CN1267722A - Engineering fungus containing large-scale salmon growth factor - Google Patents

Abstract

By using available sGH DNA as well as specified primer and plasmid, are recombined and constituted engineering colon bacillus with pETsGH plasmid expressing sGH in high efficiency and with the strain preserving number CHMCC No.0362-1. By means of reducing the cost of culture medium, simplifying, separation and purification steps, optimizing fermentation process, etc., the present invention develops growth promoting soaked agent and feed additive with the engineering bacteria for the high-yield breeding of aqueous animal and farm animal.

Description

The engineering bacteria that contains the chinook somatomedin

The present invention relates to technical field of bioengineering, is a kind of engineering bacteria that contains the Oncorhynchi somatomedin specifically.In the aquaculture industrial technology, use this engineering bacteria goods, can strengthen fish, shrimp, the immunity of shellfish organism and resistance against diseases, can produce favorable economic benefit.

In the prior art, utilize genetic engineering technique can in engineering bacteria, successfully express exogenous growth factors.Utilize that the isolating spontaneous growth factor has identical physiological function in the genetic engineering bacterium synthetic recombinant fish growth factor and the fish pituitary gland.Also exist weak point in these researchs: at first, the raw materials cost height that engineering bacteria is cultivated and fermented, the expression productive rate of the recombinant fish growth factor is lower.Secondly, the recombinant fish growth factor that is used for promotes growth experiment is the goods behind the purifying, and it separates and the complex steps of purifying, the consumption costs height, lack large scale fermentation processing condition and technology, its goods do not have practicable treatment process and utilisation technology in extensive aquaculture.

In a word, existing research only shows that the recombinant fish growth factor has tangible growth enhancing effect, but utilizes prior art recombinant fish growth factor cost synthetic and purifying too high, if use in aquaculture production, input is far longer than output, does not have any economic benefit.

Have document to record a kind of plasmid psGH4 that contains chinook somatomedin sGH gene, it is to be separated and cloned by just good laboratory, University of Toronto mound.The structure of the DNA gene library of relevant this sGH gene, the structure of this sGH Gene Sequence Analysis and plasmid psGH 4 all is published in " fish Physiology and biochemistry magazine " the 7th phase, promptly; In " molecular cloning of salmon pituitary hormone and expression " literary composition of " Fish Physiology and Biochemistry " vol.7 nos1-4 (1989) 375-380 pages or leaves.

This article has disclosed following content: the collection of (one) experiment material: from the chinook that the Washington state Colambia River is fished for, it is frozen in-70 ℃ to collect its pituitary gland, standby.

From the chinook pituitary gland, extracted total RNA, crossed the Oligo-dT post and separate total mRNA.Cut pUC13 with the SstI enzyme, dT-connects tail, and the HindIII enzyme is cut, and crosses the SEPHAROSE4B post and separates large fragment DNA (carrier DNA), and poly (T) is connected with mRNA with the pairing of poly (A) then.With mRNA is template, and with the synthesizing single-stranded cDNA of reversed transcriptive enzyme, then at archaeal dna polymerase, the effect of dna ligase and nuclease H is synthetic double-stranded down.After phenol-imitative extracting, with the cyclisation of T4 ligase enzyme.The JM103 bacterial classification of plasmid Transformed E .coli after the reorganization expands bacterium and constitutes the cDNA library.

Partial amino-acid order according to sGH, the oligonucleotide (CCYTCRAARTCRTTRAACAT, wherein R represents dG or dA, Y represents dT or dC) that has synthesized one section 20 nucleosides with dna synthesizer, behind the polyacrylamide gel electrophoresis purifying,, be the synthetic cDNA of template with mRNA as primer.This cDNA is used ³²The P mark carries out the screening first time as probe, uses then ³²The 20-Nucleotide of P mark repeats screening as probe, obtains a positive colony, go out the fragment of insertion from this clone and separate after, again be cloned into the M13 carrier, carry out dna sequence analysis, confirm to have obtained to comprise the coding sGH sGH gene cDNA sequence in proper order of signal peptide.Utilize this gene order to make up recombinant plasmid psGH4 then.

(2) the cDNA sequential analysis of sGH

From 800 clones, filter out 4 positive colonies.One of them (GH-4) carried out sequential analysis.This clone cDNA total order is classified 1148 base pairs as, 210 the amino acid whose GH precursors of encoding.Comprising 22 amino acid whose signal peptides and 188 amino acid whose ripe GH sequences.In the non-coding sequence of 3 ' end, contain the peculiar sequence A ATAAA in the site that methylates.

Comparison shows that of protein sequence, chinook GH and rainbow trout GH only have an amino acid whose difference (Val-Lew) at the 125th, although these two kinds of salmons, have than big-difference at the nucleotide sequence of 3 ' end non-coding region greater than 95% in the homology in nucleic acid sequence encoding district.

The present invention makes further research and cut-and-try work on the basis of above-mentioned prior art.

