CN104789583A

CN104789583A - Human enterotoxigenic Excherichia coli flagellin 2FliC fusion protein and application thereof

Info

Publication number: CN104789583A
Application number: CN201410024732.4A
Authority: CN
Inventors: 彭健; 王荣; 蒋思文; 潘中勉
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2014-01-20
Filing date: 2014-01-20
Publication date: 2015-07-22

Abstract

The invention discloses a human enterotoxigenic Excherichia coli flagellin 2FliC fusion protein and an application thereof. A recombinant plasmid pRSET-B-2fliC containing a human enterotoxigenic Excherichia coli fliC gene provided by the invention is converted to Escherichia coli BL21(DE3) to induce a recombined expression bacterium BL21(DE3)/pRSET-B-2fliC to express the 2FliC fusion protein. The above expressed recombinant protein is used to actively immunize first farrowing laying hens, a yolk antibody aiming at the protein is prepared, the extracted yolk antibody verifies that the recombinant protein has good antigenicity, and mouse intestinal tract toxicity attack test and in vitro experiments prove that the purified yolk antibody can inhibit adherence of bacteria to ETEC strains in mouse intestinal tracts and in vitro.

Description

A kind of people source enterotoxigenic escherichia coli flagellin 2FliC fusion rotein and application thereof

Technical field

The invention belongs to technical field of molecular biology, be specifically related to a kind of people source enterotoxigenic escherichia coli flagellin 2FliC fusion rotein, by building the flagellin gene fliC recombinant plasmid pRSET-B-2fliC of people source enterotoxic Escherichia coli, express 2FliC fusion rotein, the yolk antibody utilizing it to prepare can effectively suppress intestinal bacteria in vivo with external adhesive attraction.

Background technology

Enterotoxigenic 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.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 etal2008,2009b).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, utilizes the key gene fliC of gene engineering method clonal expression in bacterial adhesion epithelial cell process, obtains a large amount of target protein.In conjunction with IgY technology, namely produce the special yolk antibody for FliC antigen by Immune Laying Hens, be intended to develop a kind of health-care eggs food with diarrhea function with low cost.

Summary of the invention

The object of the present invention is to provide a kind of recombinant plasmid pRSET-B-2fliC containing people source ETEC fliC gene, its sequence is for shown in SEQ ID NO.1.

Another object of the present invention there are provided a kind of genetic engineering bacterium BL21 (DE3)/pRSET-B-2fliC containing recombinant plasmid pRSET-B-2fliC.Recombinant plasmid pRSET-B-2fliC is transformed into intestinal bacteria (Escherichia coli) BL21(DE3), the genetic engineering bacterium of acquisition can express flagellin 2FliC fusion rotein.

Of the present invention also have an object to there are provided a kind of people source enterotoxigenic escherichia coli flagellin 2FliC fusion rotein, and its sequence is for shown in SEQ ID NO.2.This protein immunogenic is good, and production cost is low, after this protein immunization laying hen, can obtain the egg with anti-ETEC.

A further object of the invention there are provided the refolding method of a kind of people source enterotoxigenic escherichia coli flagellin 2FliC fusion rotein, method is simple, easy, adopt SKL, Sleep-promoting factor B and reduced glutathion carry out renaturation to the target protein of expressing, and obtain the yolk antibody of more efficient valency in later stage immunologic process.

A further object of the invention there are provided yolk antibody prepared by a kind of 2FliC of utilization fusion rotein.This yolk antibody has the effect that broad spectrum suppresses ETEC to adhere to.

Last object of the present invention there are provided the application of a kind of yolk antibody in the enterotoxic Escherichia coli immune vaccine of preparation anti-human source.

In order to achieve the above object, the present invention adopts following technical measures:

The preparation method (Fig. 1,2) of a kind of recombinant plasmid pRSET-B-2fliC containing people source ETEC fliC gene, its concrete steps are as follows:

In the GenBank of NCBI, search 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 key gene flagellin gene fliC recombinant expression plasmid in bacterial adhesion epithelial cell process, wherein do with the restriction enzyme site of heterozygosis between two tumor-necrosis factor glycoproteinss and connect.Carry out enzyme to recombinant plasmid and cut qualification and order-checking, result display builds correct, and by its called after pRSET-B-2fliC, its sequence is for shown in SEQ ID NO.1.

