CN103409455A - Egg yolk antibody anti-human enterotoxigenic escherichia coli adhesion protein and application thereof - Google Patents

Abstract

The invention discloses a recombinant plasmid pET-EF of an adhesin gene etpA containing human enterotoxigenic escherichia coli and flagellar gene fliC. The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.1. The recombinant plasmid pET-EF is constructed through a genetic engineering technology, the recombinant plasmid is transformed into escherichia coli BL21 (DE3) to induce recombinant expression bacteria BL21 (DE3)/pET-EF to express fusion protein EF. An egg yolk antibody of the protein is prepared from the expressed recombinant protein active immune primary laying hens, the extracted IgY verifies the recombinant protein has good antigenicity, the stability under different pH, temperature and enzyme conditions is detected; as shown in a mouse bowel poison attack test, the purified IgY can inhibit the adhesion of ETEC strain on epithelial cells in vitro.

Description

Yolk antibody and the application thereof of anti-human source enterotoxic Escherichia coli adhesin albumen

Technical field

The invention belongs to technical field of molecular biology, the HpaA gene etpA that is specifically related to people source enterotoxic Escherichia coli merges the structure of flagellin gene fliC recombinant plasmid pET-EF and recombination bacillus coli, fusion rotein, yolk antibody and the application thereof of having expressed etpA gene and fliC gene.

Background technology

Enterotoxic Escherichia coli (Enterotoxigenic Escherichia coli, ETEC) is the Main Pathogenic Bacteria (Black1990 that causes diarrhoea; Ericsson2003).Annual ETEC can cause hundred million diarrhoea of 2.8-4 in children below 5 years old according to statistics, and state of an illness weight does not wait, and slight diarrhoea can only be arranged, and can be severe cholera sample yet, causes annual about 30-50 ten thousand Infant and child deaths (WHO2004).It also to malnutrition and relevant (the Qadri et al2007 of hypoevolutism of many infants; Petri et al2008).ETEC diarrhoea also accounts for 1/3-1/2(WHO2004 in Africa, Asia and Hispanic traveler's diarrhea).The pathogenesis of ETEC diarrhoea is to pass through adhesin; at first adhere on host's the epithelial acceptor of mucous membrane of small intestine brush border; the cleaning of opposing intestines peristalsis and content; one or both enterotoxins (enterotoxin) are grown afterwards and are discharged in realization surely; cause fluid and electrolyte balance imbalance in cell, thereby produce watery diarrhea (Natro and Kaper1998; Sack1980).Adhesin and enterotoxin are important antigen (Svennerholm and Tobias2008) indispensable in traditional E TEC vaccine research.

Think that in the past adhesin refers to that mainly the pili adhesin is thalline surface pili antigen CFAs or CS.At present in people source ETEC, identified and surpassed 22 kinds of CFAs, common are CFA/I, CS1-CS7, CS14, CS17, CS21(Qadri et al2005), but between the allos pili without cross-protection (Qadri et al2006).Research finds that adhesion process also needs the participation of non-pili adhesin in addition.Intestinal bacteria comprise that ETEC all can bear flagellum on the whole body or two ends, and quantity is few than pili, but length is the several times of thalline.Recent study shows except the pili that spreads all over the thalline surface, the flagellum that end is given birth to not only can be used as locomotive organ, and this mobility (motility) works at pathogenic courses such as some Gram-negative bacteria field planting, invasion and proinflammatory reactions, as Salmonellas (Allen-Vercoe et al1999), chicken pathogenic escherichia coli (La Ragione et al2000).

Roy etc. (2006) have found a kind of albumen EtpA of ETEC secretion with the swivel base plasmid, be one of member of two companion's secretins (TPS), by ETEC peculiar.Be distributed in the ETEC bacterial strain of 59% CFAI, 56% CFA II, 75% CS14 or CS17, and these several pili types occupy approximately 60% ETEC(Wolf1997).It is by outside born of the same parents, doing mutually with the conserved regions of flagellin (FliC) that EtpA has been illustrated in research in bacterial adhesion, (the Roy et al2009a) of the function served as bridge performance of mediation flagellum and host cell.Test cell line confirms that by disappearance and restructuring this mutual work has keying action for effective field planting of ETEC, be that etpA or fliC deletion mycopremna adherent cell ability are compared wild-type and greatly reduced, or the antibody that adds the two in substratum also can reduce the bacterial count of adhesion, and external source is added recombinant protein or this gene of homologous recombination can make gene-deleted strain regain the function (Roy et al2009a) of adherent cell.

Usually the conserved regions of flagellin is hidden in the flagellum neck, but it can be exposed to the flagellum top moment, catches the EtpA molecule for identifying the eukaryotic cell surface receptor.Flagellum mediation adheres to does not have the serotype restriction, as at flic(H11) flic(H48 recombinates in mutant strain) can regain motion and adhesive capacity.With identical anti-EtpA serum, can suppress the ETEC of multiple H serotype to epithelial adhesion in addition.In mouse model, with rEtpA and rFliC nasal feeding immune mouse, obtained the result similar in vitro tests, and the two adhesiving effect that presses down used simultaneously is better than independent use (Roy et al2008,2009b).This Novel sticky random system is present in most ETEC, and the protein molecular of other and EtpA homology also may adopt this similar mode of action in Gram-negative bacteria separately, although concrete mediated process it be unclear that, infer that two kinds of albumen can be used as the candidate antigens of New-type wide-spectrum vaccine development.

A large amount of test-results show; by to young animal, providing exogenous antibodies (comprising blood antibody and yolk antibody) to improve its passive immunization level, prevent and treat bacterium and viral infection (particularly enteron aisle diarrhoea) is effective approach (Hatta et al1993b; King's Jie 2007).Yolk immunoglobulin (Egg yolk immunoglobulin, IgY) becomes a kind of Substitutes For Antibiotic that has much potentiality.U.S. FDA is listed it in " It is generally accepted safe material " (Coleman1996).IgY exploitation for various gastrointestinal tract disease at present has broad research.The investigator is usingd pili and is prepared IgY treatment ETEC diarrhoea (Yokoyama et al1992 as antigen; Ikemori et al1992; Marquardt et al1999).Report the earliest derives from Perorally administrable antimicrobial hair yolk antibody can prevent that K88, K99 and 987P ETEC from infecting (Yokoyama et al1992).After the piglet birth, 4h namely infects ETEC, the basic all survivals for the treatment of group (oral IgY), and show dose-dependent effect, placebo and control group mortality ratio are greater than 75%, have suppressed too ETEC to the chitling cell adhesion at the external IgY of adding.Marquardt etc. (1999) also prepare yolk antibody with purification K88 pilin Immune Laying Hens, be born 3 ages in days and 21 age in days weanling pigs are attacked to the poison treatment, wherein the diarrhoea of age group piglet on the 3rd is cured entirely after oral IgY24h, and the diarrhoea of common IgY group continues and mortality ratio reaches 62.5%; Of short duration diarrhoea has appearred in weanling pig oral IgY after attacking poison, and all survival and body weight increase, and severe diarrhea and dehydration also appear in control group, and death namely occurs after infecting 48h.The oral anti-ETEC pili of king's Jie (2007) subunit yolk antibody can provide effective passive immunization protection to piglet, the infection of opposing enteron aisle germ.

