CN100537749C - Express the reorganization bacterium of enterotoxin colibacillus adhesin gene and the application in Yolk antibody feed thereof - Google Patents

Abstract

The invention belongs to the Animal Nutrition and Feed Science technical field, relate to the application of animal Protocols in Molecular Biology simultaneously.Particularly, the present invention relates to a kind of structure and application in the preparation Yolk antibody feed thereof of expressing the recombination bacillus coli of chitling toxin intestinal bacteria K88 and F18 adhesin gene faeG and fedF.The intestinal bacteria of this expression enterotoxic Escherichia coli K88ac adhesin gene faeG and F18ac adhesin gene fedF recombinant plasmid are to utilize connection peptides to make up faeG and fedF fusion gene, and structure among this fusion gene insertion prokaryotic expression carrier pET28a is formed.Recombination bacillus coli BL21 of the present invention (DE3)/pET-8818 is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:M207090.The invention also discloses preserving number is the application of recombination bacillus coli in the preparation Yolk antibody feed of CCTCC NO:M207090.

Description

Express the reorganization bacterium of enterotoxin colibacillus adhesin gene and the application in Yolk antibody feed thereof

Technical field

Animal nutrition of the present invention and forage science technical field relate to the molecular biological application of animal simultaneously.Particularly, the present invention relates to a kind of reorganization bacterium and application in the preparation Yolk antibody feed thereof of expressing chitling toxin intestinal bacteria K88 and F18 adhesin gene faeG and fedF.

Background technology

In large scale of pig farm was produced, enterotoxic Escherichia coli (being called for short ETEC) was to cause newborn piglet and the main paathogenic factor of diarrhea of weaned piglets.K88 causes (the .Rapid and specificdifferentiation of enterotoxin-producing Escherichia coli strains from other gram-negative enteric bacteriausing multiplex PCR.Berl Munch Tierarztl Wochenschr such as OsekJ of topmost a kind of pili genotype among the diarrhea of weaned piglets ETEC, 2000,113:265-70); Wherein common with the K88ac hypotype.In Denmark, (.Detection of fimbrial and toxin genes in and their Prevalence inpiglets with diarrhea.The applfication of colony hybridization assay such as Ojeniyi B such as Ojeniyi, polymerase chain reaction andphenotype assays.J Vet Med B, 1994,41:49-59); In Germany, (.Prevalence of thefimbrial antigens F18 and K88 and of enterotoxins and verotoxins among Escherichia coli isolated fromweaned pigs.Zentralbl Bakteriol such as Wittig W such as Wittig, 1995,283:95-104); Domestic one-tenth Daiei (become Daiei. the molecular epidemiology of weanling pig people from source enterobacteria virulence factor and F18 pili part The Characteristic Study .[doctorate paper]. Yangzhou: Yangzhou University Library, 2005) investigation shows, F18 is another the main pathogenic colon bacillus that causes weanling pig edema disease and diarrhoea, the 2134P relevant with causing diarrhea of weaned piglets ETEC, 8813,8199 grades belong to F18ac (.Expression and plasmid transfer ofgenes coding for the fimbrial antigen F107 in porcine Escherichia coli strains.Zentralbl Bakteriol such as Wittig W mostly, 1994,281:130-139).

With the K88 or the F18 pili immune hen of purifying, preparation is at the yolk antibody of ETEC K88 or F18, externally can block K88 or F18 bacterial strain sticking to the piglet intestinal cells, make an addition in the feed, can effectively reduce the grice diarrhoea rate, and improve growth in piglets performance (.Passive protective effect of chicken egg yolk immunoglobulinsagainst experimental enterotoxigenic Escherichia coli infection in neonatal piglets.Infect Immun such as Yokoyama H to a certain extent, 1992,60:998-1007; .Chicken egg yolk antibodies against F18ab Fimbriae of Escherichia coliinhibit shedding of F18 Positive E.coli by experimentally infected pigs.Vet Micro such as Imberechts H, 1997,54:329-341; .Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichiacoli K88 such as Marquardt R R ⁺Infection in neonatal and early-weaned piglets.FEMS Immunol Med Microbiol, 1999,23:283-288).Yet; the main method of directly separating purification from the ETEC bacterial strain that adopts prepares pilin antigen in the above-mentioned data; because the pili expression amount of general ETEC is very limited; and the faint even expression of some pili vivoexpressions; thereby it is few by the pili antigen amount of separating the method acquisition of purifying; the cost height is not suitable for the large-scale production of yolk antibody.

The primary structure albumen of faeG genes encoding K88 pili and adhesin (.The nucleotide sequence of thegene encoding the K88ab protein subunit of porcine enterotoxigenic Escherichia coli.FEMS MicrobiolLett such as Gaastra W, 1981,12:41-46).This albumen not only contains a large amount of K88 pili antigen determinants (.The nucleotidesequence of the K88ad protein subunit of porcine enterotoxigenic Escherichia coli.FEMS Microbiol Lett.1983 such as GaastraW, 18:177-183), and be main binding site (the .Characterization of the antigenicand adhesive properties of FaeG such as Bakker D of K88 acceptor, the major subunit of K88 fimbriae.Mol Microbiol, 1992,6:247-255).The fedF encoded protein is not only the synthetic necessary subunit of F18 pili, still produce major protein (the .Mapping the binding domain of the F18 Fimbrial adhesin.Infect Immune such as Smeds A of the effect of sticking, 2003,71:2163-2172).The albumen of faeG and fedF genes encoding all has antigenicity preferably. and be the preferred object gene of design ETEC subunit vaccine.