The objective of the invention is to utilize the long factor sGH of sashimi (raw fish) gene in engineering bacteria, to express and the synthetic recombinant fish growth factor; from reducing culture medium cost and simplifying purification procedures and start with; on the recombinant plasmid basis that makes up new high expression level; make up the active engineering strain of sGH gene high expression; and solution large scale fermentation technological problems and product treatment technology problem; reduce cost; improve the productive rate and the effect of product; make it finally to be effectively applied in the aquaculture industry, create certain economic benefits and social benefit.

Task of the present invention is realized by following technical scheme, developed a kind of engineering bacteria that contains the chinook somatomedin, employed chinook somatomedin, sGH cDNA gene order wherein is to make up chinook pituitary gland cDNA gene library by people such as Dr.Choy L.Hew, separate its sGH cDNA gene and made up plasmid psGH4, sGH cDNA gene order in the described engineering bacteria is obtained through pcr amplification by plasmid psGH4, and its basic step is as follows:

(1) according to the sequences Design at two ends, sGH cDNA coding region a pair of primer B-GH1 and B-GH2, synthesized the PCR reaction product that has BamHI and NcoI restriction enzyme site through pcr amplification, after cutting through these two kinds of enzyme enzymes again, obtained the sGH cDNA fragment of band BamHI and NcoI sticky end;

(2) select plasmid pET15b for use, with BamHI and NcoI double digestion, the linearization plasmid pET15bDNA fragment of separating 5.6kb;

(3) with the dna fragmentation of the plasmid pET15b in the sGH cDNA fragment in above-mentioned (1) and (2) under the effect of T4 ligase enzyme, carry out the recombinant plasmid pETsGH that recombination to construct becomes to have cyclic DNA;

(4) select intestinal bacteria (E.coli) BL21 bacterial classification for use, the competent cell of preparation BL21 bacterium;

(5), under the 1.5min heat-shocked condition, the cyclic DNA of pETsGH is converted in the BL21 competent cell at 42 ℃;

(6) cultivate at the LB solid medium upper berth ware that contains ampicillin, screen transformant, promptly obtained transforming the engineering strain of pETsGH plasmid, called after BL21/pETsGH,

Its culture presevation number: CGMCC No.0362-2, preservation date: 1998.9.29.

Described primer B-GH1, its nucleotides sequence is classified as:

5′-CAC?ACC?ATG?GGC?ATA?GAA?AAC?CAA?CGG?CTC-3′，

Described primer B-GH2, its nucleotides sequence is classified as:

5′-CAC?AGG?ATC?CTA?CAG?AGT?GCA?GTT?GGC?CTC-3′。

The expression culture condition of described this bacterial classification is; In the LB substratum, 37 ℃, fully oxygen supply is cultivated down; As somatic cells OD ₆₀₀When value reached 0.6-0.8, adding IPTG was 0.05-1mM to final concentration, and continuation was cultivated 2-3 hour at 37 ℃, induced the effect of laeUV5 promotor, carried out the sGH expression of gene.

The described BL21/pETsGH bacterial classification that transforms the pETsGH plasmid expands bacterium through routine and cultivates, and obtains OD ₆₀₀Value is 3 fermentation culture, and the processing of its thalline and the step of extracting highly active sGH gene renaturation expression product are:

(1) centrifugal collection thalline from the BL21/pETsGH bacteria culture fluid;

(2) pH7.5 of 3 times of volumes of adding, the PBS phosphoric acid buffer;

(3) above-mentioned solution is handled through ultrasonic disruption: 2-10 minute, again through 13000r.p.m, 2 minutes were centrifugal, collecting precipitation, the inclusion body of sGH;

(4) with 6M urea element, dissolve above-mentioned inclusion body;

(5) with the NH of above-mentioned solution with 30mM ₄HCO ₃Damping fluid is dialysed;

(6) get above-mentioned dialyzate and carry out lyophilize, can contain the off-white powder shape BL21/pETsGH engineering bacteria goods of the long factor of sashimi (raw fish) of sGH25-30%.

Described BL21/pETsGH engineering bacteria goods, its using method is to make soaking agent by following prescription: weight ratio is somatomedin sGH:50-70 part; Paraxin: 80-100 part; Acillin: 100-120 part; Terramycin: 50-70 part;

With above-mentioned component, be dissolved in the 20L breeding water, make soak solution;

During use, earlier aquaculture kind fish, shrimp, shellfish, crab class seedling are immersed 2.5% high salinity treatment solution, soaked 3-5 minute; This high salinity prescription for the treatment of liquid is as follows:

Weight ratio: sodium-chlor: 450-480 part; Repone K: 5-10 part; Calcium chloride: 3-5 part; Sal epsom: 2-4 part; Yellow soda ash: 5-10 part; Be dissolved in 20L and culture in the water, normal temperature uses down; Seedling is taken out from above-mentioned high salinity treatment solution again, be transferred to rapidly in the soak solution, ventilation is soaked after 30-40 minute down, transfers in the cultivating pool and cultures.