Based on above research background, this research be take flagellin FliC and EtpA and is candidate antigens, utilizes key gene etpA and fliC and the two fusion rotein of gene engineering method clonal expression in bacterial adhesion epithelial cell process, obtains a large amount of target proteins.In conjunction with the IgY technology, namely by Immune Laying Hens, produce the special yolk antibody for EtpA and FliC antigen, be intended to develop a kind of health-care eggs food with broad spectrum diarrhea function with low cost.

Summary of the invention

First purpose of the present invention is to provide the recombinant plasmid pET-EF of people source ETEC HpaA gene etpA and fliC.

Another object of the present invention is above recombinant plasmid transformed is entered to intestinal bacteria (Escherichia coli) BL21(DE3), the recombinant bacterium that can express adhesin EtpA and the two fusion rotein of flagellin FliC of acquisition.

The 3rd purpose of the present invention is to provide the fusion rotein EF that induces recombination bacillus coli BL21 (DE3)/pET-EF to express, and this fusion protein immunization originality is good, and production cost is low.

The 4th purpose of the present invention is to utilize described fusion rotein EF to prepare yolk antibody.

The 5th purpose of the present invention is to provide yolk antibody and the application in the anti-human source enterotoxic Escherichia coli ETEC diarrhoea of preparation food thereof.

The present invention is achieved by the following technical solutions:

One, the structure of recombinant plasmid pET-EF (seeing Fig. 1,2)

In the GenBank of NCBI, search the locus that sequence number is AY920525, wherein etpA is positioned at coding region 2718-8021, total length 5304bp, and the purpose fragment is the 1701bp of 5' end; Flagellin fliC(sequence number is AY906918) total length 1464, conserved regions is the 519bp of 5' end.The people source enterotoxic Escherichia coli type strain H10407 of take is test materials, the design primer, purpose fragment after pcr amplification is connected to expression vector, successfully built key gene etpA in bacterial adhesion epithelial cell process and the recombinant expression plasmid of the two fusion gene of flagellin gene fliC, wherein between fusion gene with one section hydrophobicity connection peptides (Gly ₄Ser) ₃Do connection.Recombinant plasmid is carried out to enzyme and cut evaluation and order-checking, result shows that structure is correct, by its called after pET-EF.

Two, recombination fusion protein EF abduction delivering and extraction purifying

Recombinant plasmid pET-EF is transformed into to e. coli bl21 (DE3), does abduction delivering with inductor sec.-propyl-β-d-thiogalactoside (Isopropyl-β-d-Thiogalactoside, IPTG).With different inducing temperatures and inductor concentration, progressively optimize prokaryotic expression, obtain best abduction delivering condition and be: 37 Bed is cultured to OD600 and reaches 0.5-1.0, adds IPTG to suitable concentration (0.5mM), continues to cultivate 3-4h, and final expression amount is all more than 15%, exists with the form of inclusion body.Inclusion body, through the inclusion body protein that pressure breaking, denature and renature obtain after concentrating, is measured to protein concn with the Bradford method.

Three, with the recombinant protein Immune Laying Hens, prepare yolk antibody

In renaturation, protein solution after concentrated, add sterilizing tween-80 to final concentration 4%, then in tissue refiner, mix 30min with the equal-volume white-oil adjuvant, fully emulsified to protein concentration be 0.5mg/mL.By minutes 2 groups at random of laying hens, 100 of blank group 50 (physiological saline) and immune group.Adopt chest muscle injection (every side 500 μ L).After head exempted from for 2 weeks, each group was used respectively same oil breast seedling booster immunization once, 2 weeks of interval are immunity again, two respectively organize weekly 5 pieces of eggs of random acquisition after exempting from, the purifying, collecting yolk antibody, indirect ELISA monitoring antibody titer changes, western-blot identifies the specificity of yolk antibody, treats that yolk antibody is tired to reach 1:2 ⁹Rear collection egg.The egg of collection is shelled, stir, (air inlet 150 ℃ gives vent to anger 70 to spraying drying

, in the sample sack of the sealing of packing into after cooling.

Four, the stability test of yolk antibody

Respectively 60, ℃ 65, ℃ 70, ℃ 75

In bathing, process 30min, or the pH value is that 2,3,4 damping fluid is diluted to lmg/mL by IgY, 37

Educate 3h, or process antibody 0-4h with simulated gastric fluid or intestinal juice.Result shows: temperature is lower than 70

PH>3 o'clock, the IgY activity keeping is stable; In temperature 70

Upper processing, namely lose 80% activity after 5min; After in the environment of pH=2, processing 0.5h, IgY tires and is negative.In the stomach en-environment of pH=1.2, hydrolysis is rapid, and trypsinase has no significant effect the IgY activity.

Five, the effect evaluation of yolk antibody

By setting up the field planting model of ETEC in mouse intestinal, with three kinds of clinical common different serotypes bacterial strains, attack malicious mouse, after the yolk antibody that will prepare early stage simultaneously was oral, different antibody groups had produced and has pressed down in various degree adhesiving effect bacterium.Wherein the antibody group of reference culture H10407 has reduced the quantity of infecting mouse.E519 and 44815 does not produce EtpA, but after the two attacks poison the IgY for preparing of oral fusion rotein EF all significantly anti-bacteria adhere to number (being respectively P<0.01, P<0.05).By cell adhesion, test, confirmed that yolk antibody is at the external same similar anti-adhesion effect of having brought into play.

The accompanying drawing explanation

Fig. 1 is a kind of construction of recombinant plasmid route of etpA-fliC fusion gene.In figure, pMD18-T is cloning vector, and pET-28a is expression vector.PET28a-EF is that the recombinant plasmid, XhoI, the EcoRI that have inserted etpA and two genes of fliC are the restriction endonuclease sites on plasmid.