ETEC K88 adhesin gene faeG recombinated to contain the expression vector of T7 phage strong promoter, and transform in the recipient bacterium BL21 (DE3) that can make the expressing protein stable existence, can obtain the recombinant bacterial strain of stability and high efficiency expressing K 88ac pili subunit protein, its FaeG expression amount accounts for 30% of bacterial protein, and has better immunogenicity (.K88 pilin subunit gene clone, expression and recombinant protein Antiserum Preparation such as master Zhao river.The Shanghai Agricultural journal, 2000,16:38-41).(.Characterization of the adhesin of Escherichia coli F18 Fimbriae.Infect Immune such as Smeds A such as Smeds, 2001,69:7941-7945) specificity analysis to intestinal bacteria F18 pili pili shows that anti-FedF antibody can suppress intestinal bacteria sticking the chitling cell; And FedF-MBP (maltose binding protein) fusion rotein can stick with chitling cell generation specificity (adult is flourish. the molecular epidemiology of weanling pig source intestinal bacteria virulence factor and F18 pili part The Characteristic Study.[doctorate paper].Yangzhou: Yangzhou University Library, 2005).

Based on above discovery, we utilize the fusion gene of round pcr amplification faeG and fedF, and the recombinant bacterial strain of this fusion gene of construction expression, and prepare the heavier histone of mode and the immunogenicity of native protein and the yolk antibody effect of preparation thereof of yolk antibody with immune laying hen.

Summary of the invention

First purpose of the present invention is construction expression enterotoxic Escherichia coli K88ac adhesin gene faeG and the dual-gene recombinant bacterial strain of F18ac adhesin gene fedF, obtains the lower two valency recombinant pilin antigens of the better production cost of a kind of immunogenicity

Second purpose of the present invention is the above-mentioned application of two valency recombinant pilin antigens in the anti-ETEC yolk antibody of preparation, and application wherein mainly is the application in the preparation Yolk antibody feed.

The present invention implements by the following technical programs:

A kind of expressing K 88ac and the F18ac pair of antigenic recombinant bacterial strains of valency are that the faeG of amplification acquisition and SacI and the HindIII site structure of fedF fusion gene insertion prokaryotic expression carrier pET28a are formed, intestinal bacteria Escherichia coliBL21 (the DE3)/pET-8818 that contains this plasmid, submit Chinese typical culture collection center (CCTCC) preservation on June 28th, 2007, preserving number is CCTCC NO:M207090.

Described recombinant plasmid, it is to utilize connection peptides to make up faeG and fedF fusion gene, and this fusion gene inserted makes up among prokaryotic expression carrier pET28a.This recombinant plasmid is pET-8818.

Two valency recombinant pilins of a kind of K88ac adhesin gene faeG and F18ac adhesin gene fedF are to be the expressed product of intestinal bacteria of CCTCC NO:M207090 by preserving number.

Technical scheme of the present invention following (the detailed technology step is referring to " embodiment " part):

In the following technological step without the method that specifies all with reference to J. Sa nurse Brooker (J. Sa nurse Brooker, E.E. is the Ritchie not, T. Manny A Disi work, Jin Dongyan etc. translate.The molecular cloning experiment guide.Beijing: Science Press, version in 1989).

One, the structure of expressing K 88ac adhesin gene faeG and F18ac adhesin gene fedF gene recombination plasmid pET8818

Design has the primer of restriction enzyme digestion sites according to the faeG gene order of having reported among the Genebank (accession number M29375) and fedF gene order (accession number AY970782), wherein introduces connection peptides (Gly in the upstream primer design of the downstream primer of faeG and fedF ₄Ser) ₃Sequence.Genome with intestinal bacteria K88ac reference culture C83715 and intestinal bacteria F18ac bacterial strain 2134P is a template, and the method by pcr amplification makes up by (Gly ₄Ser) ₃The fusion gene that connects faeG and fedF, called after faeG-linker-fedF.

The pcr amplification product of fusion gene faeG-linker-fedF is inserted in the pMD-18T cloning vector, makes up the cloned plasmids that contains fusion gene faeG-linker-fedF, cut evaluation confirmation structure correctly, called after pMD-8818 through PCR and enzyme.After cutting pMD-8818 with SacI and HindIII enzyme respectively, the enzyme of purifying recovery is cut product to be connected with pET28a (+) the expression vector plasmid of cutting with same enzyme, obtain the prokaryotic expression plasmid of fusion gene, cut evaluation confirmation structure correctly, called after pET-8818 through PCR and enzyme.

Two, the abduction delivering of FaeG-FedF fusion rotein and extraction purifying

The recombinant plasmid pET-8818 that order-checking is correct, be converted in the e. coli bl21 (DE3), make up reorganization bacterium BL21 (DE3)/pET-8818, and (Isopropyl-D-1-thiogalactopyranoside IPTG) carries out abduction delivering to utilize isopropyl-.By the soluble analysis of BL21 (DE3)/pET-8818 expression product is found that BL21 (DE3)/pET8818 abduction delivering product exists with the inclusion body form, take the ultrasonic disruption method to extract inclusion body, by the sex change and renaturation purifying BL21 (the DE3)/pET-8818 expression product FaeG-FedF fusion rotein of inclusion body.SDS-PAGE detects the pilin purity after purifying, and the Bradford method is measured protein concn.

Three, the preparation technology of yolk antibody

With tween-80 to the final concentration that adds sterilization in the FaeG-FedF fusion rotein solution of purifying is 4%, behind the thorough mixing again with isopyknic white-oil adjuvant (94% white oil, 6% Si Ben-80,2% aluminum stearate) mixes, and fully emulsifiedly make the oily newborn seedling that protein concentration is 500 μ g/mL.Open ages in 20 weeks laying hen chest muscle both sides respectively multi-point injection 500 μ L concentration be the newborn seedling of FaeG-FedF fusion rotein oil of 500 μ g/mL, two week the back exempt from the back with quadrat method booster immunization twice, two and one week began to collect egg.Adopt indirect enzyme-linked immunosorbent assay (ELISA) monitoring yolk antibody to tire.

