grade microorganisms, and carrier adopts the food grade selective marker, promptly can not have antibiotic marker to exist.Usually with deficient strain as recipient bacterium, carry the gene and the recipient bacterium complementation of recipient bacterium institute defective on the carrier, as selective marker.When the selection of non-resistance selective marker, often select those genes that is coded in the enzyme that plays a crucial role in the metabolic process, in the metabolism of folic acid as beta-galactosidase gene and thymidylic acid synthase gene (ThyA, it plays a crucial role) etc.The system of comparative maturity comprises at present: ThyA gene (Morona, 1994; Wang Chunfeng, Sun Zhe etc., 2001), Asd gene (Nakayama, 1988), β-Gal gene (Hashiba, 1992; Wang Chunfeng, Sun Zhe etc., 2003), SSB gene (Porter, 1990), amber mutant (Allison, 1996), ochre mutant (Chaillou, 1998), nisin (Sorensen, 1999) and other the factor gene etc. of nourishing and growing.The acidic cellulase gene is expressed in cud symbiosis milk-acid bacteria, to increase the biological function of milk-acid bacteria undoubtedly, more can increase ruminating animal and be lower than 6.0 o'clock cellulose hydrolysis in ruminal pH value, this will have great importance aspect the utilization ratio of roughage the raising ruminating animal.

Milk-acid bacteria is a main member in the biosphere; they are distributed widely in nature; as soil; water; in the plant; especially be present in people and animals' digestive tube; except that minority; the overwhelming majority is a requisite normal microflora with important physiological function in the humans and animals body; closely bound up with the life of the mankind and animal; its application in life science is subjected to people's attention and extensive exploitation utilization day by day; with the F-strain of lactobacillus as expression external source protective antigen gene; just the biological function of milk-acid bacteria and the specific immunity of external source function antigen gene can be combined; lactobacillus is as the normal physiological bacterium simultaneously; the oral safety that meets new generation vaccine again; convenient; characteristics such as cheapness, its scientific meaning and application prospect will be very wide.

3, transgenosis application prospect and limiting factor

Genetic engineering technique has been swarmed into human society forcefully, and this is one of Modern Logo of human commander and scientific and technological progress.21 century is an era gene, and genetic engineering technique is changing nature and also changing the world, and this is progressive irresistible paces of epoch, and transgenic technology will be to solve 21 century ever-increasing population to the effective means or the important channel of grain demand build-up of pressure.Yet, exist the focus of query to mainly contain two aspects to transgenic product at present: at first to be that the antibiotics resistance gene that uses in the transgenosis process exists the danger of " genetic drift "; Secondly, whether the foreign gene that changes over to can cause toxicity, the sensitivity response of humans and animals.

The antibiotics resistance that occurs in the microorganism is becoming one of biggest threat of 21 century human health.The danger of antibiotics resistance may pass to intravital bacterium by marker gene in the food chain, and this is a breakneck fact and signal to human beings'health and medical treatment.From medical angle analysis, but any bacterium that antibiosis is have resistance all may be a disaster to human and domestic animal.Antibiotics resistance gene might be transferred in the organism (comprising the germ body) that causes disease, thereby may produce resistance, makes microbiotic become invalid to some treatment of diseases.

In " the arduous problem of marker gene in the transgenosis food plant " of Northern Europe Committee of Ministers publication in 1996 three kinds of main microbiotic (kantlex, Totomycin, Streptomycin sulphate) have been made detailed analysis as the marker gene and the gus reporter gene of three kinds of weedicides.Think that Aph2 of card and coded product (NpiII) thereof are safe.A disaster is being fermented in the use of kantlex in genetically modified crops.Produce the amikacin resistance because kanamycin gene can suddenly change, and amikacin does not use as yet in medical treatment or seldom uses as the microbiotic of " reservation " in the human medicine or " urgent need ".If commercially produce those mark transgenic plant of card, then will cause communicable disease to become no medicine can use, and this is the unacceptable risk of human health.Select expression vector system can eradicate this type of threat by making up the non-antibiotic resistance!

In the nature evolutionary history, the mean lifetime cycle of species approximately is 106～107 years, often needs millions of years or the longer time and form a new species.Have microorganism in large quantities in the enteron aisle, they can catch exogenous DNA, and the exchange of genetic material can take place under condition suitably.But under the digestive tract environment condition, the DNA reorganization can not take place generally, be difficult to cause the danger that the microorganism resistance increases.Genetically modified product and metabolite thereof be during as food, should consider at first that in the whole digestion of Digestive tract and absorption process genetically modified food is whether poisonous or cause anaphylaxis.All have DNA in any theoretically food, DNA can be digested and assimilated well in Digestive tract, this means dna molecular and nontoxicity.But, can not get rid of the enhancing of the pathogeny bacterium antagonism property of medicine, thereby cause medical-risk owing to the influence of uncertain factor.

4, the proposition of this patent and practice

The seminar at the applicant place subsidizes under the subsidy of project (20060390879) in state natural sciences fund (30500364) and Chinese post-doctor's science fund early stage, the mould acidic cellulase gene of fungi wood is connected to non-antibiotic resistance expression carrier, and successfully be transformed in the competence intestinal bacteria and lactobacillus of specific gene defective, through enzyme cut, PCR identifies, obtained to contain the positive recombinant expression vector of wooden mould acidic cellulase gene, the non-resistance expression vector-CBHII of called after; Non-resistance expression vector-CBHII recombinant vectors is expressed in the intestinal bacteria of specific gene defective and lactobacillus, expression product is through the SDS-PAGE electrophoretic analysis, make negative control with empty carrier, the result shows that the CBHII gene expresses in intestinal bacteria and lactobacillus, the marking protein molecular weight is about 49.6KD; Transparent circle has clearly appearred around the flat board detection discovery bacterium colony of the flat board detection of non-resistance expression vector-CBHII intestinal bacteria transgenic engineered bacteria transformant and non-resistance expression vector-CBHII lactobacillus transgenic engineered bacteria transformant, illustrate that the engineering bacteria that changes the CBHII gene truly has the plain enzyme of eccrine fiber to the thalline surface, transgenic engineered bacteria has the ability of decomposition of cellulose enzyme; The optimum of CBHII enzyme activity determination is 50 ℃ of temperature, pH5.0, and this moment, enzyme work reached maximum value.

Summary of the invention

The object of the present invention is to provide the preparation method of the transgenic engineering milk-acid bacteria that is used for secreting acidic cellulase, comprise the steps: 1) mould recovery cultivation and the inducing culture of wood; 2) clone of wooden mould CBHII gene and order-checking; 3) expression of wooden mould CBHII gene in milk-acid bacteria; 4) the acid cellulose enzymic activity detects.

The present invention also provides the transgenic engineering milk-acid bacteria of the secreting acidic cellulase that is made by aforesaid method.

The present invention also provides the transgenic engineering milk-acid bacteria of secreting acidic cellulase, it is characterized in that described milk-acid bacteria expresses wooden mould CBHII gene.

The present invention also provides the transgenic engineering milk-acid bacteria that is used for secreting acidic cellulase in system milk, Application in Food.

The present invention also provides food or the composition that comprises above-mentioned transgenic engineering milk-acid bacteria.

This transgenic engineering milk-acid bacteria both can be used for the processing of roughages such as ensiling, can also directly feed and make it field planting in animal digestive tract, procreation to animal, secreting acidic cellulase decomposes the Mierocrystalline cellulose in the digestive tube continuously, from feed, add direct economy cost and the artificial labour cost increase that is caused thereby saved cellulase, and too much enzyme only digests the waste that is caused with protein form.This is in Animal nutrition, especially at ruminant animal nutrition, particularly at intensive culture, be significant on the Animal nutrition under the fine fodder condition at high proportion.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

Embodiment 1

1, mould recovery cultivation and the inducing culture of wood

1.1 bacterial strain

Wood mould (Trichoderma Koningii) (PDA substratum), preserving number CGMCCNo.1962.

1.2 key instrument equipment

The vertical steam sterilizer of LS-B35L type electrically heated, HD-1360 type Bechtop, constant incubator, DDHZ-300 air constant temperature oscillator, Jouan CR3i type tabletop refrigerated centrifuge, 4 ℃ and-70 ℃ of constant temperature refrigerators.

1.3 substratum preparation

PDA: potato thinly slice (peeling, cut discharge water immediately in, otherwise the easy oxidation blackening of potato), every 200g potato adds tap water 1000mL, places on the electric furnace 80 ℃ of temperature to soak 1 hour, uses filtered through gauze, be diluted to 1000mL, 15 pounds of sterilizations in 20 minutes are 20% potato and soak juice.Every 100mL potato is soaked juice and adds glucose 2g, adds agar 1.5g, and 15 pounds of autoclavings 20 minutes are solid potato agar substratum.

