CN103409455B - The yolk antibody of anti-human source enterotoxic Escherichia coli adhesin antibodies and application thereof - Google Patents

Abstract

The invention discloses a kind of containing 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.The present invention utilizes genetic engineering technique to construct recombinant plasmid pET-EF, by above-mentioned recombinant plasmid transformed to e. coli bl21 (DE3), induces recombinant expressed bacterium BL21 (DE3)/pET-EF expressed fusion protein EF.Utilize the recombinant protein active immunity just laying hen of expressing, prepare the yolk antibody for this albumen, the IgY extracted demonstrates recombinant protein and has good antigenicity, have detected the stability under the condition of different pH, temperature and enzyme, and utilize mouse intestinal challenge test, prove that the IgY purified can suppress ETEC bacterial strain in vitro to epithelial adhesion.

Description

The yolk antibody of anti-human source enterotoxic Escherichia coli adhesin antibodies and application thereof

Technical field

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

Background technology

Enterotoxic Escherichia coli (Enterotoxigenic Escherichia coli, ETEC) is the Main Pathogenic Bacteria (Black1990 causing diarrhoea; Ericsson2003).Annual ETEC can cause 2.8-4 hundred million diarrhoea less than 5 years old in children according to statistics, and not etc., only can have mild diarrhea, can not be severe cholera sample to state of an illness weight yet, causes about 30-50 ten thousand Infant and child deaths (WHO2004) every year.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 passes through adhesin; first adhere on the epithelial acceptor of mucous membrane of small intestine brush border of host; the cleaning of opposing intestines peristalsis and content; realize surely growing rear and discharging one or both enterotoxins (enterotoxin); cause fluid and electrolyte balance imbalance in cell, thus 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 adhesin mainly refers to pili adhesin and phage surface pili antigen CFAs or CS in the past.Identify more than 22 kinds of CFAs in current people source ETEC, common are CFA/I, CS1-CS7, CS14, CS17, CS21(Qadri et al2005), but without cross-protection (Qadri et al2006) between allos pili.Research finds that adhesion process also needs the participation of non-pili adhesin in addition.Intestinal bacteria comprise ETEC all can bear flagellum on the whole body or two ends, and quantity is few compared with pili, but length is the several times of thalline.Recent study shows except the pili spreading all over phage surface, the flagellum of end life not only can as locomotive organ, and this mobility (motility) works, as Salmonellas (Allen-Vercoe et al1999), chicken pathogenic escherichia coli (La Ragione et al2000) at pathogenic courses such as some Gram-negative bacteria field planting, invasion and proinflammatory reactions.

Roy etc. (2006) have found with swivel base plasmid a kind of albumen EtpA that ETEC secretes, and are one of members of two companion's secretin (TPS), by ETEC is peculiar.Be distributed in the CFAI of 59%, 56% CFA II, 75% CS14 or CS17 ETEC bacterial strain in, and this several fimbriae type occupies the ETEC(Wolf1997 of about 60%).Research is illustrated EtpA and is done mutually by conserved regions that is outer born of the same parents and flagellin (FliC), (Roy et al2009a) that the function served as bridge mediating flagellum and host cell plays.By lacking and recombinating, test cell line confirms that this mutual work has keying action for effective field planting of ETEC, namely etpA or fliC deletion mycopremna adherent cell ability is compared wild-type and is greatly reduced, or the antibody both adding in the medium also can reduce the bacterial count of adhesion, and external source adds recombinant protein or this gene of homologous recombination can make gene-deleted strain regain the function (Roy et al2009a) of adherent cell.

The conserved regions of usual flagellin is hidden in flagellum neck, but it can be exposed to flagellum top instantaneously, catches EtpA molecule for identifying eukaryotic cell surface receptor.Flagellum mediate adhesion does not have serotype to limit, as at flic(H11) recombinate in mutant strain flic(H48) motion and adhesive capacity can be regained.The ETEC of multiple H serotype can be suppressed epithelial adhesion with identical anti-EtpA serum in addition.In mouse model, with rEtpA and rFliC nasal feeding immune mouse, obtain the result similar in vitro tests, and the two adhesiving effect that presses down simultaneously used is better than being used alone (Roy et al2008,2009b).This novel adherence mechanism is present in most ETEC, and the protein molecular of other and EtpA homology also may adopt this similar mode of action in respective Gram-negative bacteria, although concrete mediated process it be unclear that, infer that two kinds of albumen can as the candidate antigens of New-type wide-spectrum vaccine development.