Four, the yolk antibody effect of FaeG-FedF fusion protein immunization preparation is estimated

By yolk antibody vitro inhibition ETEC K88ac and F18ac adherence test, the yolk antibody of checking fusion protein F aeG-FedF immunity preparation suppresses ETEC K88ac and F18ac adheres to the epithelial effect of chitling.Select 144 24 ± 3 age in days weanling pigs, be divided into 2 groups at random, every group each 72 by relative principle of correspondence such as blood relationship, weaning weight, sex and ages in days.The spraying drying height that gives the preparation of 1.5% common whole egg powder and 1.5% FaeG-FedF fusion protein immunization in the grain ration is respectively exempted from whole egg powder, carries out the weaning pig feeding test.Add 1.5% height after the off-test and exempt from whole egg powder and significantly reduced grice diarrhoea rate (P＜0.05) and diarrhoea index (P＜0.01), and the feed conversion rate (P＜0.05) of raising piglet.

Description of drawings

Fig. 1 has shown the recombinant plasmid pET-8818 design of graphics of expressing K 88ac and F18ac pilin fusion gene

Fig. 2 has shown that the enzyme of the recombinant plasmid pET-8818 of expressing K 88ac and F18ac pilin fusion gene cuts qualification result, among the figure: 1,2:pET-8818/NcoI+SacI; M1:DL2000 DNA Ladder; M2:DL15000 DNA Ladder

Fig. 3 has shown expressing K 88ac and F18ac pilin fusion gene reorganization bacterium BL21 (DE3)/pET-8818 abduction delivering result;

Among the figure: the BL21 after 1:IPTG induces (DE)/pET-8818:2:BL21 (DE)/pET-28a; M:Protein markerSM-0431 (MBI)

Fig. 4 has shown that with fusion protein F aeG-FedF be immunogen preparing yolk antibody process flow sheet

Fig. 5 has shown yolk antibody vitro inhibition ETEC (enterotoxic Escherichia coli) K88ac adherence test result, and among the figure: C is a control group; 1 is test group

Fig. 6 has shown yolk antibody vitro inhibition intestinal bacteria F18ab and F18ac adherence test result.Among the figure: C1 is the F18ac control group; C2 is the F18ab control group; 1 is the F18ac test group; 2 is the F18ab test group

Embodiment

In the following specific embodiment without the method that specifies all with reference to J. Sa nurse Brooker (J. Sa nurse Brooker, E.F. is the Ritchie not, T. Manny A Disi work, Jin Dongyan etc. translate.The molecular cloning experiment guide.Beijing: Science Press, 1989).

Embodiment 1: the structure of expressing K 88ac adhesin gene faeG and F18ac adhesin gene fedF gene recombination plasmid pET8818

1 material and method

1.1 bacterial strain and plasmid vector

Intestinal bacteria K88ac reference culture C83715 is available from China Veterinery Drug Inspection Office.Intestinal bacteria F18ac bacterial strain 2134P becomes the Daiei professor to be so kind as to give by veterinary hospital of Yangzhou University.

Bacillus coli DH 5 alpha, e. coli bl21 (DE3) and plasmid vector pET-28a are available from Novagen company.

Cloning vector pMD18-T is available from the precious biotech firm (Takara) in Dalian.

1.2 substratum

LB meat soup (LB Broth): Tryptones 10g, yeast extract 5g, NaCl 10g, distilled water is settled to 1000mL, regulates the pH value to pH7.0,121 ℃ of 1.05kg/cm ²High pressure steam sterilization 20min, room temperature preservation is standby.

LB nutrient agar medium (LB Nutrient Agar): Tryptones 10g, yeast extract 5g, NaCl 10g, distilled water is settled to 1000mL, regulates pH value to 7.0, and other adds the 15g agar powder, 121 ℃ of 1.05kg/cm ²High pressure steam sterilization.Pour plate, it is standby to treat to be inverted in after the culture medium solidifying 4 ℃ of preservations.If the preparation resistant panel when treating that then the substratum temperature is reduced to 50 ℃ of left and right sides, adds corresponding microbiotic (the concrete microbiotic that adds is determined by following implementation step) back pour plate and gets final product.

The trysinization soybean broth (Tryptone soya broth, TSB): pancreas casein peptone 17g, phytone 3g, NaCl 5g, K ₂HPO ₄2.5g glucose 2.5g is settled to 1000ml with distilled water, regulates pH value to 7.4,121 ℃ of 1.05kg/cm ²20-30min is standby in sterilization.

1.3 reagent

Taq archaeal dna polymerase, dNTPs, MgCl ₂, PCR Buffer and Protein Molecular Weight Marker be Fermentas company product.

DNA Ladder DL2000, DNA Ladder DL15000, restriction enzyme Nco I, Sac I, Sal I and HindIII, T4 dna ligase etc. are the precious biotechnology company limited (Takara) in Dalian product.

DNA reclaim test kit, penbritin (Amp), kantlex (Kan), isopropyl-(Isopropyl-D-1-thiogalactopyranoside, IPTG), Tryptones, yeast extract, agar powder etc. are Shanghai life worker biotechnology company limited product.

