KR101842130B1 - Transformed E. coli producing pili(F4, F18) and heat labile toxin(LT) for postweaing diarrhea vaccine in pigs and vaccine composition comprising the pili and LT produced by the same - Google Patents

Abstract

The present invention relates to a recombinant vector expressing F4 ciliated protein, F18 cilioprotein and LT toxin protein, a transformed E. coli capable of mass-producing the F4 ciliate protein, F18 cilioprotein and LT toxin protein by inserting the recombinant vector, A method for mass production of F4 ciliate protein, F18 cilioprotein and LT toxin protein by culturing Escherichia coli, and a vaccine composition for preventing porcine diarrhea comprising the F4 cilioprotein and LT toxin protein as an antigen.

Description

TECHNICAL FIELD [0001] The present invention relates to a vaccine composition for preventing pig diarrhea, comprising a transformed E. coli which mass-produces pig diarrhea back ciliates and toxins, and ciliates and toxins produced thereby as an antigen (Transformed E. coli producing pili (F4, F18) and heat labile toxin LT) for postweaing diarrhea vaccine in pigs and vaccine composition comprising the pili and LT produced by the same}

The present invention relates to a vaccine composition for preventing porcine diarrhea caused by the production of coliforms and toxins produced by porcine diarrheal disease, and a vaccine composition for preventing porcine diarrhea caused by the ciliates and toxins produced thereby. More particularly, A recombinant vector expressing a protein and a LT toxin protein, a transformed E. coli capable of mass-producing the F4 cili protein, F18 cilioprotein and LT toxin protein by inserting the recombinant vector, and culturing the transformed E. coli to produce F4 ciliate, F18 A method for mass production of a cilioprotein and a LT toxin protein, and a vaccine composition for preventing porcine diarrhea comprising the F4 cilioprotein and the LT toxin protein as an antigen.

Escherichia coli (Esherichia coli ) is a normal flora of mammalian digestive organ. However, some Escherichia coli acquire pathogenic factors and, when there is a stress factor such as transport of pigs and temperature changes, they multiply explosively and adhere to the small intestine mucosa and secrete toxins to cause diarrhea and edema (Fairbrother JM and Gyles CL 2012, Colibacilosis.Disease of swine.Wiley-Blackwell, West Sussex, UK, 723-749).

Escherichia coli occurs worldwide and depends on the type of toxin: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC) and E. coli (Sigma-like toxin producing E. coli) , And STEC).

Diarrhea in pigs is common in mammals and weaned pigs. In general, diarrhea vaccines are commonly used to prevent diarrhea in mammals through colostrum by immunizing an animal with an inactivated inactivating toxin factor (F4, LT) .

Prior arts relating to vaccine compositions for preventing swine diarrhea include transformants expressing an epitope of porcine epidemic diarrhea virus and mucosal immunity adjuvants and vaccine compositions comprising them (Patent Publication No. 10-2012-0066556), pigs A vaccine composition for a vaccine for epidemic diarrhea virus and a vaccine composition for oral administration comprising the vaccine composition for vaccinating a pandemic strain of diarrhea comprising the same (Patent Publication No. 10-1442493), a transformant expressing an epitope protein of a porcine epidemic diarrhea virus Publication No. 10-2003-0086062).

However, in colostrum, the colostrum antibody is almost absent and the susceptibility to the new adhesion factor (F18) is increased, preventing the toxin (LT) from being defended and causing diarrhea.

However, since F4 ciliate, F18 cilioprotein and LT toxin protein are very low in production of antigens for vaccine production by conventional culture methods, pig pod diarrhea The development of vaccine against diarrhea is not enough.

In order to solve the above problems, the inventors of the present invention have attempted to mass-culture F4, F18, and LT in a pathogenic Escherichia coli isolated from domestic pig diarrhea piglets using gene cloning technology, Through the investigation of the toxin production distribution, it was confirmed that the use of the preventive vaccine which confirmed safety by producing the vaccine antigen (F4, F18, LT) which can solve the problem of diarrhea in the domestic pig, The present invention has been completed.

Accordingly, an object of the present invention is to provide a recombinant vector capable of simultaneously expressing F4 cilioprotein, F18 cilioprotein and LT toxin protein.

Another object of the present invention is to provide a transformed E. coli capable of producing F4 ciliate, F18 cilioprotein and LT toxin protein in large quantities by transformation with the above recombinant vector.