Mendels minimum medium: KH ₂PO ₄2.0g/L, (NH ₄) ₂SO ₄1.4g/L, urea (Urea) 0.3g/L, MgSO ₄7H ₂O 0.3g/L, CaCl ₂0.3g/L, FeSO ₄7H ₂O 5.0mg/L, MnSO ₄H ₂O 1.6mg/L, ZnSO ₄H ₂O 1.4mg/L, CoCl ₂2.0mg/L, Microcrystalline Cellulose 0.75 weight %, tween (tween80) 1.0-2.0g/L, peptone (proteosepeptone) 0.5-1.0g/L with each composition mixing dissolving constant volume, is sub-packed in 250mL triangular flask 25mL substratum.15 pounds, 20 minutes autoclavings.

1.4 the mould recovery of wood is cultivated

Ampoul tube Kaifeng: clean ampoul tube with the degreasing tampon that soaked 75% alcohol,, drip a small amount of sterilized water a to heated tip and make it to break with its top of flame heating, with file or tweezers strike down break the ampoul tube top.

Bacterial strain recover to be cultivated: with aseptic straw draw 0.3-0.5mL suitable liquid nutrient medium (sterilized water), splash in the ampoul tube, vibration makes the freeze-drying thalline be dissolved into suspension gently, draw thallus suspension liquid with 20 μ L rifles in proportion and transplant on the PDA plate culture medium, 28 ℃ of constant temperature culture 5 days.

Cultured mycelium flat board is wrapped with preservative film, is placed on 4 ℃ of refrigerators and preserves, for future use.

1.5 the inducing culture that wood is mould

Keep in Bechtop under the aseptic state, in the 250mL Erlenmeyer flask that the 25mL inducing culture is housed, the tampon plug is good with the collarium spore on the transfering loop picking PDA substratum, and 200 rev/mins, 30 ℃, constant temperature oscillator cultivation 3-4 days.

Cultured bacterium liquid is collected the 50mL centrifuge tube, and 4 ℃, 4000 rev/mins, centrifugal 10 minutes.Abandon supernatant liquor, thalline of the washing with alcohol with 20% washs thalline twice with 0.9% physiological saline afterwards.Collect good thalline and be stored in-70 ℃ or be directly used in next step RNA and extract experiment.

Embodiment 2

1, the clone of wooden mould CBHII gene and order-checking

1.1 bacterial strain and carrier

Escherichia coli DH5a and pMD18-T vector are all available from Takara company.

1.2 main agents

The total RNA rapid extraction of plant test kit is Puli's Lay Bioisystech Co., Ltd product; AMV Reverse Transcriptase (AMV ThermoScript II), Ex Taq archaeal dna polymerase, Agarose Gel DNA Purification Kit (agarose DNA purification kit), DNaseI, T4DNA ligase, Sma I, Sph I, dNTP are the Takara product; Plasmid extraction kit, DNA Marker, agarose, 5*RNA Loading Buffer (5*RNA sample-loading buffer), 6*DNA Loading Buffer (6*DNA sample-loading buffer), EB are the Tiangen product; Amp, IPTG, X-Gal are the Sigma product.

1.3 key instrument equipment

Electrophoresis apparatus during DYY-6C type bistable (Beijing Liu Yichang), DYCP-33A type agarose horizontal strip electrophoresis groove (Beijing Liu Yichang), PTC-100 type regular-PCR instrument (U.S. MJ company), UV-2020B gel imaging analysis system, DDHZ-300 air constant temperature oscillator, TD5A-WS low speed desk centrifuge, hot thermostat water bath, electro-heating standing-temperature cultivator, stand alone type snowflake ice-making machine, Constant Temp. Oven, the dried bath of constant temperature, the portable ultraviolet lamp of WD-9403E, PHSJ-4A type laboratory pH meter, the heating magnetic stirring apparatus, WH-861 eddy mixer, laboratory microwave.

1.4 substratum preparation

The LB liquid nutrient medium: peptone 10g, yeast extract 5g, sodium-chlor 10g places the 1L beaker, adds the 800mL deionized water, abundant stirring and dissolving, Dropwise 5 moL/L NaOH transfers pH value to 7.0, adds deionized water and is settled to 1L, every part of 100mL of packing, 15 pounds, autoclaving 20 minutes.

The LB solid medium: every 100mL LB liquid nutrient medium adds the 1.5g agar powder, sterilizes 20 minutes for 15 pounds.

1.5 solution preparation

0.1%DEPC water: add 1mL DEPC in the 1000mL deionized water, be placed on leave standstill in the 1000mL volumetric flask after 4 hours standby.If be used for the preparation of 75% alcoholic acid, need autoclaving 30 minutes after then the solution mixing spends the night.

5*TBE:EDTA 4.65g/L, Tris-Base 54g/L, boric acid 27.50g/L.Dilution is 0.5*TBE during work.

1% sepharose: the 1g agarose joins 100mL 0.5*TBE, and microwave oven is heated to thawing (moderate heat 1 minute), is cooled to 60 ℃, adds EB (10mg/mL), and mixing is poured electrophoresis chamber into to an amount of, and room temperature was solidified more than 30 minutes, was 1% sepharose.

100mg/mL Amp: weighing 5g Amp places the 50mL centrifuge tube, adds the 40mL aqua sterilisa, after the thorough mixing dissolving, is settled to 50mL, with 0.22 micron filtering membrane filtration sterilization, and after the aliquot packing ,-20 ℃ of preservations.

24mg/mL IPTG: weighing 1.2g IPTG places the 50mL centrifuge tube, adds the 40mL aqua sterilisa, after the thorough mixing dissolving, is settled to 50mL, with 0.22 micron filtering membrane filtration sterilization, and after the aliquot packing ,-20 ℃ of preservations.

20mg/mL X-Gal: weighing 1g X-Gal places the 50mL centrifuge tube, adds 40mLDMF, after the thorough mixing dissolving, is settled to 50mL, with 0.22 micron filtering membrane filtration sterilization, and after the aliquot packing ,-20 ℃ of preservations.

1.6 design of primers

Gene order according to wooden mould CBHII among the GeneBank, utilize a pair of primer of Premier5.0 software design, upstream and downstream primer 5 ' end adds SmaI and SphI restriction enzyme site respectively, carries out pcr amplification CBHII gene ORF with the EX-Taq archaeal dna polymerase, and its product is 1413bp.

Upstream primer (SEQ ID NO:1): 5 '-ATA CCCGGG ATG ATT GTC GGCATT CTC ACC-3 '

SmaI

Downstream primer (SEQ ID NO:2): 5 '-TCG GCATGC TTA CAG GAA CGATGG GTT TGC-3 '

SphI

1.7 total RNA extracts

Total RNA extracts and is finished by plant RNA rapid extraction test kit.The test kit goods comprise: Plant RNAtrip, plant tissue cracking, isolation of RNA, in conjunction with vegetable polysaccharides and polyphenol; Plant RNA Aid further removes plant polyphenol and polysaccharide and secondary metabolite.

Method: collect the fresh mycelia of inducing culture or the freeze-dried vaccine of-70 ℃ of preservations, be put into fast in the mortar of liquid nitrogen precooling in advance, ground sample does not allow the liquid nitrogen volatilization clean in the process of lapping rapidly, and the limit adds liquid nitrogen limit mill, up to powdered; Ground sample is got 50-200mg to the 1.5mL centrifuge tube, adds 1mL Plant RNAtrip, fully shakes up, and puts 20 minutes on ice; 12000 rev/mins, 4 ℃ centrifugal 10 minutes; Get centrifugal back supernatant, transfer to new pipe, add the 0.2mL chloroform, fully put upside down mixing, hatched on ice 5 minutes; 12000 rev/mins, 4 ℃ of centrifugal 10 minutes solution are divided into two-phase.RNA is at the upper strata water, and lower floor is an organic phase, and two-phase interface is a thin film.The phase transition of sucking-off 0.5mL upper water is to new centrifuge tube.Should keep the thick water of 0.5-1mm when getting supernatant liquor, in order to avoid fragment below disturbance and the suction and organic phase.If suck, obtained supernatant liquor should be put back in the pipe and centrifugal again 10 minutes, draw water once more.Add 1/10 volume Plant RNA Aid to supernatant, mixing is hatched 15 minutes on ice with further combined with plant polyphenol and polysaccharide, and solution becomes muddiness; Centrifugal 12000 rev/mins, 4 ℃, 10 minutes; New centrifuge tube is arrived in careful sucking-off upper water phase transition, not transparent northern Shandong shape polysaccharide below disturbance and the suction and polyphenol precipitation.The about 500-600uL of supernatant that obtains is transferred to new pipe, add the equal-volume Virahol, mixing, ice bath 30 minutes; 10000 rev/mins, 4 ℃ centrifugal 10 minutes, abandon supernatant; Add 1mL 75% ethanol immediately, put upside down mixing washing precipitation for several times.12000 rev/mins, centrifugal 5 minutes; Abandon supernatant, inhale the liquid that goes on the tube wall as far as possible fully.Open wide mouth of pipe dry air and made the residual liquid volatilization in 5-10 minute, RNA slightly microdeposit drying gets final product; The autoclaving water dissolution precipitation of handling with the DEPC of 20-100uL.RNA solution can be stored in-70 ℃ or the liquid nitrogen, or adds the 75% ethanol-20 ℃ prolonged preservation of 3 times of volumes.Generally carry out next step test as early as possible, in case the RNA degraded.