1.3 the structure of fusion gene faeG-linker-fedF

1.3.1 design of primers

According to faeG gene order (accession number M29375) the design primer faeG-U and the faeG-D that have reported among the Genebank, wherein upstream primer faeG-U introduces Sac I restriction enzyme site, and downstream primer faeG-D introduces connection peptides (Gly ₄Ser) ₃Sequence, amplification 834bp N end has (Gly ₄Ser) ₃The faeG gene, called after faeG-linker.According to fedF gene order (accession number AY970782) the design primer fedF-U and the fedF-D that have reported among the Genebank, wherein upstream primer fedF-U introduces connection peptides (Gly ₄Ser) ₃Sequence, downstream primer fedF-D introduces the HindIII restriction enzyme site, and amplification 879bp C end has (Gly ₄Ser) ₃The fedF gene, called after linker-fedF. is a template with the PCR recovery product of faeG-linker and linker-fedF, with faeG-linker upstream primer and linker-fedF downstream primer method two purpose segments are together in series, finally obtain 1668bp by (Gly by pcr amplification ₄Ser) ₃The fusion gene that connects faeG and fedf, called after faeG-linker-fedF.Above-mentioned primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.Primer sequence is following, and (underscore of primer is a restriction enzyme site, and the overstriking italicized item of English alphabet is connection peptides (Gly ₄Ser) ₃Sequence):

faeG-U：5’-TTT GAGCTCATGGCTGGTGATTTCAATGG-3’

faeG-D：5’-GCTGCCGCCACCGCCGCTTCCGCCACCGCCGCTTCCACCGCCACCGTAATAAGTTATTGCTACGTTCAG-3’

fedF-U：5’-GGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGAGCCAAGTAGACAAGTCTGTT-3’

fedF-G：5’-TTT AAGCTTTTACTGTATCTCGAAAACAATGGGCAC-3’

1.3.2 the preparation of K88ac and F18ac adhesin gene dna profiling

From on the picking list bacterium colony 37 ℃ of shaken overnight to be housed respectively in the bacterium bottle of 3mL TSB substratum the TSB flat board of K88ac reference culture C83715 and intestinal bacteria F18ac bacterial strain 2134P.Respectively draw 500 μ L respectively from the bacterium liquid of 3mL C83715 and 2134P in the sterilization centrifuge tube of 2 1.5mL, the centrifugal 30s of 12000r/min abandons most supernatant.Add 200 μ L ddH respectively ₂O, with micropipet piping and druming evenly, 100 ℃ of water-bath 5min.The centrifugal 5min of 12000r/min, getting supernatant is template.

1.3.3 the pcr amplification of fusion gene faeG-linker-fedF

Utilize faeG-U and faeG-D amplification faeG-linker segment, utilize fedF-U and fedF-D amplification linker-fedF segment, PCR recovery product with faeG-linker and linker-fedF is a template then, with faeG-linker upstream primer and the amplification of linker-fedF downstream primer.The pcr amplification condition is: enter circulation after 94 ℃ of 5min sex change, loop parameter is: 94 ℃ of 40s, and 58 ℃ of 45s, 72.0 ℃ of 1min, 35 circulations are extended 10min for back 72 ℃.Reaction finishes the back and carry out electrophoresis detection in 1.0% sepharose.

1.4 the structure of expressing K 88ac adhesin gene faeG and F18ac adhesin gene fedF gene recombination plasmid

According to route shown in Figure 1, construction expression K88ac adhesin gene faeG and F18ac adhesin gene fedF gene recombination plasmid pET8818.

Fusion gene faeG-linker-fedF dna segment is inserted among the cloning vector pMD18-T, make up and restrain a pMD-8818 that degrades, cloned plasmids pMD-8818 is carried out enzyme with restriction enzyme SacI and HindIII to be cut and reclaims enzyme and cut product, the pMD-8818 enzyme that reclaims purifying is cut product and carried out pET28a (+) the expression vector plasmid that enzyme cuts with same enzyme and be connected, structure contains the recombinant plasmid pET-8818 of fusion gene faeG-linker-fedF, and carries out enzyme and cut evaluation.Enzyme is cut the correct recombinant plasmid pET-8818 of evaluation deliver to the precious biotech firm in Dalian (Takara) order-checking.

2 implementation results

Recombinant plasmid pET-8818 enzyme is cut product behind 0.8% agarose gel electrophoresis, observes two bands (Fig. 2) about about 1700bp and 5200bp, shows that construction of recombinant plasmid is correct.The samely take two-way order-checking, sequencing result and target gene sequences are carried out the BLAST comparison on NCBI (http://www.ncbi.nlm.nih.gov/BLAST/), the result shows that it is 99% that the sequence of faeG part is returned source property, and the sequence homology of linker part and fedF part reaches 100%.

The abduction delivering of embodiment 2:FaeG-FedF fusion rotein and extraction purifying

1 materials and methods

1.1 reagent

Tryptones, yeast extract, agar powder, isopropyl-(Isopropyl-D-1-thiogalactopyranoside, IPTG), beta-mercaptoethanol, sarcosyl (being called for short SKL), Macrogol 4000 (being called for short PEG4000), Sleep-promoting factor B, reduced glutathion be that worker's biotechnology company limited product is given birth in Shanghai.

1.2 antibody

Mouse-anti HisTag antibody is Novagen company product, and the sheep anti-mouse igg of horseradish peroxidase-labeled is purchased white SBA company.

1.3 damping fluid

Buffer A:50mol/L Tris, 0.5molL EDTA, 50mol/L NaCl, 5% glycerine is regulated pH to pH8.0, adds the dithiothreitol (DTT) of 0.5mmol/L during use.

TE(pH8.0)：10mmol/L?Tris·Cl，1mmol/L?EDTA。

PBS (pH7.4): PBS: take by weighing 8g NaCl, 0.2g KCl, 1.44g Na ₂HPO ₄, 0.24g KH ₂PO ₄, be dissolved in 800mL ddH ₂Among the O, transfer pH to 7.2, be settled to 1000mL.