Another kind contains the engineering bacteria of chinook somatomedin, and sGH cDNA gene order wherein also is to obtain through pcr amplification by the plasmid psGH4 of the described structure of claim 1, and its basic step is,

(1) according to two ends, sGH cDNA coding region sequences Design another to primer P-GH1 and P-GH2, cut through pcr amplification and EcoRI enzyme, obtained the sGH cDNA fragment that two ends have the EcoRI sticky end;

(2) select the plasmid pA0815 that has AX01 gene and His4 gene for use, also it is cut into linearizing carrier DNA fragment with the EcoRI enzyme;

(3), under the effect of T4 ligase enzyme, connect and be built into recombinant plasmid pAOsGH with the carrier DNA fragment of the plasmid pA0815 in the sGH cDNA fragment in above-mentioned (1) and (2);

(4) with BgLII and this recombinant plasmid of BamH I double digestion pAOsGH, separate the pAOsGHCDNA fragment of 1.92Kb, connect into the compound expression section of multi-copy gene again;

(5) the linearizing recombinant plasmid pAOsGHcDNA fragment of cutting with the BamHI enzyme and purify;

(6) with the linearizing recombinant plasmid pAOsGHcDNA fragment in the compound expression section of the multi-copy gene in above-mentioned (4) and (5) under the effect of T4 ligase enzyme, connect to be built into and hold shellfish expression of gene recombinant plasmid pAOnsGH more;

(7) select yeast (pichia pastoris) GS115 bacterial classification for use, have AOXI gene and His4 gene in the karyomit(e) of this bacterial classification;

(8) cut the His4 gene regions of the recombinant plasmid pAOnsGH in above-mentioned (6) with Stu I enzyme, make the dna fragmentation of linearizing pAOnsGH;

(9) with the His4 gene regions of BIO-RADGene PulserII electric impulser with the endocellular chromosome of linearizing pAOnsGHDNA integration importing GS115;

(10) the yeast GS115 bacterial classification after will transforming is laid on 30 ℃ of cultivations in the plate of MD substratum, screens this transformant, promptly gets the yeast engineering bacterial classification that contains exogenous growth factors sGH gene, called after GS115/pAOnsGH; Its culture presevation number: CGMCC No.0362-1, preservation date: 1998.9.29.

Described its nucleotide sequence of primer P-GH1 is as follows:

5′-CCC?GGG?GAA?TTC?CTA?CAG?AGT?GCA?GTT?GGC-3′

Described its nucleotide sequence of primer P-GH2 is as follows:

5′-CCC?GGG?GAA?TTC?ATG?GGA?CAA?GTG?TTT?CTG-3′。

The described GS115/pAOsGH bacterial classification that has transformed linearizing pAOnsGHDNA, its expression culture condition is: in improved culture medium, under 28-30 ℃, fully under the oxygen supply condition, cultivate this bacterium cell; As somatic cells OD ₆₀₀When value is 2-3, add the improvement nutrient solution to OD ₆₀₀Value is 1.0, and keeps methanol concentration 0.5%, cultivates thalline 24 hours, induces the effect of AOX I promotor, under the inducing action of methyl alcohol, starts the sGH expression of gene effectively and cultivates.

The described GS115/pAOnsGH bacterial classification that has transformed linearizing pAOnsGHDNA, it expresses the improved culture medium of cultivation and the improved culture medium of expansion bacterium cultivation all can adopt following prescription: weight ratio, glucose: 15-20 part, ammonium sulfate: 4-5 part, potassiumphosphate: 0.5-1 part, sodium-chlor: 0.1-0.2 part, ferric sulfate: 100-200PPM, zinc sulfate: 200-400PPM, vitamin H: 2-5PPM, 1000 parts of nutrient solutions of water constant volume.

Described GS115/pAOnsGH bacterial classification, the technology that expands the bacterium cultivation are to adopt the conventional bacterium culture scheme that expands, and suitably adjust culture temperature at 25-30 ℃, cultivate the OD of bacterium liquid ₆₀₀Value progressively increases to 20 by 4, and constant volume 300L improves nutrient solution in the pilot scale fermentation jar again, and stream adds methanol solution, and keeps methyl alcohol final concentration 0.5%, feeds filtrated air, stirs 12 hours, expands bacterium and is cultured to OD ₆₀₀Value=10-50, to having expressed the GS115/pAOnsGH saccharomycetes to make fermentation nutrient solution of sGH gene, adopt two kinds of treatment processs:

(1) a kind of is directly with the centrifugation of bacterium liquid, gets the freezing preservation of bright wet thallus;

(2) another kind is that bacterium liquid is handled through low temperature spray drying, makes to have active dried yeast powder; These two kinds of GS115/pAOsGH engineering bacteria goods all can be used as the fodder additives of aquatic animal and poultry animal.