The removal of genomic dna among the RNA: the above-mentioned RNA solution of producing is all transferred to the 1.5mL centrifuge tube, be mixed with following reaction solution, full dose is 50uL.

The removal of genomic dna among the total RNA of table 1

Above-mentioned reaction solution is evenly mixed, and 37 ℃ were reacted 30 minutes, added the DEPC water of 50 μ L, added the chloroform of 100 μ L, fully mixing.12000 rev/mins, 4 ℃ of centrifugal 10 minutes solution are divided into two-phase, get upper water and move to mutually in another centrifuge tube.Following steps are all done according to method for extracting total RNA and are obtained purifying RNA.

1.8RT-PCR amplification CBHII

1.8.1 reverse transcription

With total RNA is template, and Oligodt is a primer, joins mixing in the 1.5mL centrifuge tube in proportion, 65 ℃ 5 minutes, cooled on ice 2 minutes is added mixing to remaining reagent again, 42 ℃, reacts 60 minutes.The reverse transcription reaction system of 20uL such as following table 2:

Table 2cDNA produces

1.8.2PCR amplification

With reverse transcription product cDNA is template, according to the template concentrations optimizing reaction system, makes the PCR reaction system of 25uL, as following table 3:

Table 3PCR amplified reaction

The abundant above-mentioned reaction solution of mixing, carry out the PCR reaction as early as possible, reaction conditions: 95 ℃ 5 minutes, pre-sex change; 94 ℃ 45 seconds, sex change, 53 ℃ 45 seconds, annealing, 72 ℃ 90 seconds, extend totally 35 circulations; 72 ℃ 5 minutes, extend.

1.8.3PCR product agarose gel electrophoresis

Be made into 1% sepharose liquid with the 0.5*TBE damping fluid, microwave oven heating makes after agarose dissolves fully, add EB (10mg/mL) to final concentration be 0.5ug/mL, mixing is poured in the selected as required also electrophoresis chamber that sealing is good, gel thicknesses is between 3mm-5mm, insert the suitable comb of width, comb is apart from the about 0.5mm-1mm of base plate.After treating that gel solidifies fully, carefully extract comb, gel is put into the electrophoresis chamber that the 0.5*TBE electrophoretic buffer is housed, make electrophoretic buffer just not have glue face (about 1mm), get 5uL PCR product and 1uL 6*DNALoading Buffer mixing then, it is aerial with micro sample adding appliance biased sample to be added to well comb, with the voltage electrophoresis of 5-7v/cm to gel 2/3 place.Under long-wave ultra violet lamp, observe, and carry out Treatment Analysis and take pictures with the gel imaging instrument.

1.9CBHII the clone of gene

1.9.1CBHII the recovery of gene

DNA fast purifying/recovery test kit, test kit consists of: centrifugal post, solution A (6mol/L NaCLO ₄, 0.03mol/L NaAc, pH5.2, phenol red on a small quantity), solution B (3mol/L NaAc), solution C (time spent adds the 35ml dehydrated alcohol, fully mixing), solution D (TE damping fluid).

Method: RT-PCR product 5 μ L, the agarose gel electrophoresis with 1.0% cuts under ultraviolet lamp about the sepharose 100mg that contains target DNA, smashs to pieces by weight 1: 3 with suction nozzle and (cuts weight: the volume ratio of solution A) add solution A; 50 ℃ water-soluble 10 minutes, glue melts fully and gets final product, hydrotropy 2-3 time of can vibrating is therebetween waited to melt and is put room temperature and add 15 μ L solution B, fully mixing (greater than 500uL, can suitably increase the colloidal sol time as fruit volume); Solution is placed centrifugal post, left standstill 2 minutes, 12000 rev/mins of centrifugal 20-30 seconds, if once can not add, can be centrifugal at twice; Outwell liquid, add 500uL solution C 8000 rev/mins of centrifugal 20-30 seconds in centrifugal post, abandon liquid, repeat this step; 12000 rev/mins of recentrifuge one minute dry remaining liq to remove residual alcohol; Centrifugal post is placed new centrifuge tube, and room temperature is opened wide the centrifuge tube lid and was placed 5-10 minute, makes the ethanol volatilization totally; Add 20-30 μ L solution D (50 ℃ of water-baths), left standstill 2 minutes; 15000 rev/mins centrifugal 1 minute, the pipe end solution be required DNA.But DNA is stored in-20 ℃ of prolonged preservation.2.9.2CBHII be connected with pMD 18-T Simple carrier

The purpose fragment CBHII of above-mentioned recovery is connected in the pMD18-T Simple carrier 16 ℃ of connections of spending the night.Reaction system 10uL such as following table 4:

Table 4 clone ligation system

1.9.3pMD18-T-CBHII transformed into escherichia coli DH5a competence

Take out competent cell DH5a one from-80 ℃ and manage, dissolve in the ice; Above-mentioned connection product 10 μ L are added in the 100 μ L competent cells, and rotating centrifugal pipe mixing was placed 30 minutes in the ice gently; 42 ℃ of heat shock 60-90 seconds, put in the ice 2-3 minute; Add 890 μ L LB substratum, mixing, 37 ℃, 150 rev/mins of shaking culture 45 minutes; Draw a certain amount of bacterium liquid and coat on the ready Amp+LB agar plate (containing X-Gal and IPTG), place room temperature to be absorbed flat board, be inverted dull and stereotyped 37 ℃ of incubator overnight incubation, generally do not surpass 16 hours, otherwise be prone to assorted bacterium satellite group until liquid.

1.9.4 blue hickie screening positive clone

White colony on the picking LB solid medium (containing Amp+X-Gal+IPTG) and a locus coeruleus are seeded in 5mL LB+Amp liquid nutrient medium respectively, shift amplification cultivation, and 37 ℃, 200 rev/mins, incubated overnight performs numbering.

1.9.5pMD18-T-CBHII recombinant plasmid extracts

Utilize the little extraction reagent kit of plasmid (centrifugal column type), test kit consists of: RNsaeA, balance liquid BL, solution P1, solution P2, solution P3, rinsing liquid PW, elution buffer EB, adsorption column, collection tube.The concrete operations step is as follows:

Above-mentioned conversion flat-plate bacterial colony is inoculated in respectively in the 5mL LB+Amp liquid nutrient medium, 37 ℃, 200 rev/mins of incubated overnight; In adsorption column, add balance liquid BL, 12000 rev/mins, centrifugal 1 minute, outwell the collection tube waste liquid, adsorption column is relay the recovery collector; Get 1-5mL incubated overnight bacterium liquid, add in the centrifuge tube, 12000 rev/mins, centrifugal 1 minute, absorb supernatant liquor (bacterium liquid can be collected bacterial sediment in the centrifuge tube by repeatedly centrifugal more for a long time) as far as possible; In the centrifuge tube that leaves bacterial sediment, add 250 μ L solution P1 (please check earlier and added RNaseA whether), use pipettor or the vortex oscillation device bacterial precipitation that thoroughly suspends; In centrifuge tube, add solution P2, leniently spin upside down and make the abundant cracking of thalline for 4-6 time.Leniently mix, thermal agitation not in order to avoid interrupt genomic dna, causes in the plasmid of extraction and is mixed with the genomic dna segment.This moment the bacterium liquid refrigerant thickness that should become, the used time is no more than 5 minutes, in order to avoid plasmid is damaged; Add solution P3 in the centrifuge tube, leniently spin upside down 6-8 time immediately, fully mixing white flocks will occur at this moment.12000 rev/mins, centrifugal 10 minutes, formed precipitation in the centrifuge tube bottom this moment; Carefully supernatant is poured into or transferred in the adsorption column, the sucking-off precipitation of noting trying not with pipettor.Room temperature was placed 1-2 minute, and 12000 rev/mins, centrifugal 30-60 second outwell the waste liquid in the collection tube, adsorption column is relay reclaim in the collector; In adsorption column, add 700 μ L rinsing liquid PW (please checking whether add dehydrated alcohol earlier), 12000 rev/mins, centrifugal 30-60 second outwell the waste liquid in the collection tube, adsorption column is relay reclaim in the collector; In adsorption column, add 700 μ L rinsing liquid PW, 12000 rev/mins, centrifugal 30-60 second, discard the waste liquid in the collection tube; Adsorption column is relay the recovery collector, 12000 rev/mins, centrifugal 2 minutes, rinsing liquid remaining in the adsorption column is removed; Adsorption column is placed a clean centrifuge tube, to adsorption film middle part Dropwise 5 0-100uL elution buffer EB, room temperature was placed 1 minute, 12000 rev/mins, plasmid solution collected in the centrifuge tube in centrifugal 2 minutes, in order to increase the organic efficiency of plasmid, the solution that obtains can be added in the centrifugal adsorption column again, repeat this step.Get 2 μ L DNA samples and carry out 1% agarose electrophoresis evaluation, but remainder is put-20 ℃ of refrigerator prolonged preservation.