1.3 the abduction delivering of fusion protein F aeG-FedF and SDS-PAGE detect

Recombinant plasmid pET-8818 is converted in e. coli bl21 (DE3) recipient bacterium, and is inoculated in the bacterium bottle that 3mLLB (containing 30 μ g/mL Kan) substratum is housed, 37 ℃ of shaking culture 8h or spend the night with 1% amount.The culture of bacterium BL21 (DE3)/pET-8818 of will recombinating is inoculated into respectively in two bacterium bottles that 3mL LB (containing 30 μ g/mL Kan) substratum is housed with the amount of 1% volume again.One of them 37 ℃ of shaking culture 7h as not inductive contrast, begins to induce behind another 37 ℃ of shaking culture 3h, adds IPTG to final concentration 1mmol/L, continues 37 ℃ of shaking culture 4h.Respectively take out 500 μ L bacterium liquid after the end in new 1.5mL centrifuge tube, the centrifugal 30s of 12000r/min.Remove supernatant, add 50 μ L sample dissolution liquid, evenly with rifle piping and druming.100 ℃ of water-bath 5min, the centrifugal 5min of 12000r/min gets 10 μ L supernatants and carries out the SDS-PAGE detection.In addition, be one anti-with mouse-anti HisTag antibody, the sheep anti-mouse igg of horseradish peroxidase-labeled is two anti-, detects expression product with Westem blotting

1.4 expression product soluble analysis

Reorganization bacterium BL21 (the DE3)/pET-8818 culture of above-mentioned abduction delivering is inoculated in the bacterium bottle that 5mLLB (containing 30 μ g/mL Kan) substratum is housed with the amount of 1% volume, 37 ℃ of shaking culture 8h, 4 ℃ of placements are spent the night.Bacterium liquid is poured in the saline bottle that 50mLLB (containing 30 μ g/mL Kan) substratum is housed, at 37 ℃ of abduction deliverings.Bacterium liquid is changed in two 50mL centrifuge tubes, 4 ℃, the centrifugal 5min of 8000r/min.Abandon supernatant, thalline is resuspended with the Buffer A of 1/10 volume, 180w ultrasonication 8 times, 10s/ time at interval.4 ℃, the centrifugal 10min of 12000r/min changes supernatant in another new 50mL centrifuge tube over to, precipitates resuspended with isopyknic Buffer A.From last cleer and peaceful precipitation, respectively get 50 μ L and add isopyknic sample dissolution liquid, handle sample well and carry out the SDS-PAGE detection

1.5 the extraction purifying of fusion protein F aeG-FedF

The centrifugal 10min of reorganization bacterium BL21 (DE3)/pET-8818 culture 5000r/min of 200mL is collected thalline, thalline is resuspended among the Buffer A of 20mL again, and adds 0.05% N,O-Diacetylmuramidase, 37 ℃ of incubation 15min carry out ultrasonic disruption then.Ultrasound intensity is made as 180W, broken about 10 times of ice-bath ultrasonic, and each 20s-30s, 2min becomes limpid for good until solution at interval.The centrifugal 10min of mixed solution 12000r/min with after the fragmentation removes supernatant, PBS damping fluid (pH7.4) washing precipitation 3 times, collecting precipitation.

The precipitation of above-mentioned collection is resuspended among the Buffer A that contains 0.3% SKL, and room temperature leaves standstill 30min-2h behind the mixing that fully vibrates.The solution branch is filled in the dialysis tubing, the adding final concentration is 0.2% PEG4000 and 1mmol/L oxidized form: reduced glutathion (mol ratio is 1: 4) helps the protein renaturation of sex change promptly to recover native conformation, use TE (pH8.0) to carry out 4 ℃ of stirring dialysis renaturation, purifying greater than 20 times of volumes, every 4h changes liquid once, collect protein solution after changing liquid 6 times, divide device-20 ℃ frozen after the filtration sterilization.With the pilin purity after the SDS-PAGE detection purification, the Bradford method is measured protein concn.

2 implementation results

Recombinant plasmid pET-8818 is converted among the carrier bacterium BL21 (DE3), after IPTG induced, SDS-PAGE detected discovery, and located to occur a tangible protein band about 60kDa, consistent with the molecular weight size of prediction, its expression amount accounts for about 30% (Fig. 3) of bacterial protein.

In the present invention, the expression product of faeG-fedF fusion gene in e. coli bl21 (DE3) mainly exists with the inclusion body form

With mouse-anti HisTag antibody the total protein of reorganization behind e. coli bl21 (DE3)/pET-8818 abduction delivering being carried out Westemblotting detects, can detect the specificity positive reaction band that size is about 60kDa, the albumen that shows BL21 (DE3)/pET-8818 secretion inducing all can be discerned by mouse-anti HisTag antibody, and conclusive evidence faeG-fedF fusion gene is expressed successfully in e. coli bl21.Can extract the above faeG-fedF fusion rotein of 200mg in 1L reorganization bacterium BL21 (DE3)/pET8818 bacterium liquid.

Embodiment 3: with the FaeG-FedF fusion rotein is the immunogen preparing yolk antibody

1 materials and methods

1.1 adjuvant

White-oil adjuvant: the 94mL white oil, 6mL Si Ben-80, the 2g aluminum stearate, after mixing, 121 ℃ of 1.05kg/cm ²Autoclaving 30min, it is standby to be cooled to room temperature.