A kind of people source enterotoxigenic escherichia coli flagellin 2FliC fusion rotein, its preparation process is as follows:

Recombinant plasmid pRSET-B-2fliC 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 (0.1mM), continue to cultivate 3-4h, final expression amount, all about 50%, exists with the form of inclusion body.The inclusion body protein 2FliC fusion rotein obtained after pressure breaking, denature and renature concentrate by inclusion body, its sequence, for shown in SEQ ID NO.2, measures protein concn with Bradford method.

A refolding method for people source enterotoxigenic escherichia coli flagellin 2FliC fusion rotein, its step is as follows:

Inoculating strain is in 300mL substratum, abduction delivering, from nutrient solution, collect thalline, then be resuspended in the BufferA of 30ml of precooling, broken 3-5 time of pressure breaking instrument, centrifugal reservation precipitation, the resuspended precipitation of 30ml BufferA, slowly adds 10%SKL, does 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, to obtain final product.

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

Utilize a yolk antibody prepared by 2FliC fusion rotein, its preparation process is as follows:

To renaturation, concentrated after 2FliC fusion rotein 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.

The application of yolk antibody in the enterotoxigenic escherichia coli immune vaccine of preparation anti-human source, its application process is as follows:

Set up the field planting model of ETEC in mouse intestinal, malicious mouse is attacked with three kinds of clinical common different serotypes ETEC bacterial strains (H10407, E519 and 44815), 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 and E519 reduces the quantity of infecting mouse.44815 attack the poison IgY that afterwards prepared by oral fusion rotein has anti-bacteria to adhere to the trend of number.By cell adhesion experiment, confirm that yolk antibody has played similar anti-adhesion effect in vitro equally.

Compared with prior art, the present invention possesses following advantage:

1) applicant is for after conveniently expression method is expressed pig source flagellin, without the problem that target protein is expressed.By analyzing its protein characteristic, a pair isocaudarner on pRSET-B is adopted to build containing 2 copies and 3 recombinant plasmid pRSET-B-2FliC copied, find the expression having target protein at corresponding size place after testing, utilize yolk antibody prepared by target protein, can significantly suppress enterotoxigenic escherichia coli to the adhesive attraction of cell, and possess certain broad spectrum, on mouse, carry out detecting its anti-adhesion effect better simultaneously.

2) target protein of expressing in this experiment mainly exists in inclusion body, higher in order to make later stage Dispersal risk tire, and antibody effects is better, and we are by adopting SKL, and Sleep-promoting factor B and reduced glutathion carry out renaturation to the target protein of expressing.The yolk antibody of more efficient valency is obtained in later stage immunologic process.

Accompanying drawing explanation

Fig. 1 is a kind of construction of recombinant plasmid route schematic diagram of 2fliC fusion gene.

In figure, pMD18-T is cloning vector, and pRSET-B is expression vector.PRSET-B-2fliC be insert the recombinant plasmid of two fliC genes, BamHI, SacI, HindIII, Bgl II is restriction endonuclease sites on plasmid.

Fig. 2 is the agarose gel electrophoresis schematic diagram of pcr amplification fliC product.

In figure, product fliC molecular size range is 519bp; M is DNA molecular amount standard DL2000.

Fig. 3 is the double digestion qualification electrophoresis schematic diagram of recombinant expression plasmid pRSET-B-2fliC.

In figure, M is DNA molecular amount standard DL2000; Swimming lane 1:pRSET-B-2fliC, Insert Fragment 1056bp.

Fig. 4 is the SDS-PAGE electrophoresis detection schematic diagram before and after genetic engineering bacterium IPTG inducible protein is expressed.

In figure, left and right two bands represent before and after recombinant protein induction respectively, and gained molecular weight of albumen is 39kDa.

Fig. 5 is the soluble analysis schematic diagram of genetic engineering bacterium recombinant protein after induction.

Upper cleer and peaceful precipitation in figure 1, after 2:pRSET-B-2fliC BL21 (DE3) induction; M is protein molecular weight standard.Fig. 6 condition of different temperatures affects schematic diagram to 2FliC fusion protein expression.

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 affects schematic diagram to 2FliC fusion protein expression.

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 2FliC.

Figure 11 is the anti-adhesion effect schematic diagram of IgY to three-type-person source ETEC mouse intestinal.

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.

Figure 12 is that IgY is to the epithelial anti-adhesion effect schematic diagram of three-type-person source ETEC.