Fig. 2 is the agarose gel electrophoresis of pcr amplification etpA, fliC product.In figure, product etpA and fliC molecular size range are respectively 1700bp and 519bp; M is DNA molecular amount standard DL2000.

Fig. 3 is that the double digestion of recombinant expression plasmid pET-EF is identified the electrophoresis schematic diagram.In figure, M is DNA molecular amount standard DL2000; Swimming lane EF:pET-EF, Insert Fragment 2260bp.

Fig. 4 is the SDS-PAGE electrophoresis detection schematic diagram before and after the IPTG inducible protein is expressed.In figure, 1,2 represent that respectively recombinant protein EF induces front and back, and the gained molecular weight of albumen is 84kDa.

Fig. 5 is the soluble analysis schematic diagram of recombinant protein after inducing.In figure 1, the upper cleer and peaceful precipitation of 2:pET-EF BL21 (DE3) after inducing; M is protein molecular weight standard.

The impact of Fig. 6 condition of different temperatures on albumen EF expression amount.1,2:37 ℃ of upper cleer and peaceful precipitation after inducing; 3,4:25 ℃ of upper cleer and peaceful precipitation after inducing; 5,6:17 ℃ of upper cleer and peaceful precipitation after inducing; Arrow: target protein.

The impact of Fig. 7 inductor concentration on albumen EF expression amount.1: before inducing; 2,3,4,5,6:IPTG concentration be respectively 0.1,0.3,0.5,1.0,1.2mmol/L; Arrow: target protein.

Fig. 8 is the protein s DS-PAGE electrophoretic analysis schematic diagram after concentrated renaturation.

Fig. 9 for immunity for the second time after the yolk antibody rule schematic diagram over time of tiring.

Figure 10 is the antigenicity schematic diagram that Western-Blot detects recombinant protein EF.

Figure 11 is the thermostability schematic diagram that ELISA detects yolk antibody.

Figure 12 is the acid acceptance schematic diagram that ELISA detects yolk antibody.

Figure 13 is the enzymolysis stability schematic diagram that ELISA detects yolk antibody.

Figure 14,15 is respectively IgY to three-type-person source ETEC mouse intestinal and epithelial anti-adhesion effect schematic diagram.A, B, C mean respectively to attack malicious mouse with ETECH10407, E519 and 44815; Sea line means geometric mean, and * means significant difference, and * * means that difference is extremely remarkable.

Embodiment

In following specific embodiment, all (J. Pehanorm Brooker and D.W. Russell show, and Huang Peitang etc. translate with reference to beautiful J. Pehanorm Brooker and D.W. Russell without the method for special instruction.Molecular cloning experiment guide (third edition), Beijing: Science Press, 2002).

Embodiment 1: the structure of enterotoxic Escherichia coli flagellin and EtpA recombinant plasmid

1, bacterial strain and the plasmid vector of the present embodiment use see the following form 1.

Table 1 bacterial strain and plasmid

2, main agents and test kit

PCR related reagent: Taq archaeal dna polymerase, dNTP(2.5mM), 10 * Taq buffer, DNA marker(2kb and 10kb): Beijing Quanshijin Biotechnology Co., Ltd;

Restriction enzyme: Dalian precious biotechnology company limited;

Agarose: Biowest Agarose packing;

Tryptones, yeast extract: Britain OXOID;

Trysinization soybean broth: the U.S. green enlightening medicine equipment company limited;

DNA gel reclaims test kit: Shanghai Sheng Gong Bioisystech Co., Ltd;

Plasmid extraction kit: Shanghai Sheng Gong Bioisystech Co., Ltd.

3, solution preparation

3.1 DNA of bacteria is extracted related solution

PBS(0.012M): take NaCl8g, KCl0.2g, Na ₂HPO ₄12H ₂O3.58g, KH ₂PO ₄0.27g, add ddH ₂O is settled to 1L, packing 121

Bacterium 30min.

Chloroform/primary isoamyl alcohol (24:1): chloroform 480ml, primary isoamyl alcohol 20ml, mix and move in Brown Glass Brown glass bottles and jars only 4 Deposit.

Proteinase K (20mg/mL): take powder 20mg and be dissolved in ddH ₂O1mL, mix-20

Deposit.

3.2 substratum

1) LB substratum: Tryptones 1g, yeast extract 0.5g, NaCl1g, agar powder 1.5g(LB flat board), add ddH ₂O is dissolved to 100mL, stirs, and masking foil sealed high pressure steam sterilizing 30min.

2) TSB: take 0.3g TSB, add ddH ₂O is dissolved to 100mL, with 1) sterilizing.

3) penbritin liquid storage (100mg/mL): the 1g powder is added to 10mL ddH at aseptic operating platform ₂O dissolves, and sterile filters is filtered, and divides and is filled to 1.5mL EP pipe ,-20

Deposit.

4) kantlex liquid storage (50mg/mL): the 1g powder is added to 20mL ddH at aseptic operating platform ₂O dissolves, and all the other are with (3).

3.3DNA gel electrophoresis related solution

1) 50 * TAE:Tris242g, EDTA37.2g, glacial acetic acid 57.1mL, add ddH ₂O is dissolved to 900mL, regulates pH to 8.0, is settled to 1L.During use, being diluted to 1 * TAE gets final product.

2) 6 * DNA sample-loading buffer: EDTA7.4g, sucrose 40g, diformazan cyanophenyl 0.25g, tetrabromophenol sulfonphthalein 0.25g, add ddH ₂O is dissolved to 100mL, stirs, and four degree are preserved.

3) EB dye liquor (10mg/mL): take the 1g powder, carefully add 100mLddH ₂O, be stirred to fully and dissolve, tinfoil parcel body room temperature preservation.Every 200mL ddH during use ₂O adds 10 μ L EB.Notice that EB is strong mutagenic compound, during operation, note whole body protection.

4, the structure of flagellin FliC and EtpA recombinant plasmid

The construction recombination plasmid process is shown in Fig. 1.

4.1 the extraction of DNA of bacteria

1) in aseptic operating platform, from picking ETEC H10407 bacterium liquid slant medium, be coated with the TSB flat board, next day picking list bacterium colony in 5mL TSB substratum, 37

Cultivate night.

2) the centrifugal collection thalline of 10000rpm next day, PBS washing 2 times resuspended thalline to 500 μ L.

3) add 100 μ L10%SDS, 10 μ L20mg/mL Proteinase Ks, 50

Educate 3h or 37

Night.