Select six hickies and a locus coeruleus to carry out plasmid in the experiment and extracted evaluation.

2.9.6 enzyme is cut evaluation

After the plasmid of said extracted identified with 1% agarose gel electrophoresis, select to carry out enzyme with SmaI and SphI respectively and cut reaction system such as the table 5 of 20uL than the hickie plasmid that locus coeruleus lags behind:

Table 5 enzyme is cut the evaluation recombinant cloning vector

Behind the reaction solution mixing of making, 30 ℃, act on 1 hour, 37 ℃ afterwards, act on 2 hours.Reaction is got 5 μ L reaction product and carry out electrophoresis on 1% sepharose after finishing, and observation is also carried out PCR to the reorganization positive plasmid and identified.

1.9.7PCR identify

The above-mentioned male plasmid that is accredited as is heated up in a steamer 100 times of water dilutions with sterilization two, as template, carries out pcr amplification with this, reaction system such as following table 6 with the EX-Taq archaeal dna polymerase:

Table 6 recombinant cloning vector PCR identifies

The mixing reaction solution, reaction conditions is: 95 ℃ 4 minutes, pre-sex change; 94 ℃ 45 seconds, sex change, 53 ℃ 45 seconds, annealing, 72 ℃ 90 seconds, extend 30 circulations; 72 ℃ 5 minutes, extend.Reaction is got 5 μ L PCR products and is carried out 1% agarose electrophoresis, ultraviolet visualization after finishing.

1.9.8 order-checking is identified

The above-mentioned plasmid that is accredited as positive colony is checked order with universal primer, and Bioisystech Co., Ltd carries out by Beijing AudioCodes, and carries out homology analysis with DNAMAN software.Measured sequence following (SEQ ID NO:3):

ATGATTGTCG?GCATTCTCAC?CACGCTGGCT?ACGCTGGCCA?CACTGGCAGC?TAGTGTGCCT

CTAGAGGAGC?GGCAAGCTTG?CTCAAGCGTC?TGGGGCCAAT?GTGGAGGCCA?GAACTGGTCC

GGTCCAACCT?GCTGTGCTTC?CGGAAGTACA?TGCGTCTATT?CCAACGACTA?TTACTCCCAA

TGTCTTCCTG?GCGCTGCAAG?CTCAAGCTCT?TCGACACGCG?CCTCGTCGAC?GACTGCCCGA

GCATCGTCCA?CAACATCCAG?GTCTTCCGCG?ACCCCTCCCC?CTGGTTCTTC?CACCACGAGA

GTACCTCCAG?TGGGATCTGG?AACCGCTACA?TACTCGGGCA?ACCCTTTTGT?CGGGGTTACT

CCTTGGGCCA?ATGCATATTA?CGCCTCCGAA?GTTAGCAGCC?TTGCCATTCC?TAGCCTCACT

GGAGCCATGG?CCACTGCTGC?AGCAGCTGTC?GCCAAGGTTC?CCTCTTTTAT?GTGGCTGGAT

ACTTTTGACA?AGACCCCTCT?CATGGAGCAA?ACCTTGGCCG?ACATCCGCAC?TGCCAATAAG

AACGGCGGTA?ACTATGCTGG?TCAGTTTGTG?GTGTACGACC?TGCCGGATCG?CGACTGTGCC

GCCCTTGCTT?CCAATGGCGA?GTACTCCATT?GCCGATGGTG?GTGTCGACAA?GTATAAGAAC

TACATCGACA?CCATCCGTCA?GATCGTCGTT?GAATATTCCG?ATATCCGGAC?CCTCTTGGTG

ATTGAGCCTG?ACTCTCTTGC?CAACTTGGTG?ACAAACCTCG?GCACTCCAAA?GTGTGCGAAT

GCCCAGTCCG?CCTACCTCGA?GTGCATCAAC?TACGCTGTCA?CGCAGCTGAA?CCTTCCAAAC

GTGGCAATGT?ATCTGGACGC?TGGTCACGCA?GGATGGCTTG?GCTGGCCGGC?AAACCAGGAT

CCGGCCGCTC?AGCTGTTTGC?AAACGTTTAC?AAGAATGCAT?CGTCCCCGAG?AGCTCTTCGT

GGACTGGCAA?CCAATGTCGC?CAACTACAAC?GGATGGAACA?TTACCAGCCC?CCCATCATAC

ACTCAAGGCA?ACGCCGTCTA?CAACGAGCAG?CTGTACATCC?ACGCTATTGG?ACCTCTTCTT

GCCAACCACG?GCTGGTCCAA?CGCCTTCTTC?ATCACTGACC?AAGGCCGATC?TGGCAAGCAG

CCGACTGGGC?AACAACAGTG?GGGAGACTGG?TGCAATGTCA?TCGGTACCGG?ATTCGGTATT

CGCCCCTCCG?CAAACACTGG?AGACTCGCTG?CTGGATTCTT?TTGTCTGGAT?CAAGCCAGGC

GGCGAGTGTG?ACGGCACCAG?TGACAGCAGT?GCGCCACGAT?TTGACTCCCA?CTGTGCCCTA

CCTGATGCCC?TGCAACCGGC?GCCCCAAGCA?GGTGCTTGGT?TCCAAGCCTA?CTTTGTGCAG

CTCCTCACGA?ACGCAAACCC?ATCGTTCCTG?TAA

Embodiment 3

1 wooden mould CBHII genetic expression and detection

1.1 bacterial strain and carrier

ThyA gene defection type intestinal bacteria EX13, use be disclosed person among the CN1952156; Non-resistance expression vector, use be disclosed person among the CN 1482247.

1.2 main agents

SDS-PAGE electrophoresis agents useful for same and thymidylic acid are all available from Sigma company, and Xylene Brilliant Cyanine G R-250, protein molecular weight standard are Tiangen company product.All the other reagent are all same with embodiment 2.

1.3 key instrument equipment

The TU-1901 ultraviolet-visible pectrophotometer, DYCZ-24D vertical slab electrophoresis groove, TS-1 type decolorization swinging table, Other Instruments equipment and 2.2 experiments are together.

1.4 substratum preparation

φ b*broth (broth culture): take by weighing peptone 20g, yeast extract 5g, MgSO47H ₂O 5g places the 1L beaker, adds about 800mL deionized water, fully stirring and dissolving; Drip 1mol/L KOH, regulate pH value to 7.5, add deionized water and be settled to 1L; Behind the autoclave sterilization, 4 ℃ of preservations.

The SOB substratum:

2M MgCl ₂Solution: dissolving 19g MgCl in the 90mL deionized water ₂After, be settled to 100mL, autoclave sterilization.

250mM KCl solution: in the 90mL deionized water, behind the dissolving KCl 1.86g, be settled to 100mL.

Take by weighing peptone 20g, yeast extract 5g, NaCl 0.5g places the 1L beaker, adds about 800mL deionized water, fully stirring and dissolving; Measure 10mL 250mmol/L KCl solution, join in the beaker; Dropwise 5 mol/L NaOH regulates pH value to 7.0; Add deionized water substratum is settled to 1L; Behind the autoclave sterilization, 4 ℃ of preservations; The 2mol/LMgCl that adds the 5mL sterilization before using ₂Solution.

The SOC substratum:

The 1M glucose solution: the glucose of weighing 18g is dissolved in the 90mL deionized water, fully is settled to 100mL after the dissolving, with 0.22 micron filtering membrane filtration sterilization.

The 1mol/L glucose solution 2mL that in the 100mLSOB substratum, adds degerming, uniform mixing; 4 ℃ of preservations.

1.5 solution preparation

50mg/mL thymidylic acid solution: take by weighing thymidylic acid 0.25g in the 10mL centrifuge tube, add deionized water 3mL dissolving, be settled to 5mL,, be distributed into the 1mL/ pipe, place-20 ℃ of preservations with 0.22 micron filtering membrane filtration sterilization.