Engineering bacteria of the present invention---BL21/pETsGH, culture presevation number: CGMCC No.0362-2, preservation date: 1998.9.29.In building process,, so just can isolate the sGH gene like clockwork because the B-GH1 and a pair of primer of B-GH2 of design are designed according to sGH cDNA two terminal sequences.These two ends to primer will be to add BamHI and Nco I restriction enzyme site respectively, so that insert between the BamHI and NcoI enzyme site of plasmid pET15b.This all is only to the recombinant plasmid pETsGH that separates sGH gene and structure cyclic DNA.Owing to the plasmid pET15b that has selected to buy from Novagen company, it has phage t7 rna polymerase promoter (being called for short the laeUV5 promotor).This promotor can start the band BamHI of this plasmid pET15b access effectively under the inducing action of IPTG and the sGHcDNA fragment of Nco I sticky end can successfully be expressed the sGH gene.Therefore plasmid pET15b is a sGH recombinant plasmid carrier preferably.Because selected to buy from U.S. Novagen company, (E.coli) the BL21 bacterial classification is as experiencing bacterium.This bacterium has the favourable condition of pET15b and recombinant plasmid pETsGH growth and amplification, and recombinant plasmid pETsGH can increase at a high speed in this bacterium competence cell, expresses external source sGH gene effectively.Because the expression plasmid pET15b that selects is an expression type in the born of the same parents, the sGH that expresses in the BL21 cell is present in the cell with the form of inclusion body, therefore the present invention has designed a kind of extraction separation step of simplification, has obtained highly active sGH crude extract with lower extraction cost.This is wherein especially: owing to behind centrifugation dissolved sGH expression product, adopt the NH of 30mM ₄HCO ₃The renaturation buffer renaturation of dialysing makes the sGH product albumen matter renaturation of expression, thereby has improved the high reactivity of the crude extract sGH goods of this project bacterium.The pale yellow powder of this engineering bacteria BL21/pETsGH is as soaking agent the time, and at first the seedling with the aquaculture kind carries out the high salinity immersion treatment; Adopt the seedling after height oozes processing, be transferred to and then in the soak solution, under the aeration condition in the soak solution that contains this sGH soaking agent immersion treatment 30-40 branch, its infiltration that comes down to seedling is carried out high salinity is handled, the sGH active substance is infiltrated in the seedling body, reach somatotrophic purpose.This engineering bacteria goods prove through aquaculture repeatedly, have improved the surviving rate and the speed of growth of culturing fish, shrimp significantly, improve the unit of water body cultured output more than 20%.The income of volume increase is greater than using more than ten times of this engineering bacteria goods input.The effect of this engineering bacteria goods has: (1) can strengthen the feeding competitive capacity of the physique of the cultivated animals young.(2) can promote cultivated animals metabolism and food digestion receptivity, improve efficiency of feed utilization.(3) can promote carbohydrate katabolism and proteinic anabolism, improve liver and muscle cell anabolism amino acid absorption and nucleic acid.(4) can promote cultivated animals muscle tissue, cartilaginous tissue and skeletal growth.(5) leukocytic phagocytic activity be can improve, cultivated animals body's immunological function and resistance against diseases strengthened.

Another kind of engineering bacteria of the present invention---GS115/pAOsGH culture presevation number: CGMCC No.0362-2, preservation date: 1998.9.29.In building process, because the P-GH1 and the P-GH2 of design, this is designed according to sGHcDNA two terminal sequences to primer, so just can isolate the sGH gene like clockwork.These two ends to primer add the EcoRI restriction enzyme site respectively, so that insert in the EcoRI site of plasmid pA0815, this all is only for separating required sGH with the segmental recombinant plasmid PAOsGH of sGHcDNA that the required two ends of structure have the EcoRI sticky end.Because selected the plasmid p A0815 that buys from the Invitrogen company of the U.S., it has alcohol oxidase promoter, and can under the inducing action of methyl alcohol, start the sGH gene that this plasmid pA0815 connected effectively and successfully express.Therefore plasmid pA0815 is a yeast sGH recombinant plasmid carrier preferably.Owing to selected GS115 bacterial classification that the Invitrogen company of the U.S. buys as the acceptor bacterial classification.Have AOXI gene and His4 gene in the karyomit(e) of this bacterium.Can carry out homologous recombination with plasmid pA0815, can effectively recombinant plasmid pETnsGH be transformed and integrate in the cell chromosome that inserts the GS115 bacterial classification, make foreign gene sGH normal expression.After this Yeast engineering bacteria GS115/pAOnsGH makes up,, in cultivating, sGH expression of gene cultivation neutralization expansion bacterium all can use owing to improved culture medium prescription.The main raw material that needs in former this saccharomycetic culture medium prescription is: YNB (yeast nitrogen base) costs an arm and a leg, and every 500g valency is about 2000 yuan of Renminbi.The raw material cost of former substratum; 1 liter of bacterium liquid of every cultivation expense is about 56 yuan/1 liter bacterium liquid.The new culture medium prescription of improvement can make expense of raw materials be reduced to 0.25 yuan/1 liter bacterium liquid, is 1/224 of original formulation.Though new improved culture medium prescription saves the YNB raw material, the growing state of this Yeast engineering bacteria and fermentation production rate all keep previous level, even also are higher than previous level, expand bacterium and cultivate the OD that is reached ₆₀₀Value is 10-50, and its effect is quite obvious.Because selected expression plasmid pA0815 is an expression type in the born of the same parents, therefore the sGH product of expressing in GS115/pAOsGH engineering bacteria cell also all is present in the thalline.The present invention adopts two kinds of thalline treatment processs to obtain the feed of engineering bacteria goods as cultivated animals, can be absorbed and then produce promotes growth and disease-resistant effect in the hindgut of cultivated animals.In a word, the yeast expressed recombinant plasmid pAOsGH of multi-copy gene of the present invention has very high expression activity through repeatedly experimental results show that; Designed experiment condition and technological condition for fermentation make this engineering bacteria goods reach the level that production cost is low, efficient is high, through the repeatedly aquaculture test and the evidence of raising chickens, the feed that use to add this engineering bacteria goods animal of culturing of feeding can be improved its speed of growth and disease resistance ability significantly, improve the unit cultured output more than 20%, the income of volume increase is greater than using more than five times of these goods input.