A large amount of test-results shows; by providing exogenous antibodies (comprising blood antibody and yolk antibody) to improve its passive immunization level to young animal, the infection (particularly enteron aisle diarrhoea) coming prevention and therapy bacterium and virus is effective approach (Hatta et al1993b; King's Jie 2007).Yolk immunoglobulin (Egg yolk immunoglobulin, IgY) becomes a kind of Substitutes For Antibiotic having much potentiality.U.S. FDA is listed in " It is generally accepted safe material " (Coleman1996).At present for the existing extensively research of IgY exploitation of various gastrointestinal tract disease.Investigator prepares IgY and treats ETEC diarrhoea (Yokoyama et al1992 using pili as antigen; Ikemori et al1992; Marquardt et al1999).Report the earliest derives from Perorally administrable antimicrobial hair yolk antibody and can prevent K88, K99 and 987P ETEC from infecting (Yokoyama et al1992).After piglet birth, namely 4h infects ETEC, the basic all survivals for the treatment of group (oral IgY), and shows dose-dependent effect, and placebo and control group mortality ratio are greater than 75%, add IgY in vitro and inhibit ETEC to chitling cell adhesion too.Marquardt etc. (1999) also prepare yolk antibody with purification K88 pilin Immune Laying Hens, poison treatment is attacked to birth 3 age in days and 21 age in days weanling pigs, wherein the diarrhoea of age group piglet on the 3rd is entirely cured after oral IgY24h, and the diarrhoea of common IgY group continue and mortality ratio reaches 62.5%; Having there is of short duration diarrhoea in weanling pig oral IgY after attacking poison, all survive and body weight increase, and severe diarrhea and dehydration also appears in control group, and namely occur death after infecting 48h.King's Jie (2007) oral anti-ETEC pili subunit yolk antibody can provide effective passive immune protection to piglet, the infection of opposing enteron aisle germ.

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

Summary of the invention

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

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

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

4th object of the present invention is that the fusion rotein EF described in utilization prepares yolk antibody.

The application that 5th object of the present invention is to provide yolk antibody and suffers from diarrhoea in food at the anti-human source enterotoxic Escherichia coli ETEC of preparation.

The present invention is achieved by the following technical solutions:

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

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 object fragment is the 1701bp of 5' end; Flagellin fliC(sequence number is AY906918) total length 1464, conserved regions is the 519bp of 5' end.With people source enterotoxic Escherichia coli type strain H10407 for test materials, design primer, object fragment after pcr amplification is connected to expression vector, successfully construct the recombinant expression plasmid of both key gene etpA in bacterial adhesion epithelial cell process and flagellin gene fliC fusion gene, wherein between fusion gene with one section of hydrophobicity connection peptides (Gly ₄ser) ₃do and connect.Carry out enzyme to recombinant plasmid and cut qualification and order-checking, result display builds correct, by its called after pET-EF.

Two, recombination fusion protein EF abduction delivering and extraction purification

Recombinant plasmid pET-EF is transformed into e. coli bl21 (DE3), does abduction delivering with inductor sec.-propyl-β-d-thiogalactoside (Isopropyl-β-d-Thiogalactoside, IPTG).With different inducing temperatures and inducer concentrations successive optimization prokaryotic expression, obtaining best abduction delivering condition is: 37 DEG C of shaking tables are cultured to OD600 and reach 0.5-1.0, add IPTG to suitable concentration (0.5mM), continue to cultivate 3-4h, final expression amount, all more than 15%, exists with the form of inclusion body.By the inclusion body protein that inclusion body obtains after pressure breaking, denature and renature concentrate, measure protein concn with Bradford method.

Three, yolk antibody is prepared with recombinant protein Immune Laying Hens

To renaturation, concentrated after protein solution in add the tween-80 of sterilizing to final concentration 4%, then in tissue refiner, mix 30min with equal-volume white-oil adjuvant, fully emulsified to protein concentration be 0.5mg/mL.2 groups are divided at random, blank group 50 (physiological saline) and immune group 100 by laying hen.Adopt chest muscle injection (every side 500 μ L).Head to exempt from after 2 weeks each group and uses same oil breast seedling booster immunization respectively once, interval 2 weeks is again immune, two exempt from after respectively organize random acquisition 5 pieces of eggs weekly, purifying, collecting yolk antibody, the change of indirect ELISA monitoring antibody titer, western-blot identifies the specificity of yolk antibody, treats that yolk antibody is tired and reaches 1:2 ⁹rear collection egg.The egg of collection is shelled, stirs, spraying dry (air inlet 150 DEG C gives vent to anger 70 DEG C), load after cooling in the sample sack of sealing.