40mM/L DL-Threonine: take by weighing 0.048g and be dissolved in the 8mL deionized water and dissolve, be settled to 10mL, with 0.22 micron filtering membrane filtration sterilization, room temperature preservation.

The SDS-PAGE agents useful for same:

30% acrylamide mixed solution: take by weighing Acr 29g, Bis 1g adds about 80mL deionized water in beaker in the 250mL beaker, and fully stirring and dissolving adds deionized water solution is settled to 100mL, with 0.45 micron filter membrane decon; Brown bottle normal temperature is preserved.

1.5M Tris-HCl (pH8.8): weighing 18.17g Tris places the 250mL beaker, adds about 80mL deionized water, and fully stirring and dissolving is regulated pH value to 8.8 with concentrated hydrochloric acid, and solution is settled to 100ml; Behind the autoclave sterilization, room temperature preservation.

1.0M Tris-HCl (pH6.8): weighing 12.11g Tris places the 250mL beaker, adds about 50mL deionized water, and fully stirring and dissolving is regulated pH value to 6.8 with concentrated hydrochloric acid, and solution is settled to 100mL; Behind the autoclave sterilization, room temperature preservation.

10%SDS: weighing 10g SDS places the 200mL beaker, adds about 80mL deionized water, adds about 80mL deionized water, 68 ℃ of heating for dissolving; Drip concentrated hydrochloric acid adjustable value 7.2; After solution is settled to 100mL, room temperature preservation.

10% ammonium persulphate: take by weighing the 1g ammonium persulphate, stirring and dissolving behind the adding 100mL deionized water is stored in 4 ℃.

5*Tris-Glycine Buffer: the following reagent of weighing in the 1L beaker, Tris 15.1g, Glycine (glycine) 94g, SDS 5.0g; Add about 800mL deionized water, stirring and dissolving; Add deionized water solution is settled to 1L, room temperature preservation.

5*SDS-PAGE Loading Buffer: measure following reagent 1M Tris-HCl (pH6.8) 1.25mL, SDS 0.5g, BPB 25mg, glycerine 2.5mL is in the 10mL centrifuge tube, be settled to 5mL after adding deionized water dissolving, after aliquot (500uL/ part) packing, in room temperature preservation.2-ME with 25uL before using is added to mixing in every aliquot, and room temperature can be preserved about one month.

Xylene Brilliant Cyanine G R-250 staining fluid: take by weighing 0.5g Xylene Brilliant Cyanine G R-250 and place the 1L beaker; The Virahol of measuring 125mL adds in the above-mentioned beaker, stirring and dissolving, and the Glacial acetic acid of adding 50mL stirs; Add the deionized water of 325mL, stir; After removing particulate matter with filter paper, room temperature preservation.

Destainer: measure acetic acid 50mL, ethanol 25mL, deionized water 425mL places the 1L beaker, and thorough mixing uses.

The structure of non-resistance expression vector 1.6 recombinate

1.6.1SmaI cut non-resistance expression vector and recombinant plasmid pMD18-T-CBHII with the SphI enzyme

To contain the glycerol stock of non-resistance expression vector plasmid and the glycerol stock that contains recombinant clone plasmid pMD18-T-CBHII of aforementioned preservation spreads cultivation respectively, and carry out plasmid and extract, with SmaI and SphI restriction enzyme it is carried out double digestion, the reaction system of 30uL such as following table 7:

The SmaI/SphI enzyme of non-resistance expression vector of table 7 and pMD18-T-CBHII is cut

Reaction solution is with rifle mixing gently, and 30 ℃, effect earlier 1 hour, acts on 2 hours by 37 ℃ afterwards.After reaction finishes, get 2 μ L reaction product and on 1% sepharose, carry out the electrophoresis observation evaluation, remaining reaction product reclaims test kit with agarose gel and reclaims the big fragment of non-resistance expression vector and the small segment of pMD18-T-CBHII, connects usefulness to do next step.

1.6.2 non-resistance expression vector is connected with CBHII's

The required fragment of above-mentioned recovery is carried out ligation, makes 10 μ L reaction systems, reaction system such as following table 8:

The ligation system of table 8 recombinant expression vector

With above-mentioned reaction solution mixing, 16 ℃ of connections of spending the night, ready-made reaction solution preferably directly carries out next step conversion test,, can be placed on-20 ℃ of preservations if experiment condition does not allow really.

1.6.3 the preparation of intestinal bacteria EX13 competent cell

Use the Takara competent cell to prepare test kit, the goods content comprises solution A, solution B.

With inoculating needle picking intestinal bacteria EX13 (70 ℃ of glycerine are preserved bacterium), streak inoculation on LB (containing the thymidylic acid that final concentration is 50ug/mL) plate culture medium is advisable single bacterium colony can occur; The plate culture medium of above-mentioned line is inverted in 37 ℃ of incubated overnight in the constant incubator; Picking list bacterium colony is inoculated in the 100mL triangular flask that contains 20mL φ b*broth substratum; 37 ℃ of shaking culture, 120 rev/mins; Measure the OD600 value, when reaching 0.35-0.5, stop to cultivate in the placement ice; Get above-mentioned bacterial culture fluid 1mL in the 1.5mL centrifuge tube, 4000 rev/mins, 4 ℃ centrifugal 5 minutes, abandon supernatant, eliminate supernatant as far as possible; The solution A that in each centrifuge tube, adds precooling in the 100uL ice, the springing centrifuge tube suspends precipitation gently, avoids thermal agitation; 4000 rev/mins, 4 ℃ centrifugal 5 minutes, abandon supernatant, note eliminating supernatant as far as possible; The solution B that adds precooling in the 100 μ L ice in each centrifuge tube, the springing centrifuge tube suspends precipitation gently, avoids thermal agitation.Ready-made competent cell can be directly used in the DNA transformation experiment, also can be in-80 ℃ of preservations, for future use.

1.6.4 the conversion of non-resistance expression vector-CBHII in EX13

Place ice to melt 10 minutes the competent cell of above-mentioned preservation; In competent cell, add aforementioned 10 μ L and connect product, placed 30 minutes in the ice behind the mixing gently; 42 ℃ of water-bath heat shocks 90 seconds were placed 5 minutes in the ice immediately; The SOC substratum that adds 37 ℃ of preheatings of 890 μ L; 37 ℃, 200 rev/mins of shaking culture are spent the night; 4000 rev/mins, 4 ℃ centrifugal 2 minutes, abandon the part supernatant, remain a small amount of supernatant suspension thalline; The suspension of bacterium liquid all is applied on the LB plate culture medium, places 37 ℃ of incubators to cultivate 16 hours flat board; Observe and cultivate bacterium colony, to carry out next step experiment.

1.6.5 the growth function of non-resistance expression vector-CBHII remedies evaluation (preparation of thymidine+LB agar plate)

Because the EX13 F-strain is a thymidylate synthase gene defective bacterium, promptly under the situation that does not have the external source thymidine to exist, F-strain is not grown, if but the non-resistance expression vector of recombinating has been transformed in the F-strain, just the function that can make is remedied, and so just can grow on common LB substratum (promptly not containing thymidine) flat board.

1.6.6 enzyme is cut evaluation

Above-mentioned bacterium colony of growing on common LB flat board is extracted the plasmid electrophoresis, and the plasmid that picking lags behind carries out double digestion and single endonuclease digestion, and reaction system is respectively 20 μ L and 10 μ L, as following table 9 and table 10:

Table 9 recombinant expression plasmid SmaI and SphI double digestion are identified

The mixing reaction solution, 30 ℃, effect earlier 1 hour, acts on 2 hours by 37 ℃ afterwards.After reaction finishes, get 5 μ L reaction product and carry out the evaluation of 1% agarose electrophoresis.

Table 10 recombinant expression plasmid SphI enzyme is cut evaluation

The mixing reaction solution, 37 ℃ act on 3 hours, and reaction finishes, and gets the 5uL reaction product and carries out the evaluation of 1% agarose electrophoresis.

1.6.7PCR identify

The above-mentioned non-resistance expression vector of the male recombinant expression plasmid-CBHII that is accredited as is diluted 100 times,, carry out pcr amplification CBHII gene, react, reaction conditions and last same according to the PCR reaction system of standard as template.

1.7SDS-PAGE analysis expression product

12% separation gel preparation: distilled water 4.9mL, 30%Acr6.0mL, 1.5MTris-HCL3.8mL, 10%SDS 0.15mL, 10% ammonium persulphate 0.15mL, TEMED0.006mL.

5% concentrates the glue preparation: distilled water 3.4mL, 30%Acr0.83mL, 1.0MTris-HCL0.63mL, 10%SDS 0.05mL, 10% ammonium persulphate 0.05mL, TEMED0.005mL.