1.2 antibody

K88ac positive serum antibody is by Wuhan Virology Institute,Chinan academy of Sciences's Experimental Animal Center preparation, and F18 " a " single-factor serum becomes the Daiei professor to be so kind as to give by veterinary hospital of Yangzhou University, and the mouse-anti chicken IgG of horseradish peroxidase-labeled is available from Sigma company

1.3 damping fluid

PBSI damping fluid: NaCl 10.0g; KH ₂PO ₄0.25g; Na ₂HPO ₄12H ₂O 3.58g; KCL 0.25g is dissolved in the 900mL distilled water, and 1mol/L HCl transfers pH to 7.2, is settled to 1000mL with distilled water, and is standby in 121 ℃ of autoclaving 15min

PBSII damping fluid: NaCl 8.0g, KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, KCl 0.2g is dissolved in the 900mL distilled water, and 1mol/L HCl transfers pH to 7.4, is settled to 1000mL with distilled water, and is standby in 121 ℃ of autoclaving 15min

PBSIII damping fluid: NaCl 8.0g, KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, KCl, 0.2g is dissolved in the 400mL distilled water, and 1mol/L HCl transfers pH to 7.4, is settled to 500mL with distilled water, and is standby in 121 ℃ of autoclaving 15min

PBSIV damping fluid: NaH ₂PO ₄2H ₂O 4.37g, Na ₂HPO ₄12H ₂O 25.81g is dissolved in the 900mL distilled water, and 1mol/LHCl transfers pH to 7.2, is settled to 1000mL with distilled water, and is standby in 121 ℃ of autoclaving 15min

Coating buffer (0.05mol/L carbonate buffer solution): NaHCO ₃2.93g; Na ₂CO ₃1.95g; Be dissolved in the 900mL distilled water, 10mol/LNaOH transfers pH to 9.6, is settled to 1000mL with distilled water

Washings (PBS-T): the PBSII that contains 0.05% tween 20

Insulation liquid (PBS-BSA): the PBSII that contains 3% bovine serum albumin

Substrate solution: O-Phenylene Diamine 40mg is dissolved in phosphoric acid salt-citrate buffer solution (0.2mol/L Na of 100mL pH5.0 ₂HPO ₄51.4mL, a few citric acid 48.6mL of 0.1mol, distilled water 100mL mixing gets final product), adding 30%H ₂O ₂0.15mL get final product, now with the current

Stop buffer: 2mol/L H ₂SO ₄Solution (mixing gets final product for vitriol oil 22.2mL, distilled water 177.8mL)

1.4 the preparation of the newborn seedling of FaeG-FedF fusion rotein oil

With tween-80 to the final concentration that adds 121 ℃ of autoclaving 30min in the FaeG-FedF fusion rotein solution after purifying is 4%, mixes with isopyknic white-oil adjuvant behind the thorough mixing again, and fully emulsifiedly makes the oily newborn seedling that protein concentration is 500 μ g/mL.

1.5 the immunity of laying hen

Select that the brown laying hen of Hai Lan is divided into three groups, 200 of blank groups, 1000 of FaeG-FedF fusion rotein groups at random about 1700 28 ages in week.Every chicken of blank group is chest muscle injection 1mL (each 500 μ L of both sides) physiological saline respectively; FaeG-FedF fusion rotein group is the newborn seedling of chest muscle injection 1mL FaeG-FedF fusion rotein oil then.After head exempted from for 2 weeks, each group was used 1mL physiological saline and the newborn seedling booster immunization of 1mL FaeG-FedF fusion rotein oil respectively once; Adopted the same manner to carry out immunity for the third time at interval in 2 weeks again.

1.6 the preparation of spraying drying whole egg powder

Collect egg, spray-dried (140 mouthfuls of air inlets are given vent to anger for 65 mouthfuls) is processed into whole egg powder.Get the 1g powdery yolk, mix with 4mL PBSII, add the 8mL chloroform again, room temperature is placed 1h, and the centrifugal 20min of 3000 * g gets supernatant and carries out titration then.

1.7 indirect enzyme-linked immunosorbent assay (ELISA) detects antibody titer

Two respectively organize 30 pieces of egg samples of equal random acquisition weekly after exempting from, the variation that indirect enzyme-linked immunosorbent assay monitoring yolk antibody is tired.After the yolk antibody level reaches requirement, just begin to collect egg, carry out spraying drying.

Determine best antigen coated amount and antibody dilution with the square formation volumetry.Get enzyme plate (8 * 12 hole), horizontally-arranged with antigen from the 1:20 doubling dilution to 1:2560, vertical setting of types with antibody from the 1:20 doubling dilution to 1:640, carry out ELISA test.Whole experiment triplicate, comprehensive three test-results are with OD ₄₂₀The protein content of the last extent of dilution correspondence of antigen diluent degree was best antigen coated concentration when value reduced significantly, with positive antibody OD under the antigen coated concentration of the best ₄₂₀The last extent of dilution correspondence of serum dilution was the antibody optimum dilution degree when value reduced significantly.

Wrap by the K88ac pilin solution bag of concentration by 96 hole polyethylene boards with 100 μ L are best, 4 ℃ spend the night (18h-24h), discard, with washings flushing 3 times, each 3min, discard, use 37 ℃ of incubation 1h of insulation liquid of 150 μ L then, discard, as preceding washing 3 times, every hole adds 100 μ L antibody to be measured and (carries out 1:10 with PBSl, 1:20, the doubling dilution of 1:40 or the like) 37 ℃ of incubation 1h discard, as preceding washing 5 times, every hole adds the ELIAS secondary antibody diluent (1:15000 of 100 μ L horseradish peroxidase-labeled, the washings dilution), 25 ℃ of bags are discarded by 30min, as preceding washing 5 times, last every hole adds 100 μ L substrate solutions, and with 50 μ L stop buffer termination reactions, microplate reader detects OD after the colour developing ₄₅₀Value.Sample OD ₄₅₀Value is judged to the positive with the ratio of feminine gender value greater than 2.1, and is antibody titer with the high dilution that positive reaction occurs.