Four, the stability test of yolk antibody

In 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C water-baths, process 30min respectively, or pH value be 2,3,4 damping fluid IgY is diluted to lmg/mL, hatch 3h for 37 DEG C, or with simulated gastric fluid or intestinal juice process antibody 0-4h.Result shows: when temperature is lower than 70 DEG C or pH > 3, and IgY activity keeps stable; Process more than temperature 70 C, after 5min, namely lose 80% activity; Process 0.5h in the environment of pH=2 after, IgY tires and is negative.In the stomach en-environment of pH=1.2, then hydrolysis is rapid, and trypsinase has no significant effect IgY activity.

Five, the application effect appraisal of yolk antibody

By setting up the field planting model of ETEC in mouse intestinal, attack malicious mouse with three kinds of clinical common different serotypes bacterial strains, after the yolk antibody simultaneously prepared early stage is oral, different antibody groups creates bacterium and presses down adhesiving effect in various degree.Wherein the antibody group of reference culture H10407 reduces the quantity of infecting mouse.E519 and 44815 does not produce EtpA, but the IgY that after the two attacks poison prepared by oral fusion rotein EF all significantly anti-bacteria adhesion number (being respectively P<0.01, P<0.05).By cell adhesion experiment, confirm that yolk antibody has played similar anti-adhesion effect in vitro equally.

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 be insert the recombinant plasmid of etpA and fliC two genes, XhoI, EcoRI are 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 the double digestion qualification electrophoresis schematic diagram of recombinant expression plasmid pET-EF.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 IPTG inducible protein is expressed.In figure, 1,2 represent before and after recombinant protein EF induction respectively, and gained molecular weight of albumen is 84kDa.

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

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

Fig. 7 inducer concentrations is on the impact of albumen EF expression amount.1: before induction; 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 to tire rule schematic diagram over time for yolk antibody after second time immunity.

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 represent respectively and attack malicious mouse with ETECH10407, E519 and 44815; Sea line represents geometric mean, and * represents significant difference, and * * represents that difference is extremely remarkable.

Embodiment

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

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

1, the present embodiment use bacterial strain and plasmid vector 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: the precious biotechnology company limited in Dalian;

Agarose: Biowest Agarose packing;

Tryptones, yeast extract: Britain OXOID;

Trysinization soybean broth: Bi Di Medical Devices Co., Ltd. of the U.S.;

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 extract related solution

PBS(0.012M): take NaCl8g, KCl0.2g, Na ₂hPO ₄12H ₂o3.58g, KH ₂pO ₄0.27g, adds ddH ₂o is settled to 1L, packing 121 DEG C of sterilizing 30min.

Chloroform/primary isoamyl alcohol (24:1): chloroform 480ml, primary isoamyl alcohol 20ml, mixing moves into 4 DEG C of preservations in Brown Glass Brown glass bottles and jars only.

Proteinase K (20mg/mL): take powder 20mg and be dissolved in ddH ₂o1mL, mixes-20 DEG C of preservations.

3.2 substratum

1) LB substratum: Tryptones 1g, yeast extract 0.5g, NaCl1g, agar powder 1.5g(LB is dull and stereotyped), add ddH ₂o is dissolved to 100mL, stirs, 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): 1g powder is added 10mL ddH at aseptic operating platform ₂o dissolves, and sterile filters is filtered, and point is filled to 1.5mL EP and manages ,-20 DEG C of preservations.

4) kantlex liquid storage (50mg/mL): 1g powder is added 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, adds ddH ₂o is dissolved to 900mL, regulates pH to 8.0, is settled to 1L.1 × TAE is diluted to during use.

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, four degree of preservations.

3) EB dye liquor (10mg/mL): take 1g powder, carefully add 100mLddH ₂o, is stirred to and dissolves completely, 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

Construction recombination plasmid process is shown in Fig. 1.

The extraction of 4.1 DNA of bacteria

1) in aseptic operating platform, from slant medium, picking ETEC H10407 bacterium liquid is coated with TSB flat board, and next day, picking list bacterium colony was in 5mL TSB substratum, 37 DEG C of incubated overnight.

2) next day 10000rpm collected by centrifugation thalline, PBS washs 2 times and resuspended thalline to 500 μ L.

3) add 100 μ L10%SDS, 10 μ L20mg/mL Proteinase Ks, hatch 3h or 37 DEG C for 50 DEG C and spend the night.

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

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

6) 0.6(V/V is added) Virahol, put upside down mixing, leave standstill the centrifugal 5min of 5min, 10000rpm, the precipitation of collection, through 75% ethanol wash of precooling, is dissolved in 100 μ L ddH after drying ₂in O ,-20 DEG C of preservations.