4) in supernatant, add equal-volume phenol: chloroform: primary isoamyl alcohol (25:24:1), put upside down and mix 5min, the centrifugal 5min of 10000rpm, shift supernatant.Repeat once.

5) add equal-volume chloroform/primary isoamyl alcohol, put upside down and mix 5min, the centrifugal 5min of 10000rpm, shift supernatant.

6) add 0.6(V/V) Virahol, put upside down and mix, standing 5min, the centrifugal 5min of 10000rpm, the precipitation of collection, through 75% alcohol washing of precooling, is dissolved in 100 μ L ddH after drying ₂In O ,-20 Deposit.

4.2 design of primers

In the GenBank of NCBI, search the locus that sequence number is AY920525, wherein etpA is positioned at coding region 2718-8021, total length 5304bp, and the purpose fragment is the 1701bp of 5' end.Flagellin fliC(sequence number is AY906918) total length 1464, conserved regions is the 519bp of 5' end.During etpA and the fusion of fliC do, in the former reverse primer, introduce one section hydrophobicity connection peptides (Gly ₄Ser) 3(table 2 dash area), by SalI, the two is connected.Concrete primer sequence sees the following form 2.

Table 2PCR amplimer

Annotate: wavy line is the hydrophobicity connection peptides; Underscore means restriction enzyme site.

4.3PCR amplifying target genes

Add ddH ₂O first is diluted to primer 10 μ M, and genomic dna is diluted to 0.05mg/mL.Take 25 μ L reaction systems as example (unit: μ L):

Reaction conditions following (cycle number: 35):

Program	Denaturation	Sex change	Annealing	Extend	Finally extend
						Temperature (℃)	95	94	See that primer is synthetic single	72	72
Time (min)	3	3	0.5	1kb/min	8

4.4PCR product detects and reclaims

1) detect: according to clip size, prepare sepharose, PCR product and the sample solution of getting 5 μ L mix point sample, and click and enter DNAmarker in a side.After electrophoresis finishes, gel is put into to the EB dye liquor, after 10min in the gel imaging system preservation of taking pictures.

2) reclaim: the comb in the time of detecting changes wide comb into, and other electrophoresis step are consistent.Band in gel excises rapidly with blade under ultraviolet lamp, all the other steps are according to reclaiming the test kit description operation.

4.5DNA the clone of fragment

1) prepare competence DH5 α:

A. in the glycerine pipe of preserving, dip bacterium liquid and draw the LB flat board, picking list bacterium colony enlarged culturing 16-18h.

B. next day is with inoculum size enlarged culturing (37, ℃ 3-4h) in triangular flask of 1%, till the concrete time is mist with bacterium liquid.

C. in aseptic operating platform, shift bacterium liquid to the centrifuge tube of precooling, continue to place 30min on ice.Then 4

The heart is collected thalline (4000rpm, 10min).

D. abandon most supernatant, add the 0.1mol/L CaCl of 10mL precooling ₂Resuspended thalline, place 25-30min on ice.

E. above two steps repeat 2 times.

F.2mL the 0.1mol/L CaCl of precooling ₂Resuspended thalline, add sterile glycerol to final concentration 15-20%, with 100 μ L/ pipe packing ,-80

Deposit standby.

2) connect: 5 μ L linked systems are as follows, and 4

Night.

The PCR product	2
		PMD18-T	0.5
Solution?I	2.5

3) transform

A. get 10 μ L connection products and add in the DH5 α competence of just having thawed, tapped mixes, and places 30min on ice.Now open the recirculated water bath and be preheated to 42.℃

B.42

Bathe thermal shock 90s, place fast 1-2min on ice.

C. add LB substratum 400 μ L, 37

Swing and cultivate 1h.

D. centrifugal (8000rpm, 2min) concentrates bacterium liquid to 100 μ L, and it is applied on the LB flat board of Amp resistance to the constant incubator incubated overnight.

E. next day picking list bacterium colony, bacterium liquid PCR detects and to have or not positive colony, and the T that will obtain clone distinguishes called after pMD-etpA, pMD-etpAL, pMD-fliCL, pMD-fliC1, pMD-fliC2.

4.6 construction expression plasmid

1) the picking positive bacteria is dropped into capable enlarged culturing, according to plasmid, extract in a small amount the explanation of test kit and carry out plasmid extraction, and by following 20 μ L systems, the T carrier and the empty expression vector that contain the purpose fragment are done to double digestion and connection, wherein in linked system the ratio of DNA and carrier with mole ratio between 1:1-5:1:

2) by pMD-etpAL, the pET28a double digestion through EcoRI and SalI, after agarose gel electrophoresis, reclaim fragment etpAL and carrier pET28a, connection, conversion, detection obtain pET-etpAL, and now purpose fragment 3 ' is with 45bp hydrophobicity connection peptides (Gly ₄Ser) ₃With the SalI site.After this use SacI, XhoI, to pET-etpAL, pMD-fliCL double digestion, reclaims carrier pET-etpAL and fliCL after agarose gel electrophoresis, connection, conversion, detection obtain pET28a-EF, the sequencing result display sequence is identical with expection, and pET28a-EF successfully constructs.The two amalgamation and expression purpose is by laying hen immunity afterwards, can forms to have and merge the yolk antibody of tiring.

3) bacterium colony PCR selects above positive colony, and the bacterial strain enlarged culturing through checking order correct is extracted in a small amount recombinant plasmid and done enzyme and cut evaluation ,-20

Deposit.Easy for writing, recombinant expression plasmid is abbreviated as: pET-EF.With EcoRI and XhoI, recombinant plasmid is done to double digestion, the results are shown in Figure 3.

The pET-EF that the present invention obtains is after double digestion is by agarose gel electrophoresis, at about 2200bp, observe respectively the purpose fragment, the sub-sequencing result of the positive colony of gained is all consistent with base sequence and the size of expection with the sequence alignment demonstration that NCBI announces, and sequence is as shown in SEQ ID NO.1.

Embodiment 2: the abduction delivering of recombinant protein and extraction purifying

1, reagent

Tryptones, yeast extract: Britain OXOID;

Trysinization soybean broth: the U.S. green enlightening medicine equipment company limited;

Tris alkali, glycine, SDS:Amresco packing;

IPTG, Amp, Kan, Sleep-promoting factor B, reduced glutathion, PEG4000:BIOSHARP packing;

2, solution preparation

The LB substratum as shown in Example 1.