Embodiments of the invention further describe as follows in conjunction with the accompanying drawings: protection scope of the present invention is not limited only among the embodiment.

Fig. 1 is a recombinant plasmid pETsGH building process synoptic diagram.

Fig. 2 is the recombinant expression pAOsGH building process synoptic diagram of multi-copy gene.

Fig. 3 is recombinant fish growth factor expression product electrophoretic analysis figure.

Fig. 4 is a recombinant fish growth factor expression product immuning hybridization analysis chart.

Among Fig. 1, N is expressed as: the restriction enzyme site of NcoI, Ba is expressed as: the BamHI restriction enzyme site.Among Fig. 2, E is expressed as: the EcoRI restriction enzyme site, Bg is expressed as: the BgLI restriction enzyme site, Ba is expressed as: the BamHI restriction enzyme site.Fig. 3,1,2 are among Fig. 4: sGHDNA expression product in the intestinal bacteria; 3 are: contrast sGH; 4-10 is: sGHDNA expression product in the yeast.

Referring to Fig. 1 by pair of primers P-GH1 and the P-GH2 of the sequences Design at two ends, sGH cDNA code area and contain the plasmid psGH4 of sGH cDNA, through the synthetic PCR product with BamHI and NcoI restriction enzyme site of pcr amplification, this product gets through BamHI and the cutting of NcoI enzyme again: with the sGH cDNA fragment of BamHI and NcoI cohesive end; Select plasmid pET15b to be cut into equally band BamHI and the NcoI restriction enzyme site linearization plasmid pET15bDNA fragment of 5.6kb with aforementioned pair of enzyme enzyme; With above-mentioned sGH cDNA fragment and pET15bDNA fragment, under the effect of T4 ligase, carry out the recombinant plasmid pETsGH that recombination to construct has cyclic DNA at last.

, cut through pcr amplification reaction and enzyme by other pair of primers P-GH1 and the P-GH2 of the sequences Design at two ends, sGH cDNA code area and contain the plasmid psGH4 of sGH cDNA referring to Fig. 2, make two ends with the sGH cDNA fragment of EcoRI cohesive end; Choose plasmid pA0815, also be cut into linearizing carrier DNA fragment with the EcoRI enzyme; Again with above-mentioned with the sGH cDNA fragment of EcoRI cohesive end and the carrier DNA fragment of plasmid pA0815, under the effect of T4 ligase, be built into recombinant plasmid pAOsGH; Use other two kinds of enzyme: BgLII and this recombinant plasmid of BamHI double digestion pAOsGH, separation makes again: the pAOsGHcDNA fragment of 1.92kb is in turn connected into aforementioned pAOsGHcDNA fragment the two compound expression pA02sGHcDNA sections of holding Bei Jiyin again; The linearizing recombinant plasmid pAOsGHcDNA fragment of also cutting with the BamHI enzyme and purify; The most aforementioned two compound expression section and the linearizing recombinant plasmid pAOsGHcDNA fragments of holding Bei Jiyin under the effect of T4 ligase, connect the recombinant expression pA02sGH that makes up the two copies gene.

Embodiments of the invention are as follows:

(1) transformed step and the condition example that the expansion bacterium of the BL21/pETsGH engineering bacteria of pETsGH plasmid is cultivated.

(1) inoculation contains the single bacterium colony of BL21 of pETsGH in the 5mlLB nutrient solution;

(2) 37 ℃, about 16 hours of 250rpm shaking culture is to OD ₆₀₀=2;

(3) in 1 liter of triangular flask, expanded bacterium and cultivate sub-100mILB nutrient solution with 1: 20;

(4) 37 ℃, about 8 hours of 250rpm shaking culture is to OD ₆₀₀=2;

(5) in 5 liters of Bio-FI03000 type bio-reactors, expanded the bacterium ratio and be incubated at 4 liters respectively with 1: 50,4.5 liters, 5 liters, in the LB nutrient solution;

(6) 37 ℃, 400rpm stirs and cultivated respectively about 6 hours, to OD ₆₀₀=2-2.1;

(7) add IPTG to inducing final concentration 1mM, continued cultivation and abduction delivering 2 hours, to OD ₆₀₀=3.