Sample preparation: on the picking LB flat board single recombinant expressed colony inoculation to 5mL LB liquid nutrient medium, 37 ℃, 250 rev/mins of incubated overnight; Get the LB liquid nutrient medium of the above-mentioned bacterium liquid of 5mL to the new preparation of 100mL, 37 ℃, 200 rev/mins of joltings are cultivated, and make its OD600 value between 0.6-0.8; Add the DL-Threonine; Per hour get bacterium liquid one time, the unified at last OD600 value to 7.0 of transferring, empty carrier is induced in contrast with the same manner by bacterium.12000 rev/mins centrifugal 10 minutes, abandon supernatant, add twice of 500uL physiological saline washing thalline, 12000 rev/mins centrifugal 10 minutes, abandon supernatant, in thalline, add 80uL two and heat up in a steamer water, mixing, add 20uL 5*SDS sample-loading buffer again, 100 ℃, boiled 5-10 minute, 15000 rev/mins centrifugal 10 minutes, get supernatant, the last sample of 10uL, low molecular weight protein Marker goes up sample 10uL.-20 ℃ of preservations after all the other packing.Elder generation carries out electrophoresis with 80 volts voltage, waits to run concentrated glue and enters after the separation gel, and voltage is brought up to 120 volts, finishes up to electrophoresis.Take out gel, discard concentrated glue, dyeing, decolouring, observations.

1.8 expression and the detection of recombinant plasmid in lactobacillus

1.8.1 the recovery of lactobacillus freeze-dried vaccine is cultivated

In Bechtop, keep under the aseptic state, DOMLas107 lactobacillus with freeze dried thymine synthetase (thyA) genetic flaw, transfer with 20 μ L rifles absorption thallus suspension liquid in proportion and in the MRS liquid nutrient medium, (be added with thymidine 50 μ g/ml among the MRS), 37 ℃ of constant temperature, the 200rpm shaking table is cultivated 5d.

Get cultivation bacterium liquid and activate (being added with thymidine 50 μ g/ml among the MRS) on the MRS flat board, connect bacterium with the inoculating needle line, 37 ℃ of constant temperature culture 2d have the lactobacillus bacterium colony to grow on the MRS flat board.

Picking list bacterium colony is connected in the MRS liquid nutrient medium of 10ml (being added with thymidine 50 μ g/ml among the MRS), 37 ℃ of constant temperature, 200rpm shaking table overnight incubation, glycerine with 60% is protected bacterium (final concentration is 15%), mix, for future use, but put-80 ℃ of refrigerator-freezer prolonged preservation.

1.8.2 the competent preparation of lactobacillus

Picking ThyA gene defection type lactobacillus Las107 bacterium colony, overnight incubation (about 16h) in 3mLMRS (being added with thymidine 50 μ g/ml among the MRS) with dilution in 1: 50, is cultivated 3.5-4.0h (the OD600 value is about 0.5) with overnight culture for 37 ℃.Ice bath stops growing bacterium, 4 ℃ of centrifugal 10min of 8000rpmp, abandon supernatant under the aseptic condition, ice-cold equal-volume sterilization washing 3 times, to remove the ion component in the substratum, centrifugal collection thalline after ice-cold 1/10 volume, 10% glycerine suspends again, 10% glycerine of last ice-cold 1/100 original volume suspends again.

1.8.3 the electric shock of lactobacillus transforms

The mixing under condition of ice bath with 40 μ l competence bacteriums and 4 μ L plasmids is placed 20min on ice, then it is joined in the 0.25cm electric shock cup of ice precooling, places 10min on ice.Use Bio-Rad company electric shock conversion instrument electricity to change electric commentaries on classics condition: 1800V, 25F, 200 Ω.After this add 400 μ lMRS rapidly, change in the 1.5mL centrifuge tube, 37 ℃ of incubation 2h.Get 20 μ l coated plates.37 ℃ of incubations are cultivated 40h and can be seen bacterium colony.

1.8.4 change the screening and the evaluation of the lactobacillus of non-resistance expression vector-CBHII over to

The above-mentioned single bacterium colony liquid of growing on common MRS flat board of picking spreads cultivation, and carries out plasmid and extract.(part is with the form-80 ℃ preservation of glycerol stock, for future use.)

Utilize the little extraction reagent kit of plasmid (centrifugal column type), the concrete operations step is as follows:

(1) the non-resistance expression vector-CBHII glycerol stock with above-mentioned preservation is inoculated in the 5mlMRS liquid nutrient medium, and 37 ℃, 200rpm cultivates more than the 16h;

(2) in adsorption column, add 500 μ l balance liquids, 12000rpm, centrifugal 1min outwells the collection tube waste liquid, and adsorption column is relay the recovery collector;

(3) get 4ml incubated overnight bacterium liquid, add in the centrifuge tube, 12000rpm, centrifugal 1min absorbs supernatant liquor (bacterium liquid can be collected bacterial sediment in the centrifuge tube by repeatedly centrifugal more for a long time) as far as possible;

(4) in the centrifuge tube that leaves bacterial sediment, add 250 μ l solution I, use pipettor or the vortex oscillation device bacterial precipitation that thoroughly suspends;

(5) adding N,O-Diacetylmuramidase, to make its final concentration be 20mg/ml, handles 30min for 37 ℃;

(6) in centrifuge tube, add solution II, leniently spin upside down and make the abundant cracking of thalline for 4-6 time.Leniently mix, thermal agitation not in order to avoid interrupt genomic dna, causes in the plasmid of extraction and is mixed with the genomic dna segment.This moment the bacterium liquid refrigerant thickness that should become, the used time is no more than 5 minutes, in order to avoid plasmid is damaged;

(7) add solution III in the centrifuge tube, leniently spin upside down 6-8 time immediately, fully mixing white flocks will occur at this moment.12000rpm, centrifugal 10min, form precipitation in the centrifuge tube bottom this moment;

(8) carefully supernatant is poured into or transferred in the adsorption column, the sucking-off precipitation of noting trying not with pipettor.Room temperature is placed 1-2min, 12000rpm, and centrifugal 30-60s outwells the waste liquid in the collection tube, adsorption column is relay reclaim in the collector;

(9) in adsorption column CB3, add 500 μ l protein liquid removal PD, 12000rpm, centrifugal 30-60s discards the waste liquid in the collection tube, adsorption column is relay reclaim in the collector;

(10) in adsorption column, add 700 μ l rinsing liquids, 12000rpm, centrifugal 30-60s outwells the waste liquid in the collection tube, adsorption column is relay reclaim in the collector; In adsorption column, add 700 μ l rinsing liquids again, 12000rpm, centrifugal 30-60s discards the waste liquid in the collection tube;

(11) adsorption column is relay the recovery collector, 12000rpm, centrifugal 2min removes rinsing liquid remaining in the adsorption column; Adsorption column is placed a clean centrifuge tube, to adsorption film middle part Dropwise 5 0-100 μ l elution buffer, room temperature is placed 1min, 12000rpm, centrifugal 2min collects plasmid solution in the centrifuge tube, in order to increase the organic efficiency of plasmid, the solution that obtains can be added in the centrifugal adsorption column again, repeat this step.

(12) get 2 μ lDNA samples and carry out 1% agarose electrophoresis evaluation, but remainder is put-20 ℃ of refrigerator prolonged preservation.

Identify same 3.6.6 1.8.5 the evaluation enzyme of the non-resistance expression vector of transgenic engineered bacteria-CBHII recombinant clone plasmid is cut, PCR identifies same 3.6.7.

1.8.6SDS-PAGE expression product analysis

Sample preparation: on the picking MRS flat board single colony inoculation to the 5mlMRS liquid nutrient medium, 37 ℃, the 250rpm incubated overnight; Get the MRS liquid nutrient medium of the above-mentioned bacterium liquid of 5ml to the new preparation of 100ml, 37 ℃, the 200rpm jolting is cultivated, and makes its OD600 between 0.6-0.8; Add the DL-Threonine; Get bacterium once before adding, every afterwards 1h gets bacterium liquid one time, unified at last tone pitch to 0.7, and empty carrier is induced in contrast with the same manner by bacterium.The centrifugal 10min of 12000rpm abandons supernatant, adds 1ml physiological saline washing thalline, the centrifugal 10min of 12000rpm abandons supernatant, adds 80 μ l two and heat up in a steamer water in thalline, adding final concentration is the N,O-Diacetylmuramidase of 10mg/ml, places 1h, makes the bacterium cracking for 37 ℃, add 20 μ l5*SDS Loading Buffer again, 100 ℃, boil 10min, the centrifugal 10min of 15000rpm, get sample on the supernatant 10 μ l, low molecular weight protein Marker goes up sample 10 μ l.-20 ℃ of preservations after all the other packing.Voltage with 100v carries out electrophoresis earlier, waits to run concentrated glue and enters after the separation gel, and voltage is brought up to 200v, finishes up to electrophoresis.Take out gel, dyeing, decolouring, observations.