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 object fragment is the 1701bp of 5' end.Flagellin fliC(sequence number is AY906918) total length 1464, conserved regions is the 519bp of 5' end.When etpA and fliC is done to merge, in the former reverse primer, introduce one section of 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

Note: wavy line is hydrophobicity connection peptides; Underscore represents restriction enzyme site.

4.3PCR amplifying target genes

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

Reaction conditions following (cycle number: 35):

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

4.4PCR product detects and reclaims

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

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

The clone of 4.5DNA fragment

1) competence DH5 α is prepared:

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

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

C. in aseptic operating platform, transfer bacterium liquid, in the centrifuge tube of precooling, continues to place 30min on ice.Then 4 DEG C of collected by centrifugation thalline (4000rpm, 10min).

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

E. above two steps repeat 2 times.

F.2mL the 0.1mol/L CaCl of precooling ₂resuspended thalline, adds sterile glycerol to final concentration 15-20%, and with 100 μ L/ pipe packing ,-80 DEG C save backup.

2) connect: 5 μ L linked systems are as follows, and 4 DEG C are spent the night.

PCR primer	2
		PMD18-T	0.5
Solution I	2.5

3) transform

A. get 10 μ L to connect products and add in the DH5 α competence of just having thawed, beat mixing gently, place 30min on ice.Now open recirculated water bath and be preheated to 42 DEG C.

B.42 DEG C water-bath thermal shock 90s, places 1-2min on ice fast.

C. add LB substratum 400 μ L, 1h is cultivated in 37 DEG C of concussions.

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

E. next day picking list bacterium colony, bacterium liquid PCR detects with or without positive colony, and T clone respectively called after pMD-etpA, pMD-etpAL, pMD-fliCL, pMD-fliC1, the pMD-fliC2 that will obtain.

4.6 construction expression plasmids

1) picking positive bacteria drops into row enlarged culturing, plasmid extraction is carried out according to the explanation of plasmid Mini Kit, and do double digestion and connection by following 20 μ L systems to containing the carrier T of object fragment and empty expression vector, wherein in linked system the ratio of DNA and carrier with mole ratio between 1:1-5:1:

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

3) bacterium colony PCR selects above positive colony, the bacterial strain enlarged culturing through checking order correct, and extraction recombinant plasmid does enzyme and cuts qualification ,-20 DEG C of preservations in a small amount.Easy for writing, recombinant expression plasmid is abbreviated as: pET-EF.With EcoRI and XhoI, double digestion is done to recombinant plasmid, the results are shown in Figure 3.

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

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

1, reagent

Tryptones, yeast extract: Britain OXOID;

Trysinization soybean broth: Bi Di Medical Devices Co., Ltd. of the U.S.;

Tris alkali, glycine, SDS:Amresco packing;

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

2, solution preparation

LB substratum as shown in Example 1.

IPTG liquid storage (1mol/L): the powder of 1g is added 4.2mL sterilizing ddH at aseptic operating platform ₂o, sterile filters is filtered rear point and is filled in 1.5mLEP pipe.

SDS-PAGE electrophoresis related solution:

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

2) 5 × electrophoretic buffer: Tris15.10g, Gly72.06g, SDS5g, adds ddH ₂o is settled to 1L.5 times are diluted during use.

3) 30% polyacrylamide: take N successively, N'-methylene diacrylamide 1g, acrylamide 29g, adds ddH ₂o is settled to 100mL, proceeds to four degree of preservations in brown bottle.Weighing process notes protection.

4) separation gel damping fluid: Tris18.17g, adds ddH ₂o90mL, dense HCl regulates pH to 8.8, is settled to 100mL, four degree of preservations.

5) concentrated glue damping fluid: Tris12.11g, adds ddH ₂o90mL, dense HCl regulates pH to 6.8, is settled to 100mL, four degree of preservations.

6) 10%SDS: take sodium lauryl sulphate 10g, adds 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 of preservations, used in two weeks.

8) 1%TEMED: draw N, N, N', N'-Tetramethyl Ethylene Diamine 1mL, add ddH ₂o to 100mL, proceeds to four degree of preservations in brown bottle.Note environment ventilation.

9) coomassie brilliant blue staining liquid: coomassie brilliant blue R_250 1.25g, methyl alcohol 450mL, glacial acetic acid 50mL, adds ddH ₂o450mL.

10) destainer: methyl alcohol 50mL, glacial acetic acid 75mL, adds ddH ₂o875mL mixes.