IPTG liquid storage (1mol/L): the powder of 1g is added to the sterilizing ddH of 4.2mL at aseptic operating platform ₂O, sterile filters is divided and is filled in the 1.5mLEP pipe after filtering.

SDS-PAGE electrophoresis related solution:

1) 2 * albumen sample solution: Tris-HCl(1M, pH=6.8) 10mL, SDS4g, tetrabromophenol sulfonphthalein 200mg, glycerine 20mL, add ddH ₂O is settled to 100mL, during use, adds 2-3% beta-mercaptoethanol (now with the current).

2) 5 * electrophoretic buffer: Tris15.10g, Gly72.06g, SDS5g, add ddH ₂O is settled to 1L.During use, dilution is 5 times.

3) 30% polyacrylamide: take successively N, N'-methylene diacrylamide 1g, acrylamide 29g, add ddH ₂O is settled to 100mL, proceeds to four degree in brown bottle and preserves.The weighing process is noted protection.

4) separation gel damping fluid: Tris18.17g, add ddH ₂O90mL, dense HCl regulates pH to 8.8, is settled to 100mL, and four degree are preserved.

5) concentrated glue damping fluid: Tris12.11g, add ddH ₂O90mL, dense HCl regulates pH to 6.8, is settled to 100mL, and four degree are preserved.

6) 10%SDS: take sodium lauryl sulphate 10g, add ddH ₂O is settled to 100mL, and normal temperature is preserved.

7) 10% ammonium persulphate: take in ammonium persulphate 0.1g to EP pipe, add 1mLddH ₂O, four degree are preserved, and use in two weeks.

8) 1%TEMED: draw N, N, N', N'-Tetramethyl Ethylene Diamine 1mL, add ddH ₂O to 100mL, proceed to four degree in brown bottle and preserve.Note environment ventilation.

9) coomassie brilliant blue staining liquid: coomassie brilliant blue R_250 1.25g, methyl alcohol 450mL, glacial acetic acid 50mL, add ddH ₂O450mL.

10) destainer: methyl alcohol 50mL, glacial acetic acid 75mL, add ddH ₂O875mL mixes.

The inclusion body purification related reagent:

1) BufferA:Tris6.055g, EDTA0.186g, NaCl2.925g, glycerine 50mL, add ddH ₂O is settled to 1L.Before use, now add DTT to final concentration 0.5mmol/L.

2) 10%SKL: take dodecyl creatine sodium 1g, add ddH ₂O10mL, be stirred to dissolve complete.

3) powder of 50mM Sleep-promoting factor B: 1g packing adds ddH ₂O32.65mL, pressure-vaccum is to dissolving.

4) powder of 100mM reduced glutathion: 1g packing adds ddH ₂O32.54mL, the same.

5) 20%PEG4000: take Macrogol 4000 20g, add ddH ₂O is settled to 100mL.

6) dialysis tubing treatment solution A(2% sodium bicarbonate, 1mMEDTA, pH8.0): take sodium bicarbonate 20g, EDTA0.372g, add ddH ₂O to 900mL, regulate pH to 8.0, is settled to 1L.

7) dialysis tubing treatment solution B(1mM EDTA, pH8.0): in A liquid, remove sodium bicarbonate and get final product.

3, the abduction delivering of recombinant protein and SDS-PAGE detect

The expression plasmid successfully constructed is transformed to expression strain BL21(DE3), picking list bacterium colony incubated overnight.Next day, bacterium liquid joined in the fresh culture that 5mL contains resistance by 1% inoculum size, and 37 ℃ of shaking tables are cultivated 3-4h.When OD600 reaches 0.5-1.0, add IPTG to final concentration 1mmol/L, continue to cultivate 3-4h.Get the centrifugal collection thalline of 500 μ L bacterium liquid, 50 μ LPBS are resuspended, and equal-volume adds sample solution, and boiling water bath 5min gets 10 μ L supernatants and carries out the SDS-PAGE electrophoresis detection.Contrast is not added inductor and is cultivated same time.

4, the soluble analysis of recombinant protein

As above recombinant strains is seeded to enlarged culturing in the LB of 50mL, collects the thalline of abduction delivering, the abundant resuspended thalline of 5mLbufferA, be placed on ice.Treat that pressure breaking instrument temperature is down to 4 ℃, start broken thalline, repeat 3 times.The centrifugation supernatant, the abundant resuspended precipitation of 5mLbufferA, respectively get 50 μ L to carry out the SDS-PAGE electrophoretic analysis.

5, the inductive condition optimization of recombinant protein

In the protein expression process, the concentration of inductor and inducing temperature are the important factors that affects expression amount.Therefore by design differing temps (37,25,17 ℃) and IPTG concentration gradient (0.1,0.3,0.5,1.0,1.2mM), study its impact on protein expression.As above inoculation culture 5mL bacterium liquid, all add inductor when the OD value reaches 0.5-1.0 under different condition, and removing 25 ℃ and 17 ℃ needs to continue incubated overnight, and all the other collect thalline after all cultivating 3-4h, carry out the SDS-PAGE electrophoresis detection as 3.

6, the extraction purifying of recombinant protein

Inoculate recombinant expressed bacterium to the 300mL substratum, with the condition abduction delivering after optimizing.From nutrient solution, collecting thalline, then be resuspended in the BufferA of 1/10 volume of precooling, broken 3-5 time of pressure breaking instrument, note whole process maintenance low temperature.Centrifugal reservation precipitation, with the abundant resuspended precipitation of the BufferA of equal above-mentioned volume, slowly add 10%SKL, do not stop to be stirred to resolution of precipitate, solution clarification, 4

Put 2h.Continue to add PEG4000,600 μ L Sleep-promoting factor B and the reduced glutathions of final concentration 0.2%, 4

Put 2h, the centrifugal 15min of 10000rpm discards undissolved thalline impurity.Dialysis tubing treatment process: dialysis tubing is cut out by the length of 20-30cm.Boiling water bath 10min in A liquid, distilled water cleans.Boiling water bath 10min in B liquid, distilled water cleans, 4 ℃ of preservations.While taking, note being with gloves.The dialysis tubing that the protein liquid of renaturation is packed into and handled well, totally 2 days, during change liquid for several times, PEG embedding dialysis tubing is concentrated into desired concn.SDS-PAGE electrophoresis detection purity of protein, the Bradford method is measured protein concentration.