Embodiment 4

1 acid cellulose enzymic activity detects

1.1 bacterial strain

Non-resistance expression vector-CBHII intestinal bacteria transgenic engineered bacteria; Non-resistance expression vector-CBHII lactobacillus transgenic engineered bacteria.

1.2 main agents

3, the 5-dinitrosalicylic acid, sodium carboxymethyl-cellulose, Congo red, absorbent cotton, p-nitrophenol (p-NPH), p-nitrophenol-β-D-cellobioside (p-NPC) (sigma company).

1.3 key instrument

Electro-heating standing-temperature cultivator, TU-1900 twin-beam ultraviolet-visible photometer, thermostat water bath, the accurate pH meter of PHS-3B, stopwatch.

1.4 substratum preparation

LB-CMC substratum: add 0.5g sodium carboxymethyl-cellulose, 15 pounds, autoclaving 20min in every 100mlLB solid medium.

MRS-CMC substratum: add 0.5g sodium carboxymethyl-cellulose, 15 pounds, autoclaving 20min in every 100mlMRS solid medium.

1.5 solution preparation

Standard glucose formulations prepared from solutions: take by weighing in advance prior to being dried to the glucose 100mg of constant weight under 105 ℃, with being settled to 100ml behind the dissolved in distilled water.Be mixed with concentration then and be respectively 0,1.11, the standard glucose of 2.22,3.33,4.44,5.55,6.66 μ mol/ml uses solution to make typical curve.

P-nitrophenol (p-NPH) standard solution preparation: take by weighing p-nitrophenol (be called for short p-NPH, sigma company produces) 55.644mg,, and be settled to 100mL, be mixed with the mother liquor of 4mM with 0.1mol/L glycine-sodium hydrate buffer solution dissolving of pH10.Be diluted to concentration then and be respectively 0,0.04, the p-nitrophenol standardized solution of 0.12,0.24,0.36,0.6 μ mol/ml is to make typical curve.

P-nitrophenol-β-D-cellobioside (p-NPC) solution (2.5mmol/L) preparation: take by weighing p-nitrophenol-β-D cellobioside (4-nitrophenyl-β-D-cellobioside is called for short p-NPC sigma company and produces) 57.93mg, use water dissolution, be settled to 50ml.

DNS formulations prepared from solutions: take by weighing Seignette salt 182g, be dissolved in the 500ml distilled water, heating.In hot solution, add 3 successively, 5-dinitrosalicylic acid 6.3g, sodium hydroxide 10g, phenol 5g, sodium sulphite anhydrous 99.3 5g is stirred to dissolving, and the cooling back is settled to 1000ml with distilled water, and mixing filters, and stores in the brown reagent bottle, places 1 all backs in the dark place and uses.

1%Na2CO3 solution: weighing sodium carbonate 1g places the 100ml beaker, adds about 80ml sterilization deionized water, and fully stirring and dissolving is settled to 100ml with solution, mixing, room temperature preservation.

1mg/ml congo red staining liquid: take by weighing Congo red 0.5g, place the 500ml beaker, add about 400ml sterilization deionized water, fully stirring and dissolving is settled to 500ml with solution, and mixing filters room temperature preservation.

1mol/LNaCl solution: take by weighing sodium-chlor 29.22g, place the 500ml beaker, add about 400ml sterilization deionized water, fully stirring and dissolving is settled to 500ml with solution, mixing, room temperature preservation.

0.05mol/L the citrate buffer solution of pH5.0: take by weighing 21.014g citric acid (C6H8O7H2O), use dissolved in distilled water, be settled to 1000ml, be made into the 0.1mol/L citric acid solution; Take by weighing 29.41g Trisodium Citrate (Na3C6H5O72H2O), use dissolved in distilled water, be settled to 1000ml, be made into the 0.1mol/L sodium citrate solution; Measure citric acid 20.5ml and Trisodium Citrate 29.5ml, be settled to 100ml, mixing.

0.05mol/L the acetate buffer solution of pH5.0: draw Glacial acetic acid 289 μ l, be placed on constant volume in the 100ml volumetric flask; Take by weighing the 0.8203g sodium acetate, anhydrous, use dissolved in distilled water, be settled to 200ml; Measure Glacial acetic acid 74ml and sodium-acetate 176ml mixing.

1.25% (w/v) CMC: take by weighing the 1.25g Xylo-Mucine, the acetate buffer solution dissolving with 0.05mol/L pH5.0 is settled to 100ml.

The 0.1mol/L glycine of pH=10-sodium hydrate buffer solution preparation: 50ml 0.1mol/L glycine+32ml 0.2mol/L NaOH thin up is regulated pH=10 to 100ml.

1mol/LNa2CO3 solution: weighing sodium carbonate 10.599g places the 100ml beaker, adds about 80ml sterilization deionized water, and fully stirring and dissolving is settled to 100ml with solution, mixing, room temperature preservation.

1.6 the preparation of crude enzyme liquid

Non-resistance expression vector-CBHII intestinal bacteria transgenic engineered bacteria is inserted in the 50mlLB substratum with 1% inoculum size, be cultured to logarithmic growth in earlier stage, add DL-Threonine (final concentration is 40mmol/L) [95] and induce 16h, contrast not inductive and do same treatment, use 5ml behind the centrifugal 3min of 12000rpm, PH is the acetate buffer solution suspension thalline of 5.0 0.05mol/L, and adding final concentration is the N,O-Diacetylmuramidase of 10mg/ml, 4 ℃ of placements are spent the night, and get supernatant and obtain crude enzyme liquid.

Detect 1.7 the thalline plate is active

Detect 1.7.1 non-resistance expression vector-CBHII intestinal bacteria transgenic engineered bacteria plate is active

Adopt the congo red staining method: the intestinal bacteria transformant is transferred on the LB agar plate will (to contain non-resistance expression vector-CBHII recombination) with toothpick, (the coating final concentration is the N,O-Diacetylmuramidase of 5mg/mL on the LB agar plate) 37 ℃ of insulation 4d; With the congo red staining 1h of 1mg/ml, discard dyeing after, with the dull and stereotyped 1h of the NaCl solution washing of 1mol/L.

Detect 1.7.2 non-resistance expression vector-CBHII lactobacillus transgenic engineered bacteria plate is active

Adopt the congo red staining method: the lactobacillus transformant is transferred on the MRS-CMC agar plate will (to contain non-resistance expression vector-CBHII recombination) with toothpick, (the coating final concentration is the N,O-Diacetylmuramidase of 5mg/mL on the MRS agar plate) 37 ℃ of insulation 4d; With the congo red staining 1h of 1mg/ml, discard dyeing after, with the dull and stereotyped 1h of the NaCl solution washing of 1mol/L.

1.8 acid cellulose enzyme activity determination

1.8.1 the influence that temperature is lived to cellulase CBHII enzyme

Reaction system is by the CMC of 0.4ml1.25% (w/v), and the citrate buffer solution of 0.1ml0.05mol/L pH5.0 and 1.5ml suitably the enzyme liquid of dilution form.At 30 ℃, 40 ℃, 50 ℃, 60 ℃, add 2mlDNS reagent termination reaction in 70 ℃ of water-baths behind the reaction 30min, and boil 5min, after the flowing water cooling at boiling water bath, add distilled water to 20ml, measure the light absorption value under the 540nm, determine the optimal reactive temperature of cellulase CBHII.

1.8.2pH to the influence alive of cellulase CBHII enzyme

Reaction system is by the CMC of 0.4ml1.25% (w/v), and 0.1ml0.05mol/LpH is respectively pH=3, pH=4, and pH=5, pH=6, the citrate buffer solution of pH=7 and 1.5ml suitably the enzyme liquid of dilution form.In 50 ℃ of water-baths, add 2mlDNS reagent termination reaction behind the reaction 30min, and boil 5min, after the flowing water cooling, add distilled water, measure the light absorption value under the 540nm, determine the optimal reaction pH of cellulase CBHII to 20ml at boiling water bath.

1.8.3 adopt carboxymethyl cellulose (CMC) to carry out the mensuration of CBHII vigor for substrate

Reaction system is by the CMC of 0.4ml1.25% (w/v), and the citrate buffer solution of 0.1ml0.05mol/LpH5.0 and 1.5ml suitably the enzyme liquid of dilution form.In 50 ℃ of water-baths, add 2mlDNS reagent termination reaction behind the reaction 30min, and boil 5min at boiling water bath, after the flowing water cooling, add distilled water to 20ml, measure the light absorption value under the 540nm, determine the reducing sugar amount that the enzyme reaction process is discharged according to the glucose typical curve.The per minute hydrolysis substrate generates the pairing enzyme work of 1 μ mol glucose and is defined as enzyme unit alive.