Inclusion body purification related reagent:

1) BufferA:Tris6.055g, EDTA0.186g, NaCl2.925g, glycerine 50mL, adds ddH ₂o is settled to 1L.DTT is now added to final concentration 0.5mmol/L before using.

2) 10%SKL: take dodecyl creatine sodium 1g, add ddH ₂o10mL, is stirred to and dissolves completely.

3) powder that 50mM Sleep-promoting factor B: 1g packs adds ddH ₂o32.65mL, pressure-vaccum is to dissolving.

4) powder that 100mM reduced glutathion: 1g packs 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, regulates pH to 8.0, is settled to 1L.

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

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

The expression plasmid successfully constructed is transformed expression strain BL21(DE3), picking list bacterium colony incubated overnight.Next day, bacterium liquid joined 5mL containing in the fresh culture of resistance by 1% inoculum size, and 3-4h cultivated by 37 DEG C of shaking tables.When OD600 reaches 0.5-1.0, add IPTG to final concentration 1mmol/L, continue to cultivate 3-4h.Get 500 μ L bacterium liquid collected by centrifugation thalline, 50 μ LPBS are resuspended, and equal-volume adds sample solution, boiling water bath 5min, get 10 μ L supernatants and carry out 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, collect the thalline of abduction delivering, the abundant resuspended thalline of 5mLbufferA, is placed on ice.Treat that pressure breaking instrument temperature is down to 4 DEG C, start broken thalline, repeat 3 times.Centrifugation supernatant, the abundant resuspended precipitation of 5mLbufferA, respectively gets 50 μ L to carry out SDS-PAGE electrophoretic analysis.

5, the optimum induction of recombinant protein

In protein expression process, the concentration of inductor and inducing temperature are the important factors affecting expression amount.Therefore by design differing temps (37,25,17 DEG C) 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 adds inductor when OD value reaches 0.5-1.0 at different conditions, and except 25 DEG C and 17 DEG C need be continued incubated overnight, all the other collect thalline after all cultivating 3-4h, as 3 carry out SDS-PAGE electrophoresis detection.

6, the extraction purification of recombinant protein

Inoculate recombinant expressed bacterium in 300mL substratum, with the condition abduction delivering after optimizing.From nutrient solution, collect thalline, then be resuspended in the BufferA of 1/10 volume of precooling, broken 3-5 time of pressure breaking instrument, notice that whole process keeps 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 is clarified, 4 DEG C of standing 2h.Continue the PEG4000,600 μ L Sleep-promoting factor B and the reduced glutathions that add final concentration 0.2%, 4 DEG C of centrifugal 15min of standing 2h, 10000rpm discard undissolved thalline impurity.Dialysis tubing treatment process: the length of dialysis tubing by 20-30cm is cut out.Boiling water bath 10min in A liquid, distilled water cleans.Boiling water bath 10min in B liquid, distilled water cleans, 4 DEG C of preservations.Note when taking being with gloves.The protein liquid of renaturation loads the dialysis tubing handled well, totally 2 days, and period changes liquid for several times, and PEG embeds dialysis tubing and is concentrated into desired concn.SDS-PAGE electrophoresis detection purity of protein, Bradford method measures protein concentration.

7, test-results

IPTG induces recombination bacillus coli BL21 (DE3)/pET-EF to express, and the results are shown in Figure 4.The albumen that corresponding positions is equipped with expection size occurs, EF molecular weight is about 84KD respectively, expression amount about 15%.Soluble analysis display (see figure 5) albumen all major part is present in precipitation with the form of inclusion body, few in supernatant.Fig. 5 display determines that recombinant protein inductive condition is that 37 DEG C 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 recombinant protein Immune Laying Hens

1, reagent

White oil, stearic acid aluminium, Si Ben-80, tween-80: Wuhan Ke Qian biological products limited liability company presents.

The anti-chicken IgG:sigma of HRP rabbit

Nitrite ion Supersignal west pico test kit: Thermo

2, solution preparation

ELISA related reagent:

PBS(0.012M): take NaCl8g, KCl0.2g, Na ₂hPO ₄12H ₂o3.58g, KH ₂pO ₄0.27g, adds ddH ₂o is settled to 1L, packing 121 DEG C of sterilizing 30min.

Coating buffer (0.05M carbonate buffer solution): NaHCO ₃2.93g, Na ₂cO ₃1.59g, adds ddH ₂o900mL dissolves completely and regulates pH to 9.6, and be settled to 1L, normal temperature is placed.

Washings: add the tween 20 that final concentration is 0.05% in PBS.