Wherein the composition of LB substratum (liquid) is referring to " molecular cloning guide " 1992 p908 of second edition Science Press.

(2) the BL21/pETsGH engineering bacteria expands the thalline processing of bacterium cultivation back and extracts active sGH gene product examples of articles as follows:

(1) expands the bacteria culture fluid from above-mentioned three batches of BL21/pETsGH engineering bacterias, distinguish centrifugal 10 minutes, collect thalline and get: 57g, 57g, the wet thallus of 60g with 7000rpm;

(2) add PBS phosphoric acid buffer: 240ml, 200ml, 250ml respectively;

(3) above-mentioned solution was handled 6,6,7 minutes through ultrasonic disruption respectively, through the 1300rpm centrifugal treating, collecting precipitation gets 200,200 again, the 250ml inclusion body;

(4) the above-mentioned inclusion body of the plain dissolving of usefulness 6M urea is distinguished centrifugal 10 minutes with 7000rpm after 1 hour again, gets supernatant liquor;

(5) with the NH of above-mentioned supernatant liquor with 30mM ₄HCO ₃Damping fluid is dialysed respectively;

(6) get above-mentioned dialyzate and carry out lyophilize respectively, get 1.50g respectively, 1.45g, the faint yellow powder BL21/pETsGH engineering bacteria goods of 1.60g dry weight record these goods and contain sGH26-30%.

(3) BL21/pETsGH engineering bacteria goods are applied to the culture experiment example:

(A) height of seedling oozes processing: (weight unit: table 1 gram):

	Sodium-chlor	Repone K	Calcium chloride	Sal epsom	Yellow soda ash
						????(1)	????450	????5	????5	????2	????5
????(2)	????480	????10	????3	????4	????10
						????(3)	????460	????8	????4	????3	????8

With (1) in the table 1-(3) prescriptions, be dissolved in 20l and culture in the water, with seedling immerse in the high sepage normal temperature height ooze handle 3-5 minute after, be transferred to rapidly in the soak solution, under ventilation, soaked 30-40 minute; (B) the soak solution example of seedling: (weight unit: table 2 milligram):

	Somatomedin sGH	Paraxin	Ampicillin	Terramycin
					????(1)	????50	????80	????100	????50
????(2)	????60	????80	????120	????70
					????(3)	????70	????100	????100	????60

The seedling that above-mentioned soak solution is handled is transferred to culture experiment in the cultivating pool: experimental result is as follows, table 3:

(1) from 94.6	Culture experiment	After 4 months	Carp output increases by 28%/mu than control group
				(2) from 96.4	Culture experiment	After 4 months	Shrimp seedling output increases by 65%/mu surviving rate than control group and improves 30%
(3) from 97.4	Culture experiment	After 4 months	Shrimp seedling output increases by 72%/mu surviving rate than control group and improves 35%

(4) transformed the GS115/pAOsGH engineering bacteria application example table 4 of linear pAOsGH DNA: improved culture medium is used for the GS115/pAOsGH engineering bacteria and expands bacterium and cultivate example:

	Glucose	Ammonium sulfate	Potassiumphosphate	Sodium-chlor	Ferric sulfate	Zinc sulfate	Vitamin H	Culture temperature ℃	OD 600Value	The temperature biomass
											?4	??15kg	??4kg	??1kg	?200g	??100mg	??200mg	??2ppM	????25	????10.5	??9.3kg
?5	??20kg	??5kg	??0.5kg	??100g	??200mg	??300mg	??5ppM	????28	????12.2	??11.5kg
											?6	??18kg	??5kg	??1kg	??200g	??200mg	??400mg	??3ppM	????30	????14.5	??13.2kg

Standby by above formulated 1000kg nutrient solution.Respectively in 97 years 5,6,7 three months, with 700 liters of fermentor tank constant volume → 300 liter fermented liquid inoculation GS115/pAOsGH engineering bacteria, carry out fermenting experiment, expand bacterium through routine and cultivate, by the processing condition of above-mentioned three examples, stream adds methanol solution, ventilate and stir fermentation after 12 hours, the sGH that thalline is expressed can reach the sGH100mg/l fermented liquid, the wet thallus of above-mentioned three examples, freezing preservation.

Above-mentioned Yeast engineering bacteria wet thallus is applied to the fish farming experiment.Experimental result such as following table 5:

(1) from June, 97	Culture experiment	After one month	Paralichthys olivaceus output increases by 12% than control group
				(2) from July, 97	Culture experiment	After one month	Paralichthys olivaceus output increases by 10% than control group
(3) from August, 97	Culture experiment	After five months	Perch output is than control group volume increase 28.3%

The detection of the sGH product of (five) two kinds of engineering bacterium expression:

The detection method basically identical of two kinds of expression products, step is as follows:

(1) centrifugation thalline;

(2) broken thalline, the isolated protein crude extract;

(3) with SDS-polyacrylamide gel electrophoresis analysing protein samples;

(4) protein belt is carried out the bright blue dyeing of Maas, the result as shown in Figure 3;

(5) the sGH antibody of use purifying carries out western blotting-immune hybrid method (Westerm-Immunoblot) analysis, and the result as shown in Figure 4.