7, test-results

IPTG induces recombination bacillus coli BL21 (DE3)/pET-EF to express, and the results are shown in Figure 4.Corresponding positions is equipped with the big or small albumen of expection and occurs, the EF molecular weight is about respectively 84KD, expression amount approximately 15%.Soluble analysis shows that the equal most of form with inclusion body of (see figure 5) albumen is present in precipitation, few in supernatant.Fig. 5 shows and determines that the recombinant protein inductive condition is that 37 ℃ of shaking tables are cultured to OD600 and reach 0.5-1.0, adds IPTG to suitable concentration 0.5mM, continues to cultivate 3-4h, collects thalline.

The aminoacid sequence of the EF albumen that the present invention builds is as shown in SEQ ID NO.2.

Embodiment 3: prepare yolk antibody with the recombinant protein Immune Laying Hens

1, reagent

White oil, stearic acid aluminium, Si Ben-80, tween-80: biological products limited liability company present before the section of Wuhan.

The anti-chicken IgG:sigma of HRP rabbit

Nitrite ion Supersignal west pico test kit: Thermo

2, solution preparation

The ELISA related reagent:

PBS(0.012M): take NaCl8g, KCl0.2g, Na ₂HPO ₄12H ₂O3.58g, KH ₂PO ₄0.27g, add ddH ₂O is settled to 1L, packing 121

Bacterium 30min.

Coating buffer (0.05M carbonate buffer solution): NaHCO ₃2.93g, Na ₂CO ₃1.59g, add ddH ₂After the O900mL dissolve complete, regulate pH to 9.6, be settled to 1L, normal temperature is placed.

Washings: in PBS, adding final concentration is 0.05% tween 20.

Confining liquid: 3% bovine serum albumin (BSA) is dissolved in washings.

OPD nitrite ion: O-Phenylene Diamine 80mg, 0.1mol/L citric acid 4.86mL, 0.2mol/L Na ₂HPO ₄5.14mL, ddH ₂O10mL, finally add 30%H ₂O ₂30 μ L.Now with the current.

Stop buffer (2M H ₂SO ₄): vitriol oil 22.2mL, distilled water 182mL, acid slowly is added to the water along wall of cup, does not stop to stir evenly.

The western-blot related reagent:

Film transfering buffering liquid: Tris5.8g, glycine 2.9g, SDS0.37g, methyl alcohol 200mL, ddH ₂O is settled to 1L.

TBS:Tris2.42g, NaCl8.78g, ddH ₂O900mL, salt acid for adjusting pH to 8.0, constant volume 1L.

In TBST:TBS, adding final concentration is 0.05% tween 20.

Confining liquid: the TBST that contains 1%BSA.

Nitrite ion: A liquid, B liquid are mixed with the ratio of 1:1, now with the current.

3, the preparation of the newborn seedling of recombinant protein oil

White oil 94mL, aluminum stearate 2g, stir, and is heated to 80 degree left and right, and continue stirring until the solution clarification.The span-80 that slowly adds 6mL, continue to stir 20min, is white-oil adjuvant, 4 ℃ of preservations after cooling.In renaturation, protein solution after concentrated, add sterilizing tween-80 to final concentration 4%, then in tissue refiner, mix 30min with the equal-volume white-oil adjuvant, fully emulsified to protein concentration be 0.5mg/mL.

4, the immunity of laying hen

Select to raise in cages for 20 ages in week 450 of Luo Man laying hens, by minutes 5 groups at random of laying hens, 100 of blank group 50 (physiological saline) and immune group.Adopt chest muscle injection (every side 500 μ L).After head exempted from for 2 weeks, each group was used respectively same oil breast seedling booster immunization once, and 2 weeks of interval are immunity again, and two respectively organize weekly random acquisition egg ELISA monitoring after exempting from sees that antibody titer changes, and treated that yolk antibody is tired to reach 1:2 ⁹Rear collection egg, cryopreservation.

5, spraying drying whole egg powder

The egg of collection is shelled, stir, spraying drying (150 ℃ of air inlets are given vent to anger 70 ℃), in the sample sack of the sealing of packing into after cooling.Gather the whole egg powder of different time sections in spraying drying, the same Fresh Egg of antibody method of purification, indirect ELISA detects antibody titer.

6, the purifying of yolk antibody

Water dilution method, concrete steps are as follows:

Separate yolk, namely at eggshell one end, break an aperture, egg white is flow to end, enlarge the broken shell scope, yolk is positioned over rollback on filter paper and moves, until egg white blots.Puncture membrane of yolk, collect egg yolk liquid in centrifuge tube.Egg yolk liquid, with the distilled water diluting of 8 times of volumes, stirs, and the salt acid for adjusting pH is to 5.0-5.2, and 4

Put 6h or spend the night.The centrifugal 20min of 10000rpm, collect supernatant and obtain water soluble ingredient WSF(water-solve fract).Supernatant adds ammonium sulfate to 50% saturation ratio, stirs, and 4 Put 2h or spend the night, fully precipitating, the centrifugal 10min of 10000rpm, abandon supernatant, and precipitation is dissolved in PBS to former egg yolk liquid volume.Add ammonium sulfate to 33% saturation ratio, repeat above step.4

Analyse 24h ,-20

Deposit standby.

7, indirect ELISA mensuration yolk antibody is tired

Two exempt from a week to start to gather egg afterwards, and weekly at random from 100 laying hens, collecting 5 pieces of eggs, after extracting antibody, with the recombinant protein envelope antigen, indirect ELISA mensuration two is exempted from the antibody titer in rear 1-7 week.Section sample before, during and after spray-drying process, in kind detect and tire after extraction antibody.

The square formation volumetry is determined best antigen coated concentration.Antigen is done to doubling dilution (1:400,1:800,1:1600,1:3200,1:6400) with coating buffer respectively, and every hole 100 μ L are coated with polystyrene reactant plate, two parallel holes of each extent of dilution, 4

Night.During working sample, by sample doubling dilution on demand, the anti-chicken IgG of enzyme mark rabbit does the 1:10000 dilution with PBST, carries out the ELISA detection, and concrete sample determination step is as follows:

1) antigen is coated with to polystyrene reactant plate, 4 by every hole 100 μ L

Night;

2) discard liquid, the PBST washing pats dry for 3 times, adds the every hole 100 μ L of confining liquid, 37

Educated 2 hours;

3) as above washing, add the every hole 100 μ L(of antibody to be measured to dilute with PBST), 37

Educated 1 hour;

4) as above washing, add the every hole 100 μ L of ELIAS secondary antibody (PBST dilution), 37

Educated 1 hour;

5) as above washing, add the OPD nitrite ion 100 μ L that now join, 37

Light is hatched 15min;

6) every hole adds stop buffer 50 μ L, under the 450nm wavelength, measures the OD value in each hole, returns to zero with blank well.The ratio of antibody OD value to be measured and negative control is greater than at 2.1 o'clock and is judged to the positive, and antibody titer is the high dilution while positive reaction occurring.