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).Agents useful for same of the present invention, cell or bacterial strain, unless otherwise noted, all derive from commercial channel.

Embodiment 1:

The structure of enterotoxic Escherichia coli flagellin 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 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

According to people source ETEC flagellum (fliC) gene order (sequence number is AY906918) the design and synthesis primer (see table 2) of report on NCBI.Concrete primer sequence sees the following form 2.

Table 2PCR amplimer

Note: F, upstream primer; R, downstream primer; Boldface type is protectiveness base; 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

Obtain two object fragments, the sequence increased with fliC-F and fliC-R1, without terminator codon fliC, contains terminator codon fliC-T by the sequence of fliC-F and fliC-R2 amplification.

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 is pMD-fliC, pMD-fliC-T by the T clone designation of acquisition.

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-fliC, pMD-fliCT, pRSET-B double digestion through BamHI and SacI, reclaim fragment fliC and fliCT and carrier pRSET-B after agarose gel electrophoresis, connection, conversion, detection obtain pRSET-B-fliC, pRSET-B-fliCT.After this carry out double digestion with Bgl II, HindIII and BamHI, HindIII two pairs of enzymes to the two, using the former as carrier, connection, conversion, detection obtain pRSET-B-2fliC, and sequencing result display sequence is identical with expection, and pRSET-B-2fliC successfully constructs.

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, be abbreviated as by recombinant expression plasmid: pRSET-B-2fliC, the 2fliC fusion gene sequence that it contains is for shown in SEQ ID NO.3.With BamHI and SacI, double digestion is done to recombinant plasmid, the results are shown in Figure 3.

The present invention obtain pRSET-B-2fliC after double digestion after agarose gel electrophoresis, object fragment is observed respectively at about 1000bp, 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, recombinant plasmid sequence is for 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)/pRSET-B-2fliC to express, and the results are shown in Figure 4.The albumen that corresponding positions is equipped with expection size occurs, 2FliC molecular weight is about 3KD respectively, expression amount about 50%.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 0.1mM, continues to cultivate 3-4h, collects thalline.After purifying, protein concentration is 0.581g/L.

The aminoacid sequence of the 2FliC fusion rotein that the present invention builds is as shown in SEQ ID NO.2.

Embodiment 3:

Yolk antibody is prepared 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:

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 150 of raising in cages 20 week age, divide 2 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 1:800 by concentration, i.e. 0.12 microgram/hole.After exempting from two, the antibody titer of one week 2FliC reaches 3200 respectively, and two exempt from rear second week occurs rising of tiring, and reaches 12800 respectively, for there is downward trend of tiring to the 7th week antibody titer.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.

Embodiment 4:

The application of yolk antibody in the enterotoxic Escherichia coli immune vaccine of preparation anti-human source, its application process is as follows:

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; CS, coli surface antigen

Yolk antibody is that embodiment 3 prepares, and it is 64000 that the yolk antibody of FliC group is tired by FliC fusion protein immunization laying hen gained, and concentration is 10mg/mL.

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 OD ₆₀₀reach 1.0(about 5 × 10 ⁸cFU/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 yolk antibody 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, 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 *.

Mouse experiment is divided into 2 groups, and do not add yolk antibody group and add yolk antibody group, each treatment group 8 mouse, adhesion situation adopts dilution plate counting statistics and averages.In mouse challenge test, when not adding yolk antibody, the adhesion number of H10407 is 8.1 × 10 ⁴, the adhesion number of E519 is 2.2 × 10 ⁵, the adhesion number of 44815 is 1.6 × 10 ⁵; The adhesion number adding H10407 during yolk antibody is 1.3 × 10 ⁴, the adhesion number of E519 is 3.1 × 10 ⁴, the adhesion number of 44815 is 8.3 × 10 ⁴.

In test cell line, adopt dilution plate counting statistics to adhere to situation, each process 3 repetition, averages and compares.When not adding yolk antibody, the adhesion rate of H10407 to be the adhesion rate of 0.0022%, E519 be 0.0023%, 44815 adhesion rate be 0.0025%; The adhesion rate adding H10407 during yolk antibody to be the adhesion rate of 0.0016%, E519 be 0.0017%, 44815 adhesion rate be 0.0019%.

Each group of mouse intestinal bacterial adhesion the results are shown in Figure 11, wherein after H10407 attacks poison, fusion rotein 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 H10407 and E519, yolk antibody prepared by fusion rotein 2FliC all significantly suppresses (P<0.01, P<0.05) ETEC in the field planting of mouse intestinal.