2 implementation results

FaeG-FedF has immunogenicity by laying hen immunity test proof recombinant protein, can cause the laying hen immune response, and can induce the special yolk antibody that produces anti-ETEC K88 and F18.

Embodiment 4: the yolk antibody effect is estimated

1 materials and methods

1.1 bacterial strain

Intestinal bacteria K88ac reference culture C83715 is available from China Veterinery Drug Inspection Office.Intestinal bacteria F18ac bacterial strain 2134P becomes the Daiei professor to be so kind as to give with F18ab bacterial strain F107/86 by veterinary hospital of Yangzhou University.

1.2 solution

Giemsa dye liquor: claim the Giemsa powder 0.5 gram, add several glycerine and grind, add glycerine (the glycerine total amount that makes adding is 33mL) again.56

Middle insulation 90-120min.Add 33mL methyl alcohol, put in the brown bottle and preserve, this is a Giemsa stoste.Dilute 10 times with PBS during use.

1.3 spraying drying whole egg powder

Common whole egg powder (SDEP): the egg of collecting kind and raising the not immune laying hen of term harmonization, the spraying drying whole egg powder that is prepared from.

Height is exempted from whole egg powder: contain the spraying drying whole egg powder by the special yolk antibody of FaeG-FedF fusion rotein antigen prepd after the renaturation, the anti-K88 1:17959 that tires, the anti-F18 1:8000 that tires.

1.4 yolk antibody vitro inhibition ETEC K88ac and F18ac adherence test

Yolk antibody vitro inhibition ETEC K88ac and F18ac adherence test are to adopt (.Passiveprotective effect of chicken egg yolk immunoglobulins against experimental enterotoxigenic Escherichiacoli infection in neonatal piglets.Infect Immun such as Yokoyama H such as Yokoyama, 1992,60:998-1007) method.At first separating preparation new existence pig and weanling pig jejunal epithelium cell, is 10 with 1mL concentration ⁶The intestinal epithelial cells suspension of individual/mL and 1mL cell concentration are 10 ⁸After the ETEC C83715 of CFU/mL or 2134P or F107/86 thalline suspension mixed, slide seasoning, ice ethanol were fixed, the dyeing of Giemsa dye liquor, observed under 1000 times of opticmicroscopes.Measure adherent total plate count on 20 epithelial cells, calculate the bacterial count of average each cell adhesion, in contrast group.With the 1mL cell concentration is 10 ⁸The ETEC C83715 of CFU/mL or 2134P or F107/86 thalline suspension and 1mL are by the yolk antibody liquid of FaeG-FedF fusion protein immunization preparation (the anti-K88 1:17959 that tires, the anti-F18 1:8000 that tires) mixes, 37 ℃ of temperature are bathed 30min, add 1mL epithelial cell suspension again, 37 ℃ of temperature are bathed 30min, adherent bacterial count on 20 epithelial cells of same mensuration is as test group record result.Above-mentioned test repeats 3 times.

1.5 prevention diarrhea of weaned piglets test

1.5.1 selection of laboratory animal and grouping

Select 144 24 ± 3 age in days three way cross Du * length * big weanling pigs, by blood relationship, go into the relative principle of correspondence such as test mass, sex and age in days and be divided into 2 groups at random, every group each 72, divide the raising of 6 hurdles, be repetition with the hurdle, 12 on every hurdle, male and female ratio unanimity

1.5.2 test daily ration

The single-factor design is adopted in this test, and the common whole egg powder (SDEP) of interpolation 1.5% is a control group in the daily ration, and it is test group that the height of interpolation 1.5% is exempted from a powdered egg

Test duration 28 days was divided into for two stages: be wean back 0-14 early stage, the later stage is wean back 15-28d.Early stage, each organized the daily ration of the different treatment of feeding, daily ration unanimity of each group of later stage.Wean back 0-14d, with reference to the preparation of NRC (1998) 3-5kg growing swine feeding standard, and with reference to the ideal protein pattern.Wean back 15-28 days is with reference to NRC (1998) 5-10kg growing swine feeding standard.Two groups of diets of different nutrition level basically identicals.

1.5.3 the feeding and management of test pig

All feeding experiments are all carried out at the child care house, are slatted floor, and the well-ventilated has insulation or anti-administration equipment preferably, and envrionment temperature maintains 20-28 ℃ substantially.Feed every day 4 times, exceed with the slightly surplus material of having enough at every turn, freely drink water.Carry out anti-administration or insulation measure, all the other Routine Managements and immune programme for children are undertaken by large-scale pig farm breed program.In time find that sick pig also treat and record, the course of disease surpass serious symptom on the 3rd more the person in time reject overbit and the body weight of the superseded pig of record.

1.5.4 the mensuration of diarrhoea situation record and growth performance

Write down piglet ight soil situation morning every day test early stage, is divided into 0,1,2,3 level Four records by dried, soft, rare, water sample, does during statistics just reaching soft stool for normally, rarely just is designated as diarrhoea with watery stool.

Grice diarrhoea head number of times in diarrhea rate=trial period/(test fate * pig's head number).

Diarrhoea index=stools scored sum/total number of confession examination pig, this index is high more, and representative diarrhoea is serious more.

The ight soil score value is calculated as follows: 0 minute: ight soil drying (normally); 1 minute: ight soil was soft and be shaped; 2 minutes: ight soil was yellow liquid (moderate diarrhoea); 3 minutes: ight soil was water sample spurting (severe diarrhoea).