Confining liquid: the bovine serum albumin (BSA) of 3% 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 adds 30%H ₂o ₂30 μ L.Now with the current.

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

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.

The tween 20 that final concentration is 0.05% is added in TBST:TBS.

Confining liquid: containing the TBST of 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, stirs, and is heated to about 80 degree, and continues stirring until solution clarification.Slowly add the span-80 of 6mL, continue to stir 20min, be white-oil adjuvant after cooling, 4 DEG C of preservations.To renaturation, concentrated after protein solution in add the tween-80 of sterilizing to final concentration 4%, then in tissue refiner, mix 30min with equal-volume white-oil adjuvant, fully emulsified to protein concentration be 0.5mg/mL.

4, the immunity of laying hen

Select Roman egghen 450 of raising in cages 20 week age, divide 5 groups at random by laying hen, blank group 50 (physiological saline) and immune group 100.Adopt chest muscle injection (every side 500 μ L).Head to exempt from after 2 weeks each group and uses same oil breast seedling booster immunization respectively once, and interval 2 weeks is again immune, two exempt from after respectively organize weekly random acquisition egg ELISA monitoring and see that antibody titer changes, treat that yolk antibody is tired and reach 1:2 ⁹rear collection egg, cryopreservation.

5, spraying dry whole egg powder

The egg of collection is shelled, stirs, spraying dry (air inlet 150 DEG C gives vent to anger 70 DEG C), load after cooling in the sample sack of sealing.Gather the whole egg powder of different time sections in spraying dry, 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:

Be separated yolk, namely break an aperture in eggshell one end, flow to end by egg white, expand 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.The egg yolk liquid distilled water diluting of 8 times of volumes, stirs, and salt acid for adjusting pH, to 5.0-5.2, is placed 6h or spends the night for 4 DEG C.The centrifugal 20min of 10000rpm, collects supernatant and obtains water soluble ingredient WSF(water-solve fract).Supernatant adds ammonium sulfate to 50% saturation ratio, stirs, and place 2h or spend the night for 4 DEG C, fully precipitate, the centrifugal 10min of 10000rpm, abandons supernatant, and precipitation is dissolved in PBS to former egg yolk liquid volume.Add ammonium sulfate to 33% saturation ratio, repeat above step.4 DEG C of dialysis 24h ,-20 DEG C save backup.

7, indirect ELISA mensuration yolk antibody is tired

Two exempt from after within one week, start to gather egg, at random weekly from 100 laying hens, collect 5 pieces of eggs, extract after antibody, with recombinant protein envelope antigen, indirect ELISA measures the antibody titer that two exempt from rear 1-7 week.Section sample before, during and after spray-drying process, in kind detects after extracting antibody and tires.

Square formation volumetry determines best antigen coated concentration.Antigen is done doubling dilution (1:400,1:800,1:1600,1:3200,1:6400) with coating buffer respectively, and every hole 100 μ L wraps by polystyrene reactant plate, and each extent of dilution two parallel holes, 4 DEG C are spent the night.By sample doubling dilution on demand during working sample, enzyme mark rabbit anti-chicken IgG PBST does 1:10000 dilution, and carry out ELISA detection, concrete sample determination step is as follows:

1) wrapped by polystyrene reactant plate by every hole 100 μ L by antigen, 4 DEG C are spent the night;

2) discard liquid, PBST washs 3 times and pats dry, and adds confining liquid every hole 100 μ L, hatches 2 hours for 37 DEG C;

3) as above wash, add test antibodies every hole 100 μ L(PBST and dilute), hatch 1 hour for 37 DEG C;

4) as above wash, add the every hole 100 μ L of ELIAS secondary antibody (PBST dilution), hatch 1 hour for 37 DEG C;

5) as above wash, add the OPD nitrite ion 100 μ L now joined, 37 DEG C of lucifuges hatch 15min;

6) every hole adds stop buffer 50 μ L, measures the OD value in each hole, return to zero with blank well under 450nm wavelength.The ratio of test antibodies OD value and negative control is judged to the positive when being greater than 2.1, and antibody titer is most high dilution when there is positive reaction.

8, western-blot detects recombinant protein immunogenicity

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

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

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

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

5) plastic plate black flour, sponge, 2 metafiltration paper, gel, pvdf membrane, 2 metafiltration paper, sponge alignment are put well successively, note the bubble of driving away 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) close: take out pvdf membrane TBST rinsing a little, be immersed in the plate containing confining liquid and slowly sway 2h.