1.8.4pNPC method

With the pNPC solution of the sodium-acetate buffer of 50mmol/LpH5.0 preparation 2.5mmol/L, 1ml adds the 1.5ml crude enzyme liquid, 50 ℃ of insulation 30min, the Na with 1% ₂CO ₃The 1ml termination reaction, the 410nm colorimetric.Growing amount with p-nitrophenol is represented enzyme activity, and an enzyme activity unit is defined as per minute hydrolysis pNPC and produces the needed enzyme amount of 1 μ mol p-nitrophenol.

The preparation of transgenic engineering bacteria preparation and preservation

Vacuum lyophilization is one of preparation and the effective means of the activity that preserves microbial cells, and is freezing and combination dry technology.The factor that influences the lactic acid bacteria freeze drying effect has a lot; as strain growth stage and speed, initial cell concentration, pH value, protective material system, pre-freeze temperature, rate of temperature fall, lyophilize condition, cell water content, cytolemma composition and rehydration condition etc., wherein protective material is the most outstanding to freeze dried influence.Because cell sustains damage in freeze-drying process easily, if when freeze-drying, add suitable protective material, the damage that can alleviate to a great extent or avoid the lyophilize pair cell to bring.

Embodiment 5

1.1 freeze-dried preparation freeze-dry process

Pure-blood ferment liquid---centrifugal---thalline enriched material---pre-freeze pre-treatment---pre-freeze---lyophilize---freeze-dried preparation

1.2 the preparation of lyophilized vaccine

With physiological saline solution bacterial classification protective material, the protective material of choosing has trehalose, glycerine (edibility), sorbyl alcohol (solution of concentration 70%), oligomeric isomaltose, skimmed milk.

The preparation of composite frozen drying protectant: every kind of single protective material is respectively with the physiological saline preparation, and sterilization is converted in proportion before the use and made composite protectant separately.

1.3 the calculating of freeze dried fermenting preparation cell survival rate (%)

Freeze-drying survival rate=(viable count that the sample unit volume records after the freeze-drying)/(viable count that the sample unit volume records before the freeze-drying) * 100%.

1.4 dull and stereotyped numeration

Get thalline enriched material 1ml after fermented liquid 6000rpm30min in aseptic centrifuge tube is centrifugal, the numeration of gradient dilution rear plate is done three parallel laboratory tests for every group and is averaged; Frozen fermented liquid numeration: centrifugal thalline enriched material and etc. the frozen-dried protective agent solution of quality mix, vibration makes and mix half an hour, puts to preserve below-40 ℃ in the refrigerator and thaws after 24 hours, dull and stereotyped numeration is done three parallel laboratory tests for every group and is averaged.

1.5 the preparation of freeze-dried preparation

Sour milk behind the pure-blood ferment behind the centrifugal 30min of 6000rpm and etc. put in-75 ℃ of refrigerators pre-freeze behind the complex protection liquid thorough mixing of quality 24 hours; put lyophilize in the lyophilizer then;-62 ℃; 0.03mbar freeze-drying 24-35 hour; again with the ferment-fermented sterilization breast after the freeze-drying; inoculum size is pressed 7/106-10/106 (w/w); cultivate after 16 hours for 30 ℃ tunning is carried out physico-chemical analysis; and and the physical and chemical index of freeze-drying primary fermentation product compare; do three repetitions at every turn, average.

SEQUENCE?LISTING

＜110〉Institute of Animal Sciences, Chinese Academy of Agricultural Sciences of Northeast Normal University

＜120〉transgenic lactobacillus of secreting acidic cellulase and its production and application

<160>3

<210>1

<211>30

<212>DNA

＜213〉artificial sequence

<400>1

ATACCCGGGA?TGATTGTCGG?CATTCTCACC 30

<210>2

<211>30

<212>DNA

＜213〉artificial sequence

<400>2

TCGGCATGCT?TACAGGAACG?ATGGGTTTGC 30

<210>3

<211>1413

<212>DNA

＜213〉wood mould (Trichoderma Koningii)

<400>3

ATGATTGTCG?GCATTCTCAC?CACGCTGGCT?ACGCTGGCCA?CACTGGCAGC?TAGTGTGCCT 60

CTAGAGGAGC?GGCAAGCTTG?CTCAAGCGTC?TGGGGCCAAT?GTGGAGGCCA?GAACTGGTCC 120

GGTCCAACCT?GCTGTGCTTC?CGGAAGTACA?TGCGTCTATT?CCAACGACTA?TTACTCCCAA 180

TGTCTTCCTG?GCGCTGCAAG?CTCAAGCTCT?TCGACACGCG?CCTCGTCGAC?GACTGCCCGA 240

GCATCGTCCA?CAACATCCAG?GTCTTCCGCG?ACCCCTCCCC?CTGGTTCTTC?CACCACGAGA 300

GTACCTCCAG?TGGGATCTGG?AACCGCTACA?TACTCGGGCA?ACCCTTTTGT?CGGGGTTACT 360

CCTTGGGCCA?ATGCATATTA?CGCCTCCGAA?GTTAGCAGCC?TTGCCATTCC?TAGCCTCACT 420

GGAGCCATGG?CCACTGCTGC?AGCAGCTGTC?GCCAAGGTTC?CCTCTTTTAT?GTGGCTGGAT 480

ACTTTTGACA?AGACCCCTCT?CATGGAGCAA?ACCTTGGCCG?ACATCCGCAC?TGCCAATAAG 540

AACGGCGGTA?ACTATGCTGG?TCAGTTTGTG?GTGTACGACC?TGCCGGATCG?CGACTGTGCC 600

GCCCTTGCTT?CCAATGGCGA?GTACTCCATT?GCCGATGGTG?GTGTCGACAA?GTATAAGAAC 660

TACATCGACA?CCATCCGTCA?GATCGTCGTT?GAATATTCCG?ATATCCGGAC?CCTCTTGGTG 720

ATTGAGCCTG?ACTCTCTTGC?CAACTTGGTG?ACAAACCTCG?GCACTCCAAA?GTGTGCGAAT 780

GCCCAGTCCG?CCTACCTCGA?GTGCATCAAC?TACGCTGTCA?CGCAGCTGAA?CCTTCCAAAC 840

GTGGCAATGT?ATCTGGACGC?TGGTCACGCA?GGATGGCTTG?GCTGGCCGGC?AAACCAGGAT 900

CCGGCCGCTC?AGCTGTTTGC?AAACGTTTAC?AAGAATGCAT?CGTCCCCGAG?AGCTCTTCGT 960

GGACTGGCAA?CCAATGTCGC?CAACTACAAC?GGATGGAACA?TTACCAGCCC?CCCATCATAC 1020

ACTCAAGGCA?ACGCCGTCTA?CAACGAGCAG?CTGTACATCC?ACGCTATTGG?ACCTCTTCTT 1080

GCCAACCACG?GCTGGTCCAA?CGCCTTCTTC?ATCACTGACC?AAGGCCGATC?TGGCAAGCAG 1140

CCGACTGGGC?AACAACAGTG?GGGAGACTGG?TGCAATGTCA?TCGGTACCGG?ATTCGGTATT 1200

CGCCCCTCCG?CAAACACTGG?AGACTCGCTG?CTGGATTCTT?TTGTCTGGAT?CAAGCCAGGC 1260

GGCGAGTGTG?ACGGCACCAG?TGACAGCAGT?GCGCCACGAT?TTGACTCCCA?CTGTGCCCTA 1320

CCTGATGCCC?TGCAACCGGC?GCCCCAAGCA?GGTGCTTGGT?TCCAAGCCTA?CTTTGTGCAG 1380

CTCCTCACGA?ACGCAAACCC?ATCGTTCCTG?TAA 1413

Claims (6)

1. the preparation method of the transgenic engineering milk-acid bacteria of secreting acidic cellulase comprises the steps: 1) mould recovery cultivation and the inducing culture of wood; 2) clone and the order-checking of wooden mould cellobiohydrolase II gene; 3) expression of wooden mould cellobiohydrolase II gene in milk-acid bacteria; 4) the acid cellulose enzymic activity detects.

2. the transgenic engineering milk-acid bacteria of secreting acidic cellulase is characterized in that described milk-acid bacteria expresses wooden mould cellobiohydrolase II gene.

3. the transgenic engineering milk-acid bacteria of the described secreting acidic cellulase of claim 2 is characterized in that, it is prepared from by the described method of claim 1.

4. the application of transgenic engineering milk-acid bacteria in food, fodder additives of claim 2 or 3 described secreting acidic cellulases.

5. the food that contains claim 2 or 3 described milk-acid bacterias

6. the feed composition that contains claim 2 or 3 described milk-acid bacterias.