The weaned piglet recast is for going into test mass, weighs by head before raising morning every other week after the wean and writes down body weight.Trial period is interior to be unit record feed consumption every day with the hurdle.

1.5.5 data analysis

Data such as average daily gain, food consumption and feedstuff-meat ratio are represented with X ± SD, linear model as the employing SAS statistical study, carry out one-way analysis of variance (GLM program), and the average of the significant difference factor (P＜0.05) level is carried out Deng Kenshi multiple comparisons diarrhea rate all adopt χ with the diarrhoea index ²Check (FREQ program).

2 implementation results

2.1 yolk antibody is to the restraining effect of the external adherent cell of ETEC

Observe discovery at microscopically (* 1000), intestinal epithelial cells is more rounded, and Giemsa dyeing back shows purple; And the very for a short time rod-short that is of bacterium shows blue.Behind the ETEC K88ac bacteria suspension and intestinal epithelial cell effect without the yolk antibody processing, more K88ac bacterium is attached on the cell: and behind the K88ac bacteria suspension and intestinal cells effect after height is exempted from the whole egg powder processing, only see an odd 1-2 bacterial adhesion on cell, on the cell that has even do not see bacterial adhesion (Fig. 5).

Table 1 yolk antibody is to the adherent influence of enterotoxic Escherichia coli K88ac cell in vitro

¹⁾Capitalization is represented the significance of difference on α=0.01 level

3 revision tests, in 60 intestinal epithelial cellss that every group is observed altogether, average each epithelial cell of the control group of no antibody adheres to 7.6 ± 0.4 K88ac; And test group has all extremely significantly suppressed the adhesive attraction of K88ac to intestinal epithelial cells than control group, and on average each epithelial cell only adheres to 1.0 ± 0.2 bacteriums (p＜0.01) (seeing Table 1) respectively.The yolk antibody powder that is obtained by the FaeG-FedF fusion protein immunization all has remarkable restraining effect to the bacterial adhesion intestinal epithelial cells.

Table 2 yolk antibody is to F18ab and the adherent influence of F18ac cell in vitro

¹⁾Capitalization is represented the significance of difference on α=0.01 level

²⁾Highly in this test exempt from tiring of the anti-F18ac of whole egg powder and be 1:8000

It is by the spraying drying whole egg powder of fusion protein F aeG-FedF immunity laying hen preparation that height is exempted from whole egg powder, has anti-K88ac FaeG yolk antibody and anti-F18ac FedF yolk antibody.The bacteria suspension that height is exempted from F18ab bacterial strain F107/86 after whole egg powder is handled and F18ac bacterial strain 2134P respectively with weanling pig jejunal epithelium cytosis after, microscopically is observed and is found (Fig. 6), adherent F18ab and F18ac bacterial count are all less on the cell.

Carry out revision test equally 3 times, calculate every group of 60 epithelial bacterial adhesion numbers observing, found that (seeing Table 2), compare that the numbers of poles that the test group F18ac after height is exempted from the whole egg powder processing adheres on the intestinal epithelial cells significantly reduces (P＜0.01) with the F18ac control group; Test group F18ab after height is exempted from the whole egg powder processing also is starkly lower than F18ab control group (P＜0.01) to the adhesion of intestinal epithelial cells equally.Show that thus height is exempted from the adhesive attraction that whole egg powder not only can suppress homologous strain F18ac, can also block the adhesion of allos bacterial strain F18ab intestinal epithelial cells.

2.2 yolk antibody is to the influence of diarrhea of weaned piglets situation

Just as shown in table 3, test 0-14d, has added the test group diarrhea rate and the diarrhoea index of exempting from whole egg powder by the height of FaeG-FedF immunity preparation and all extremely significantly has been lower than control group (P＜0.01) at i.e. wean back 1-2 week.Then the result with 0-14d is consistent to test full phase result.

Table 3 is respectively organized the influence of grice diarrhoea

¹⁾Lowercase is represented the significance of difference on α=0.05 level, and capitalization is represented the significance of difference on α=0.01 level

2.2 yolk antibody is to the influence of weanling pig growth performance

As can be seen from Table 4, two groups of each stages all show the speed of growth preferably, and piglet body weight in 7 age in week all can reach more than the 15kg, and there are not significant difference (P〉0.05) in average daily gain and food consumption between two groups.Yet in various test stages, the feedstuff-meat ratio of test group all significantly is lower than control group (P＜0.05).

Table 4 is respectively organized the influence of piglet growth performance

¹⁾Lowercase is represented the significance of difference on α=0.05 level, and capitalization is represented the significance of difference on α=0.01 level

Claims (3)

1, a kind of intestinal bacteria (Escherichia coli) BL21/pET-8818 of recombinant plasmid pET-8818 that contains the adhesin gene faeG of enterotoxic Escherichia coli K88ac and contain the adhesin gene fedF of enterotoxic Escherichia coli F18ac, be deposited in Chinese typical culture collection center, its preserving number is CCTCC NO:M207090; Described construction of recombinant plasmid step is, earlier the adhesin gene faeG of pig source enterotoxic Escherichia coli K88ac and the adhesin gene fedF of enterotoxic Escherichia coli F18ac are utilized connection peptides gene fusion construct faeG-linker-fedF, be inserted into the SacI of plasmid pET28a and Hind III site again and construct and form, its structure is shown in the pET8818 of Fig. 1.

2, comprising claim 1 described is the Yolk antibody feed of the colibacillary expression product FaeG-FedF fusion rotein preparation of CCTCC NO:M207090 by preserving number.

3, the application of the described intestinal bacteria of claim 1 in the preparation Yolk antibody feed.

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