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

9) two anti-to hatch: TBST rinsing film three times, each 5-10min.Two anti-confining liquids dilute with 1:10000, and pvdf membrane room temperature shaker hatches 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 on pvdf membrane, preservation picture of taking pictures in time.

9, test-results

The best antigen coated concentration of table 3

After laying hen immunity, prove that recombinant protein has good immunogenicity by the detection of antibody titer, the immune response of laying hen can be caused, and corresponding specific antibody can be produced.The best bag of antigen is 1:3200 by concentration, i.e. 0.03 microgram/hole.After exempting from two, the antibody titer of one week EF reaches 3200 respectively, and two exempt from rear second week occurs rising of tiring, and reaches 64000 respectively, for there is downward trend of tiring to the 7th week antibody titer.And fusion rotein is induction of the specific antibody producing anti-2FliC and EtpA two kinds of albumen, reaches the object preparing fusion rotein.Spray-dired whole egg powder is tired and is respectively 32000 after testing.

The yolk antibody molecular weight that SDS-PAGE electrophoresis showed is purified is about 180, and purity reaches 70-80%.Western-blot detected result shows the good immunogenicity of recombinant protein, has again proved laying hen immunity and has obtained successfully.

The stability study of embodiment 4 yolk antibody

1, the THERMAL STABILITY of yolk antibody

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

2, the acid acceptance research of yolk antibody

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

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

Prepare treatment solution: the whole egg powder two parts taking 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 PH to 3.5.

Simulated gastric fluid (Simulated gastric fluid, SGF): take NaCl2g, stomach en-3.2g, adds 950ml deionized water and makes it to dissolve, regulate pH to 3.5, be settled to l L with dense HCI.

Treatment solution (with the estimation of IgY10mg/ every g whole egg powder) mixes with above-mentioned solution, enzyme: substrate=1:100(mass ratio), 37 DEG C of reaction 0-2h, add NaHC03 solution (0.1M, pH9.6) termination reaction, make its pH about 6.0.PH to 5.0 is regulated, centrifuging and taking supernatant after standing 4-6h by second part.Then its remaining activity is detected with ELISA.

4, test results and analysis

1) arrange differing temps to after IgY process, dilute antibody with PBST1:1000, detect the change of OD450 value with indirect ELISA, wherein negative control OD450 is 0.111.Test-results is as Figure 11, and when result is presented at 60 DEG C and 65 DEG C, tiring of IgY declines slowly, and namely after half an hour, activity still keeps 98% and 90%, now has good thermostability, illustrates and adopts pasteurization to be feasible to IgY; 70 DEG C time, 10min is heated to IgY, activity decrease 22%, and when exceeding 70 DEG C, active beginning declines rapidly, namely loses most of vigor after 5min.Therefore higher than under 70 DEG C of conditions, the activity very easily inactivation of IgY.

2) different pH is to after IgY process, carries out 1:2000 and dilutes antibody, detect the change of OD450 value with indirect ELISA with PBST, wherein yolk antibody stoste is tired is 8000, and negative control OD450 is 0.175, and test-results is as Figure 12, under pH=4 condition, the activity of IgY is substantially unaffected; But when pH value drops to 3, activity just starts slow decline, but to detect after 3h that residue tires be 2000; When pH is 2, after 0.5h process, IgY is not positive under this extent of dilution.Experiment shows that IgY has certain acid resistance, is more stable when pH>3.

3), in the simulated gastric fluid of pH3.5, after IgY hatches different time at 37 DEG C, IgY is detected active.Negative control is that what show in 0.070, figure is the OD value under 1:800 extension rate.Result is as Figure 13, and the OD value that stomach en-37 DEG C process 0.5h, ELISA detect IgY activity is respectively 0.48 and 0.29, and in whole process, the OD value of whole egg powder is all than the height of purifying, and the OD value of 2h is respectively 0.25 and 0.094.The hydrolysis of other compositions to yolk antibody in visible whole egg powder serves provide protection.

The application effect appraisal of embodiment 5 yolk antibody

1, subjects

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

2, test materials

Table 4 bacterial strain

Note: CFA(colonization factor antigen, colonization factor antigen); CS(coli surface antigen, surface of E. coli antigen)

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

3, mouse challenge test

1) mouse arrives rear adaptive phase 2-3 days, and random packet is often organized 8, freely drunk water and search for food.

2) before attacking poison, 24-48h mouse drinks water the sterilized water changed to containing Streptomycin sulphate (5g/L), to eliminate normal intestinal flora.