It is a further object of the present invention to provide a method for producing F4 ciliated protein, F18 ciliated protein and LT toxin protein in large quantities by culturing the transformed E. coli.

It is still another object of the present invention to provide a vaccine composition for preventing pig diarrhea, which comprises F4 cilioprotein and LT toxin protein simultaneously produced by the above-mentioned transformed E. coli as an antigen.

In order to accomplish the above object, the present invention provides a gene encoding an MBP fusion protein, a gene encoding the F4 ciliate protein comprising the amino acid sequence of SEQ ID NO: 1, a gene encoding the F18 cili protein comprising the amino acid sequence of SEQ ID NO: 2 Gene and a gene encoding the LT toxin protein consisting of the amino acid sequence of SEQ ID NO: 3 are operably linked.

In addition, the present invention provides a transformed E. coli having the recombinant vector inserted therein.

The present invention also provides a method for mass production of F4 ciliate, F18 cilioprotein and LT toxin protein, comprising culturing the transformed E. coli in a medium containing lactose as an inducer.

In addition, the present invention provides a vaccine composition for preventing porcine diarrhea caused by the above-mentioned transforming E. coli, which comprises as an antigen a LT toxin protein consisting of the F4 ciliate protein consisting of the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3 .

In addition, the present invention provides a vaccine composition for preventing swine diarrhea, comprising the F4 quadruple protein consisting of the amino acid sequence of SEQ ID NO: 1 and the LT toxin protein consisting of the amino acid sequence of SEQ ID NO: 3 produced by the above method as an antigen .

The present invention uses a F4 ciliate protein and a LT toxin protein produced by the strain as a preventive vaccine antigen by using the transformed E. coli which mass-produce F4 ciliate protein, F18 cilioprotein and LT toxin protein, Can be effectively prevented and the mortality of pigs can be reduced.

1 shows a cleavage map of a recombinant vector pET28 MBP-F4-F18-LT according to an embodiment of the present invention.
FIG. 2 shows the results of confirming that the F4 cilioprotein and LT toxin protein expressed by Example 2 are expressed in a water-soluble manner.
FIG. 3 shows the results of confirming that F4 cilioprotein and LT toxin protein are antigenic.

The term "vector" as used herein refers to an expression vector capable of expressing a desired protein in a suitable host cell, and which comprises an essential regulatory element operably linked to the expression of the gene insert. As used herein, the term " regulatory element "includes promoters for performing transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling transcription and translation termination.

The term "operably linked" in the present invention means that the nucleic acid sequence having the promoter activity functions as a functional linkage between the transcription initiation and promoter sequence of the target gene encoding the protein, such as the attachment factor F4, the toxin factor LT, That is, it means that a gene necessary for expression and its regulatory sequence are operatively linked to each other in such a manner as to enable gene expression.

In the present invention, the term "host cell" means a cell in which a vector is transformed into a host cell to have various genetic or molecular effects in the host cell.

In addition, the term "transformed" in the present invention means that DNA is introduced into a host and the DNA becomes replicable as a factor other than a chromosome or by insertion into a chromosome.

The present invention relates to a gene encoding a maltose binding protein (MBP) fusion protein, a gene encoding the F4 ciliate protein comprising the amino acid sequence of SEQ ID NO: 1, a gene encoding the F18 cilioprotein comprising the amino acid sequence of SEQ ID NO: 2, Lt; RTI ID = 0.0 > LT < / RTI > toxin protein comprising the amino acid sequence of SEQ ID NO: 3.

The recombinant vector of the present invention may be a recombinant vector pET28 MBP-F4-F18-LT having a cleavage map of Fig.

The recombinant vector of the present invention is characterized in that it can express F4 ciliate protein, F18 cilioprotein and LT toxin protein in a large amount.

The present invention also relates to a transformed E. coli having the recombinant vector inserted therein.

The transformed E. coli of the present invention is Escherichia coli (Eschrerichia coli ) pET28 MBP F4-F18-LT (KCTC18478P).

From the transformed E. coli of the present invention, the host cell to be used is a well-known E. coli, in the case of the transformed E. coli of the, e.g., E. coli BL21 (DE3) (E. coli BF - dcm opmT hsdS (rB - mB -a ) gal [ lambda] novagen, USA) can be used.