Claims (10)

1, a kind of engineering bacteria that contains the chinook somatomedin, employed chinook somatomedin, sGH cDNA gene order wherein is to make up chinook pituitary gland cDNA gene library by people such as Dr.Choy L.Hew, separate its sGH cDNA gene and made up plasmid psGH4, it is characterized in that: the sGH cDNA gene order in the described engineering bacteria is obtained through pcr amplification by plasmid psGH4, and its basic step is as follows:

(1) according to the sequences Design at two ends, sGHcDNA coding region a pair of primer B-GH1 and B-GH2, synthesized the PCR reaction product that has BamHI and NcoI restriction enzyme site through pcr amplification, after cutting through these two kinds of enzyme enzymes again, obtained the sGH cDNA fragment of band BamHI and NcoI sticky end;

(2) select plasmid pET15b for use, with BamHI and NcoI double digestion, the linearization plasmid pET15bDNA fragment of separating 5.6kb;

(3) with the dna fragmentation of the plasmid pET15b in the sGH cDNA fragment in above-mentioned (1) and (2) under the effect of T4 ligase enzyme, carry out the recombinant plasmid pETsGH that recombination to construct becomes to have cyclic DNA;

(4) select intestinal bacteria (E.coli) BL21 bacterial classification for use, the competent cell of preparation BL21 bacterium;

(5), under the 1.5min heat-shocked condition, the cyclic DNA of pETsGH is converted in the BL21 competent cell at 42 ℃;

(6) cultivate at the LB solid medium upper berth ware that contains ampicillin, screen transformant, promptly obtained transforming the engineering strain of pETsGH plasmid, called after BL21/pETsGH,

Its culture presevation number: CGMCC No.0362-2, preservation date: 1998.9.29.

2, according to the described engineering bacteria that contains the chinook somatomedin of claim 1, it is characterized in that: described primer B-GH1, its nucleotides sequence is classified as:

5′-CAC?ACC?ATG?GGC?ATA?GAA?AAC?CAA?CGG?CTC-3′，

Described primer B-GH2, its nucleotides sequence is classified as:

5′-CAC?AGG?ATC?CTA?CAG?AGT?GCA?GTT?GGC?CTC-3′。

3, the engineering bacteria that contains the chinook somatomedin according to claim 1 is characterized in that: the expression culture condition of this bacterial classification is; In the LB substratum, 37 ℃, fully oxygen supply is cultivated down; As somatic cells OD ₆₀₀When value reached 0.6-0.8, adding IPTG was 0.05-1mM to final concentration, and continuation was cultivated 2-3 hour at 37 ℃, induced the effect of laeUV5 promotor, carried out the sGH expression of gene.

4, it is characterized in that according to claim 1 or the 3 described engineering bacterias that contain the chinook somatomedin: the described BL21/pETsGH bacterial classification that transforms the pETsGH plasmid, expand bacterium through routine and cultivate, obtain OD ₆₀₀Value is 3 fermentation culture, and the processing of its thalline and the step of extracting highly active sGH gene renaturation expression product are:

(1) centrifugal collection thalline from the BL21/pETsGH bacteria culture fluid;

(2) pH7.5 of 3 times of volumes of adding, the PBS phosphoric acid buffer;

(3) above-mentioned solution is handled through ultrasonic disruption: 2-10 minute, again through 13000r.p.m, 2 minutes were centrifugal, collecting precipitation, the inclusion body of sGH;

(4) with 6M urea element, dissolve above-mentioned inclusion body;

(5) with the NH of above-mentioned solution with 30mM ₄HCO ₃Damping fluid is dialysed;

(6) get above-mentioned dialyzate and carry out lyophilize, can contain the off-white powder shape BL21/pETsGH engineering bacteria goods of the long factor of 25-30% sashimi (raw fish).

5, the engineering bacteria that contains the chinook somatomedin according to claim 1, it is characterized in that: described BL21/pETsGH engineering bacteria goods, its using method is to make soak solution by following prescription: weight ratio is somatomedin sGH:50-70 part; Paraxin: 80-100 part; Acillin: 100-120 part; Terramycin: 50-70 part;

With above-mentioned component, be dissolved in the 20L breeding water, make soak solution;

During use, earlier aquaculture kind fish, shrimp, shellfish, crab class seedling are immersed 2.5% high salinity treatment solution, soaked 3-5 minute; This high salinity prescription for the treatment of liquid is as follows:

Weight ratio: sodium-chlor: 450-480 part; Repone K: 5-10 part; Calcium chloride: 3-5 part; Sal epsom: 2-4 part; Yellow soda ash: 5-10 part; Be dissolved in 20L and culture in the water, normal temperature uses down; Seedling is taken out from above-mentioned high salinity treatment solution again, be transferred to rapidly in the soak solution, ventilation is soaked after 30-40 minute down, transfers in the cultivating pool and cultures.