3) prepare bacterium: the mono-bacterium colony of picking ETEC in fresh TBS substratum, 37 DEG C of shaking table overnight incubation.Attack poison next day and inoculated fresh culture with 1/100 amount again the same day, treat that OD600 reaches 1.0(about 5 × 108CFU/mL), bacterium liquid is centrifugal obtains bacterial sediment, is resuspended in the long-pending aseptic PBS of 1/10 bacteria liquid, 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 hydrochloric acid in gastric juice to the impact of thalline.

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

7) 12h fasting before anesthesia, normal water.

8) after attacking poison, after 48-72h, anesthesia is put to death and dissects mouse, and intercept 3cm ileum (returning blind junction upwards 6cm), longitudinally cut enteron aisle open, 2mL PBS dilutes content, and vortex is incubated at room 10min after 5 seconds, again vortex 5 seconds.Gained supernatant is coated with maconkey agar plate count (Allen et al2006) to get 100 μ L after 10-1 and 10-2 dilution.

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

4, cell adhesion experiment

Method: in 10mL fresh LB, by 1/100 inoculated bacteria, reaches growth logarithmic phase in 37 DEG C of 225r/min shaking culture to bacteriums.Add 600 μ L bacterium liquid respectively 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, treatment group is added to the yolk antibody of 100 μ L.Join in Tissue Culture Plate with the infection multiplicity of 100:1 (MOI), control group (common yolk antibody) and treatment group are all placed in 37 DEG C and hatch 1h.After incubation 1h, with aseptic PBS wash-out 3 times, remove the bacterium do not adhered to, 5-10min is hatched through 200 μ L0.1%Triton-X-100 phosphoric acid salt with the bacterium of cell adhesion, after 100 μ L suspensions are diluted 1000 times, even spread LB is dull and stereotyped, the bacterial count that namely colony number (CFU) of secondary day growth adheres to.Adhesion rate=1000* colony number/total bacteria (each process in triplicate).

This is tested each group of data acquisition GraphPad Prism5.0 and adds up, and the often group colony number of mouse adherence test represents with geometrical mean, and cell adhesion experiment then represents with mean+SD, compares between two through distribution free Mann-Whitney one tailed test.P<0.05 judges significant difference.

5, test results and analysis

Attack malicious mouse with three kinds of common people source ETEC, then yolk antibody prepared by the different albumen of gavage, the bacterium that enteron aisle adheres to recovers in maconkey agar substratum, and picking 20 bacterium colonies are PCR detects.Positive colony number/20 of the colony number adhered to=plate count mean value * extent of dilution *.

Each group of mouse intestinal bacterial adhesion 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 adhesion being detected, and what also show fusion rotein antibody presses down adhesive attraction.Attack in malicious group at E519 and 44815, yolk antibody prepared by fusion rotein EF all significantly suppresses (P<0.01, P<0.05) ETEC in the field planting of mouse intestinal.

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

Claims (1)

1. be used for the treatment of a preparation method for the yolk antibody of people source enterotoxic Escherichia coli diarrhoea, it is characterized in that comprising the following steps:

1) with the DNA of people source enterotoxic Escherichia coli type strain H10407 for material, design primer, pcr amplification object fragment, object fragment after amplification is connected to expression vector, build containing the HpaA gene etpA of people source enterotoxic Escherichia coli and the recombinant plasmid pET28a-EF of flagellin gene fliC fusion gene, the nucleotide sequence of described fusion gene is as shown in SEQ ID NO.1, and pcr amplification primer is:

2) recombinant plasmid pET28a-EF is transformed into e. coli bl21 (DE3), with inductor IPTG abduction delivering, best abduction delivering condition is: 37 DEG C of shaking tables are cultured to OD600 and reach 0.5-1.0, adding inductor to concentration is 0.5mM, continue to cultivate 3-4h, collect thalline, obtain recombination bacillus coli BL21 (the DE3)/pET-EF expressing described recombinant plasmid pET28a-EF;

3) separation and purification fusion rotein from recombination bacillus coli BL21 (DE3)/pET-EF, its aminoacid sequence is as shown in SEQ ID NO.2;

4) to renaturation, concentrated after fusion rotein solution in add the tween-80 of sterilizing to final concentration 4%, in tissue refiner, 30min is mixed again with equal-volume white-oil adjuvant, fully emulsified to protein concentration be 0.5mg/mL, immunity is carried out to laying hen, employing chest muscle is injected, head exempt from 2 weeks afterwards booster immunization once, interval 2 weeks is again immune, two exempt from after weekly random acquisition egg ELISA monitor antibody titer change, treat that yolk antibody is tired and reach 1:2 ⁹rear collection egg, purifying, collecting yolk antibody from egg.