As shown in figure 12, carry out cell adhesion with ETEC H10407, compared with control group, the bacterial adhesion number of bacterial strain H10407 and 44815 after adding special yolk antibody all significantly reduces.And bacterial strain E519 is in adhesion process, treatment group compared with the control without significant difference, but presents certain reduction trend (P>0.05).Therefore the also similar anti-adhesion effect demonstrating yolk antibody on a cellular level.

SEQUENCE LISTING

<110> Hua Zhong Agriculture University

<120> people source enterotoxigenic escherichia coli flagellin 2FliC fusion rotein and application thereof

<130> people source enterotoxigenic escherichia coli flagellin 2FliC fusion rotein and application thereof

<160> 3

<170> PatentIn version 3.1

<210> 1

<211> 3944

<212> DNA

<213> artificial sequence

<400> 1

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatccgat ggcacaagtc attaatacca acagcctgtc gctgttgacc 240

cagaataacc tgaacaaatc tcagtcttct ctgagctccg ccattgaacg tctctcttct 300

ggcctgcgta ttaacagtgc taaagatgac gcagcaggtc aggcgattgc taaccgtttt 360

acagcaaata ttaaaggtct gactcaggct tcccgtaacg cgaatgatgg tatttctgtt 420

gcgcagacca ctgaaggtgc gctgaatgaa attaacaaca acctgcagcg tgtacgtgaa 480

ctgactgttc aggcaactaa cggtactaac tctgacagcg atctttcttc tatccaggct 540

gaaattactc aacgtctgga agaaattgac cgtgtatctg agcaaactca gtttaacggc 600

gtgaaagtcc ttgctgaaaa taatgaaatg aaaattcagg ttggtgctaa tgatggtgaa 660

accatcacta tcaatctggc aaaaattgat gcgaaaactc tcggcctgga cggttttcga 720

gctcgagatc cgatggcaca agtcattaat accaacagcc tgtcgctgtt gacccagaat 780

aacctgaaca aatctcagtc ttctctgagc tccgccattg aacgtctctc ttctggcctg 840

cgtattaaca gtgctaaaga tgacgcagca ggtcaggcga ttgctaaccg ttttacagca 900

aatattaaag gtctgactca ggcttcccgt aacgcgaatg atggtatttc tgttgcgcag 960

accactgaag gtgcgctgaa tgaaattaac aacaacctgc agcgtgtacg tgaactgact 1020

gttcaggcaa ctaacggtac taactctgac agcgatcttt cttctatcca ggctgaaatt 1080

actcaacgtc tggaagaaat tgaccgtgta tctgagcaaa ctcagtttaa cggcgtgaaa 1140

gtccttgctg aaaataatga aatgaaaatt caggttggtg ctaatgatgg tgaaaccatc 1200

actatcaatc tggcaaaaat tgatgcgaaa actctcggcc tggacggttt ttgagagctc 1260

gagatctgca gctggtacca tggaattcga agcttgatcc ggctgctaac aaagcccgaa 1320

aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct 1380

ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tctggcgtaa 1440

tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg 1500

ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 1560

cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 1620

cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 1680

tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 1740

gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 1800

tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 1860

ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 1920

taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat 1980

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 2040

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 2100

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 2160

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 2220

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 2280

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 2340

gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 2400

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 2460

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 2520

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 2580

ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 2640

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 2700

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 2760

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 2820

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 2880

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 2940

cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 3000

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 3060

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 3120

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 3180

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 3240

agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc 3300

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 3360

gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 3420

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 3480

tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 3540

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 3600

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 3660

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 3720

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 3780

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 3840

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata 3900

cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcag 3944

<210> 2

<211> 351

<212> PRT

<213> artificial sequence

<400> 2

Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn

1 5 10 15

Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg Leu

20 25 30

Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln

35 40 45

Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala

50 55 60

Ser Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly

65 70 75 80

Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr

85 90 95

Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Ser Ser Ile

100 105 110

Gln Ala Glu Ile Thr Gln Arg Leu Glu Glu Ile Asp Arg Val Ser Glu

115 120 125

Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ala Glu Asn Asn Glu Met

130 135 140

Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asn Leu

145 150 155 160

Ala Lys Ile Asp Ala Lys Thr Leu Gly Leu Asp Gly Phe Arg Ala Arg

165 170 175

Asp Pro Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr

180 185 190

Gln Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu

195 200 205

Arg Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala

210 215 220

Gly Gln Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr

225 230 235 240

Gln Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr

245 250 255

Glu Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu

260 265 270

Leu Thr Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Ser

275 280 285

Ser Ile Gln Ala Glu Ile Thr Gln Arg Leu Glu Glu Ile Asp Arg Val

290 295 300

Ser Glu Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ala Glu Asn Asn

305 310 315 320

Glu Met Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile

325 330 335

Asn Leu Ala Lys Ile Asp Ala Lys Thr Leu Gly Leu Asp Gly Phe

340 345 350

<210> 3

<211> 1056

<212> DNA

<213> artificial sequence

<400> 3

atggcacaag tcattaatac caacagcctg tcgctgttga cccagaataa cctgaacaaa 60

tctcagtctt ctctgagctc cgccattgaa cgtctctctt ctggcctgcg tattaacagt 120

gctaaagatg acgcagcagg tcaggcgatt gctaaccgtt ttacagcaaa tattaaaggt 180

ctgactcagg cttcccgtaa cgcgaatgat ggtatttctg ttgcgcagac cactgaaggt 240

gcgctgaatg aaattaacaa caacctgcag cgtgtacgtg aactgactgt tcaggcaact 300

aacggtacta actctgacag cgatctttct tctatccagg ctgaaattac tcaacgtctg 360

gaagaaattg accgtgtatc tgagcaaact cagtttaacg gcgtgaaagt ccttgctgaa 420

aataatgaaa tgaaaattca ggttggtgct aatgatggtg aaaccatcac tatcaatctg 480

gcaaaaattg atgcgaaaac tctcggcctg gacggttttc gagctcgaga tccgatggca 540

caagtcatta ataccaacag cctgtcgctg ttgacccaga ataacctgaa caaatctcag 600

tcttctctga gctccgccat tgaacgtctc tcttctggcc tgcgtattaa cagtgctaaa 660

gatgacgcag caggtcaggc gattgctaac cgttttacag caaatattaa aggtctgact 720

caggcttccc gtaacgcgaa tgatggtatt tctgttgcgc agaccactga aggtgcgctg 780

aatgaaatta acaacaacct gcagcgtgta cgtgaactga ctgttcaggc aactaacggt 840

actaactctg acagcgatct ttcttctatc caggctgaaa ttactcaacg tctggaagaa 900

attgaccgtg tatctgagca aactcagttt aacggcgtga aagtccttgc tgaaaataat 960

gaaatgaaaa ttcaggttgg tgctaatgat ggtgaaacca tcactatcaa tctggcaaaa 1020

attgatgcga aaactctcgg cctggacggt ttttga 1056

Claims

1. one kind contains people source enterotoxigenic escherichia coli fliCthe recombinant plasmid pRSET-B-2fliC of gene, its sequence is for shown in SEQ ID NO.1.

2. the genetic engineering bacterium containing recombinant plasmid described in claim 1.

3. the fusion rotein of genetic engineering bacterium expression described in claim 2, its sequence is for shown in SEQ ID NO. 2.

4. utilize yolk antibody prepared by the fusion rotein described in claim 3.

5. yolk antibody according to claim 4 suppresses the application in E. coli adhesion in vitro.

6. the application of yolk antibody according to claim 4 in the enterotoxic Escherichia coli immune vaccine of preparation anti-human source.

7. fusion rotein according to claim 3 is preparing the application in diarrhea eggs.

8. the refolding method of fusion rotein described in claim 3, its step is as follows:

Inoculating strain is in 300mL substratum, abduction delivering, thalline is collected from nutrient solution, be resuspended in the BufferA of the 30ml of precooling again, broken 3-5 time of pressure breaking instrument, centrifugal reservation precipitation, the resuspended precipitation of 30ml BufferA, slowly adds 10%SKL, does 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 standing 2h, the centrifugal 15min of 10000rpm discards undissolved thalline impurity, to obtain final product;

The formula of described BufferA: Tris 6.055g, EDTA 0.186g, NaCl 2.925g, glycerine 50mL, adds ddH ₂o is settled to 1L, now adds DTT to final concentration 0.5mmol/L before using;

Described bacterial strain is the genetic engineering bacterium containing recombinant plasmid pRSET-B-2fliC, and pRSET-B-2fliC sequence is for shown in SEQ ID NO.1.