8, western-blot detects the recombinant protein immunogenicity

1) SDS-PAGE electrophoresis: protein sample is pressed to example 2 method electrophoresis.

2) transferring film: adopt wet method to shift, electricity turns liquid precooling on ice

3) remove sheet glass, take out gel, according to marker and molecular size range, near the glue of the 1cm width purpose band is scaled off and is immersed in electricity and turns in liquid.

4) according to the size of glue, cut out filter paper and the pvdf membrane of formed objects.Filter paper is immersed in electricity and turns in liquid, and film first infiltrates about 30s in methyl alcohol, be transferred to ddH2O rinsing 1-2min, is immersed in afterwards electricity and turns in liquid.

5) successively plastic plate black flour, sponge, 2 metafiltration paper, gel, pvdf membrane, 2 metafiltration paper, sponge alignment are put well, note driving away the bubble between film and glue, plastic plate fine flour and black flour clamp, and put into electric turn trough.

6) power-on, 100V constant voltage electrophoresis 1-1.5h(is according to molecular weight of albumen).

7) sealing: take out pvdf membrane with TBST rinsing a little, be immersed in the plate that contains confining liquid and slowly sway 2h.

8) primary antibodie is hatched: primary antibodie is diluted with 1:1000 with confining liquid, and wherein, 4 ℃ are spent the night in the pvdf membrane submergence.

9) two anti-hatching: TBST rinsing film three times, each 5-10min.Two anti-ly dilute with 1:10000 with confining liquid, and pvdf membrane room temperature shaking table is hatched 1h.

10) ECL colour developing: TBST rinsing film 3 times, TBS rinsing film 2 times, each 5-10min.Open imaging system, precooling 30min.By the nitrite ion uniform fold that mixes to pvdf membrane, the preservation picture of taking pictures in time.

9, test-results

The best antigen coated concentration of table 3

After the laying hen immunity, the detection proof recombinant protein by antibody titer has good immunogenicity, can cause the immune response of laying hen, and can produce corresponding specific antibody.The coated concentration of the best of antigen is 1:3200, i.e. 0.03 microgram/hole.Two exempt from after the antibody titer of all EF reached respectively 3200, two and exempted from the rising that occurs tiring of rear second week, reach respectively 64000 to the 7th all antibody titers, for the downward trend of tiring occurring.And fusion rotein has been induced the specific antibody that produces anti-2FliC and two kinds of albumen of EtpA, has reached the purpose for preparing fusion rotein.Spray-dired whole egg powder is tired and is respectively after testing 32000.

The yolk antibody molecular weight that the SDS-PAGE electrophoresis showed is purified is in 180 left and right, and purity reaches 70-80%.The western-blot detected result has shown the immunogenicity that recombinant protein is good, has again proved the laying hen immunity and has achieved success.

The stability study of embodiment 4 yolk antibodies

1, the THERMAL STABILITY of yolk antibody

By IgY PBS(pH=7.2) be diluted to 1mg/mL, minute be filled to the EP pipe, every pipe 1mL, get wherein a pipe and measure it and tire in contrast.Get the IgY that above-mentioned dilution is good, in 60 ℃, 65 ℃, 70 ℃, 75 ℃ water-baths, process 30min respectively, 0,10,20, the 30min each point is cooling in taking out and putting frozen water immediately, envelope antigen ELISA measures its absorbance.Draw under differing temps and tire-time changing curve.

2, the acid acceptance of yolk antibody research

With the PBS damping fluid that the pH value is 2,3,4, IgY is diluted to lmg/mL, hatches 3h for 37 ℃, 0,0.5,1,2,3h, then PBS does 10 times of dilutions, makes the pH value be 7.0 left and right, and ELISA measures and tires.Draw under different pH and tire-time changing curve.

3, the enzymolysis stability study before and after the yolk antibody purification

Prepare treatment solution: take two parts of the whole egg powders of 5g, a with the abundant stirring and evenly mixing of 30mL deionized water, regulate PH to 5.0, centrifuging and taking supernatant after standing 4-6h, now second part is also diluted with consubstantiality ponding.Two parts for the treatment of solutions are all regulated to PH to 3.5.

Simulated gastric fluid (Simulated gastric fluid, SGF): take NaCl2g, stomach en-3.2g, add the 950ml deionized water to make it to dissolve, and regulates pH to 3.5 with dense HCI, is settled to l L.

Treatment solution (with the every g whole egg powder estimation of IgY10mg/) mixes enzyme with above-mentioned solution: substrate=1:100(mass ratio), 37 ℃ of reaction 0-2h, add NaHC03 solution (0.1M, pH9.6) termination reaction, makes its pH in 6.0 left and right.By second part, regulate PH to 5.0, centrifuging and taking supernatant after standing 4-6h.Then with ELISA, detect its remaining activity.

4, test results and analysis

1) after differing temps being set IgY being processed, dilute antibody with PBST1:1000, with indirect ELISA, detect the variation of OD450 value, wherein negative control OD450 is 0.111.Test-results such as Figure 11, when result was presented at 60 ℃ and 65 ℃, tiring of IgY descended slowly, i.e. and after half an hour, activity still keeps 98% and 90%, now has good thermostability, illustrate that to IgY employing pasteurization be feasible; In the time of 70 ℃, IgY is heated to 10min, activity has descended 22%, and while exceeding 70 ℃, the active beginning descends rapidly, after 5min, namely loses most of vigor.Therefore under higher than 70 ℃ of conditions, the activity of IgY is inactivation very easily.

2) after different pH process IgY, carry out 1:2000 dilution antibody with PBST, with indirect ELISA, detect the variation of OD450 value, wherein to tire be 8000 to yolk antibody stoste, and negative control OD450 is 0.175, test-results such as Figure 12, under the pH=4 condition, the activity of IgY is substantially unaffected; But when the pH value dropped to 3, activity just started slow decreasing, be 2000 but after 3h, detect that residue tires; When pH was 2, after 0.5h processed, IgY was not positive under this extent of dilution.Experiment shows that IgY has certain acid resistance, at pH > 3 o'clock be more stable.