6, another kind contains the engineering bacteria of chinook somatomedin, and sGHcDNA gene order wherein also is to obtain through pcr amplification by the plasmid psGH4 of the described structure of claim 1, it is characterized in that: its basic step is,

(1) according to two ends, sGH cDNA coding region sequences Design another to primer P-GH1 and P-GH2, cut through pcr amplification and EcoRI enzyme, obtained the sGH cDNA fragment that two ends have the EcoRI sticky end;

(2) select the plasmid pA0815 that has AXO1 gene and His4 gene for use, also it is cut into linearizing carrier DNA fragment with the EcoRI enzyme;

(3), under the effect of T4 ligase enzyme, connect and be built into recombinant plasmid pAOsGH with the carrier DNA fragment of the plasmid pA0815 in the sGH cDNA fragment in above-mentioned (1) and (2);

(4) with BgLII and this recombinant plasmid of BamH I double digestion pAOsGH, separate the pAOsGHDNA fragment of 1.92Kb, connect into the compound expression section of multi-copy gene again;

(5) the linearizing recombinant plasmid pAOsGHcDNA fragment of cutting with the BamHI enzyme and purify;

(6) with the linearizing recombinant plasmid pAOsGHcDNA fragment in the compound expression section of the multi-copy gene in above-mentioned (4) and (5) under the effect of T4 ligase enzyme, connect the recombinant expression pAOnsGH that is built into multi-copy gene;

(7) select yeast (pichia pastoris) GS115 bacterial classification for use, have AOXI gene and His4 gene in the karyomit(e) of this bacterial classification;

(8) cut the His4 gene regions of the recombinant plasmid pAOnsGH in above-mentioned (6) with Stu I enzyme, make the dna fragmentation of linearizing pAOnsGH;

(9) with the His4 gene regions of BIO-RADGene PulserII electric impulser with the endocellular chromosome of linearizing pAOnsGHDNA integration importing GS115;

(10) the yeast GS115 bacterial classification after will transforming is laid on 30 ℃ of cultivations in the plate of MD substratum, screens this transformant, promptly gets the yeast engineering bacterial classification that contains exogenous growth factors sGH gene, called after GS115/pAOnsGH; Its culture presevation number: CGMCC No.0362-1, preservation date: 1998.9.29.

7, according to the described engineering bacteria that contains the chinook somatomedin of claim 6, it is characterized in that: described its nucleotide sequence of primer P-GH1 is as follows:

5′-CCC?GGG?GAA?TTC?CTA?CAG?AGT?GCA?GTT?GGC-3′

Described its nucleotide sequence of primer P-GH2 is as follows:

5′-CCC?GGG?GAA?TTC?ATG?GGA?CAA?GTG?TTT?CTG-3′。

8, according to the described engineering bacteria that contains the chinook somatomedin of claim 6, it is characterized in that: the described GS115/pAOsGH bacterial classification that has transformed linearizing pAOnsGHDNA, it expresses culture condition: in improved culture medium, under 28-30 ℃, fully under the oxygen supply condition, cultivate this bacterium cell; As somatic cells OD ₆₀₀When value is 2-3, add the improvement nutrient solution to OD ₆₀₀Value is 1.0, and keeps methanol concentration 0.5%, cultivates thalline 24 hours, induces the effect of AOX I promotor, under the inducing action of methyl alcohol, starts the sGH expression of gene effectively and cultivates.

9, according to claim 6 or the 8 described engineering bacterias that contain the chinook somatomedin, it is characterized in that: the described GS115/pAOnsGH bacterial classification that has transformed linearizing pAOnsGHDNA, it expresses the improved culture medium of cultivation and the improved culture medium of expansion bacterium cultivation all can adopt following prescription: weight ratio, glucose: 15-20 part, ammonium sulfate: 4-5 part, potassiumphosphate: 0.5-1 part, sodium-chlor: 0.1-0.2 part, ferric sulfate: 100-200PPM, zinc sulfate: 200-400PPM, vitamin H: 2-5PPM, 1000 parts of nutrient solutions of water constant volume.

10, according to the described engineering bacteria that contains the chinook somatomedin of claim 6, it is characterized in that: described GS115/pAOnsGH bacterial classification, the technology that expands the bacterium cultivation is to adopt the conventional bacterium culture scheme that expands, and suitably adjusts culture temperature at 25-30 ℃, cultivates the OD of bacterium liquid ₆₀₀Value progressively increases to 20 by 4, and constant volume 300L improves nutrient solution in the pilot scale fermentation jar again, and stream adds methanol solution, and keeps methyl alcohol final concentration 0.5%, feeds filtrated air, stirs 12 hours, expands bacterium and is cultured to OD ₆₀₀Value=10-50, to having expressed the GS115/pAOnsGH saccharomycetes to make fermentation nutrient solution of sGH gene, adopt two kinds of treatment processs:

(1) a kind of is directly with the centrifugation of bacterium liquid, gets the freezing preservation of bright wet thallus;

(2) another kind is that bacterium liquid is handled through low temperature spray drying, makes to have active dried yeast powder; These two kinds of GS115/pAOsGH engineering bacteria goods all can be used as the fodder additives of aquatic animal and poultry animal.

Images