3) in the simulated gastric fluid of pH3.5, IgY 37 ℃ hatch different time after, detect the IgY activity.Negative control is that what in 0.070, figure, show is the OD value under the 1:800 extension rate.Result such as Figure 13,37 ℃ of stomach en-s are processed 0.5h, and the OD value that ELISA detects the IgY activity is respectively 0.48 and 0.29, and in whole processing, the OD value of whole egg powder is all than the height of purification, and the OD value of 2h is respectively 0.25 and 0.094.Other compositions in visible whole egg powder have played provide protection to the hydrolysis of yolk antibody.

The effect evaluation of embodiment 5 yolk antibodies

1, subjects

6-8 Kunming kind SPF female mice in age in week;

2, test materials

Table 4 bacterial strain

Annotate: CFA(colonization factor antigen, colonization factor antigen); CS(coli surface antigen, the intestinal bacteria surface antigen)

Yolk antibody is that embodiment 3 prepares, the EF protein immunization laying hen gained (concentration is 10mg/mL, and tiring is 64000) that the yolk antibody of EF group is merged by EtpA and FliC respectively.

3, mouse challenge test

1) mouse arrives rear adaptive phase 2-3 days, and random packet,, freely drink water and search for food by 8 every group.

2) attack the front 24-48h mouse drinking-water of poison and change to the sterilized water that contains Streptomycin sulphate (5g/L), to eliminate normal intestinal flora.

3) prepare bacterium: the mono-bacterium colony of picking ETEC in fresh TBS substratum, 37 ℃ of shaking table overnight incubation.Attack poison same day next day again with 1/100 amount inoculation fresh culture, treat that OD600 reaches 1.0(approximately 5 * 108CFU/mL), the centrifugal bacterial sediment that obtains of bacterium liquid, be resuspended in the aseptic PBS that 1/10 bacteria liquid amasss, stand-by.

4) attack the front 12h fasting of poison, drinking-water changes to distilled water.

5) attack the front 1-3h abdominal injection Cimitidine Type A/AB (50mg/kg body weight) of poison, reduce the impact of hydrochloric acid in gastric juice on thalline.

6) every mouse is filled with and feeds the fresh bacterium liquid of 200 μ L through the gavage pin, the corresponding gavage 50 μ L IgY solution of each treatment group after 1h, normal feeding and drinking-water afterwards.

7) 12h fasting before anesthesia, normal drinking-water.

8) attack after poison anesthesia after 48-72h and puts to death and dissects mouse, intercept 3cm ileum (time blind junction make progress 6cm), vertically cut enteron aisle open, 2mL PBS dilutes content, and vortex is incubated at room 10min after 5 seconds, and vortex is 5 seconds again.The gained supernatant is got 100 μ L coating maconkey agar plate counts (Allen et al2006) after with 10-1 and 10-2, diluting.

9) 20 bacterium colonies of each dull and stereotyped picking, the primer of the distinctive heat-stable toxin LT of design ETEC (forward: 5'-CTAGTTTTCCATACTGAT-3' and reverse: 5'-CCCCAGTCTATTACAGAA-3') carry out the PCR detection, take and identify that whether bacterium colony is ETEC, calculates positive bacterium colony ratio.

4, cell adhesion test

Method: in the fresh LB substratum of 10mL, by 1/100 inoculated bacteria, reach the growth logarithmic phase in 37 ℃ of 225r/min shaking culture to bacteriums.Add respectively 600 μ L bacterium liquid in the centrifuge tube of 1.5mL, at the centrifugal 5min of 5000r/min, remove supernatant, add the resuspended thalline of cell culture medium of 600 μ L, for treatment group, add the yolk antibody of 100 μ L.Infection multiplicity (MOI) with 100:1 joins in Tissue Culture Plate, control group (common yolk antibody) and treatment group all is placed in to 37 ℃ and hatches 1h.After incubation 1h, with aseptic PBS wash-out 3 times, remove the bacterium do not adhered to, with the bacterium of cell adhesion, hatch 5-10min through 200 μ L0.1%Triton-X-100 phosphoric acid salt, 1000 times of 100 μ L suspension dilutions evenly are coated with to the LB flat board, the bacterial count that the colony number of inferior day growth (CFU) namely adheres to afterwards.Adhesion rate=1000* colony number/total bacteria (each processes triplicate).

This test is respectively organized the data GraphPad Prism5.0 and is added up, and every group of colony number of mouse adherence test is with the geometric mean value representation, and the cell adhesion test means with mean+SD, in twos relatively through distribution free Mann-Whitney one tailed test.Significant difference is judged in P<0.05.

5, test results and analysis

With three kinds of common people source ETEC, attack malicious mouse, then the yolk antibody for preparing of the different albumen of gavage, the bacterium that enteron aisle adheres to recovers in the maconkey agar substratum, and 20 bacterium colonies of picking are PCR and are detected.The positive colony number of the colony number adhered to=plate count mean value * extent of dilution */20.

Each is organized the mouse intestinal bacterial adhesion and the results are shown in Figure 14, wherein after H10407 attacks poison, fusion rotein EF group does not reach conspicuous level difference with contrasting, but it should be noted that, 8 mouse of attacking poison only have 3 ETEC that adhesion detected, have also shown the adhesive attraction that presses down of fusion rotein antibody.In E519 and 44815 attacks malicious group, the field planting of ETEC at mouse intestinal that yolk antibody prepared by fusion rotein EF has all significantly suppressed (P<0.01, P<0.05).

As shown in figure 15, carry out cell adhesion with ETEC H10407, compare with control group, bacterial strain H10407 and the E519 bacterial adhesion number after adding special yolk antibody all significantly reduces.And bacterial strain 44815 is in adhesion process, treatment group is compared with the control without significant difference, but presented certain reduction trend (P=0.06).Therefore the also similar anti-adhesion effect of verifying yolk antibody on cell levels.

Claims (6)

1. contain the HpaA gene etpA of people source enterotoxic Escherichia coli and the recombinant plasmid pET-EF of flagellin gene fliC, its nucleotide sequence is as shown in SEQ ID NO.1.

2. express the recombination bacillus coli BL21 (DE3) of the described recombinant plasmid pET-EF of claim 1/pET-EF.

3. the fusion rotein of a separation, its aminoacid sequence is as shown in SEQ ID NO.2.

4. the application of fusion rotein claimed in claim 3 in preparing yolk antibody.

5. yolk antibody, it is to take fusion rotein claimed in claim 3 to be antigen, laying hen is carried out to immunity is prepared from.

6. the application of yolk antibody claimed in claim 5 in the enterotoxic Escherichia coli diarrhoea food of preparation treatment people source.

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