The transformed E. coli of the present invention is characterized by being capable of producing a large amount of LT toxin protein consisting of F4 ciliate protein, F18 cilioprotein, and amino acid sequence of SEQ ID NO: 3 consisting of the amino acid sequence of SEQ ID NO: 1.

The present invention also relates to a method for mass production of F4 cilioprotein and LT toxin protein, comprising culturing the transformed E. coli in a medium containing lactose as an inducer.

The cultivation of the above-mentioned transformed E. coli can be carried out according to a conventional method known in the art.

The medium to be used for culturing the transformed E. coli should meet the requirements of a specific strain in an appropriate manner. Examples of the saccharide that can be used for the medium include saccharides such as glucose, sucrose, lactose, fructose, maltose, starch, And oils and fats such as carbohydrates, soybean oil, sunflower oil, castor oil, coconut oil and the like, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol and organic acids such as acetic acid. These materials may be used individually or as a mixture.

In addition, nitrogen sources that may be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may also be used individually or as a mixture.

Potassium which may be used in the culture medium include potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth. In addition, essential growth materials such as amino acids and vitamins can be used in addition to the above materials. In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) can be injected into the culture to maintain aerobic conditions.

In the production method of the present invention, the medium may be, for example, an auto induction medium constituted by 50 μg of kanamycin and the components and contents described in Table 1 of Example 2 below.

In the production method of the present invention, the culturing temperature of the transformed E. coli is 20 占 폚 to 45 占 폚, preferably 23 占 폚 to 30 占 폚, more preferably 24 占 폚 to 26 占 폚, the culturing time is 10 to 40 hours, Preferably 10 to 20 hours, more preferably 15 to 18 hours.

The present invention also relates to a vaccine composition for the prevention of porcine diarrhea caused by E. coli producing the F4 ciliate protein comprising the amino acid sequence of SEQ ID NO: 1 and the LT toxin protein comprising the amino acid sequence of SEQ ID NO: 3 as the antigen.

The present invention also relates to a vaccine composition for preventing swine diarrhea, which comprises the F4 ciliate protein comprising the amino acid sequence of SEQ ID NO: 1 and the LT toxin protein comprising the amino acid sequence of SEQ ID NO: 3, produced by the above method .

The present invention also relates to a vaccine composition for preventing swine diarrhea, which comprises the F4 ciliate protein comprising the amino acid sequence of SEQ ID NO: 1 and the LT toxin protein comprising the amino acid sequence of SEQ ID NO: 3, produced by the above method .

The vaccine composition for preventing diarrheal diseases of pigs according to the present invention may be a vaccine live vaccine or an inactivated vaccine, and the vaccine composition may be prepared by a conventional method for preparing a vaccine composition. .

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

≪ Example 1 > (Construction of Recombinant Gene and Expression Vector)

One. Attachment factor Toxin factor  Construction of Simultaneous Expression Vector

A vector capable of expressing E. coli was prepared by codon-optimizing the genes coding for the attachment factors F4 (FaeG), F18 (FedF), and the toxin factor LT (heat labile toxin, elt A linker elt B).

The one with the genetic information used Separation diarrhea in pigs toxin producing E. coli (Enterotoxigenic E. coli) E. coli (Eschrerichia coli) KEFS1057 (F4: LT: STb) (Accession No: We refer to 197th amino acids in the first of the sequence of F4 KCTC18476P) (SEQ ID NO: 1), even if a toxin producing E. coli, the E. coli isolated from pigs (Eschrerichia The 60th to 109th amino acids of the F18 sequence of E. coli KEFS218 (F18: LT: STa: STX2e) (Accession No .: KCTC18475P) were referred to (SEQ ID NO: 2), and the LT gene was introduced into Escherichia coli we refer to the 60th amino acid and 103 amino acid sequence in the first of eltB (GenBank Accession No. WP_024168673) in the first of (Eschrerichia coli) as a template and the KEFS1057, eltA (GenBank Accession No. ABV01336) (SEQ ID NO: 3) . The termination codon was removed from each adherence gene and toxin gene, and codon optimization for E. coli was performed in DNA2.0 of USA.

The codon-optimized F4-F18-LT had EcoRI at the 5 'end and Xho at the 3' I restriction enzyme site was inserted and inserted into pJ201 vector (USA DNA2.0), and was named pJ201-F4-F18-LT.

The plasmid was then digested with EcoR I and Xho I restriction enzyme treatment and then introduced into the pET28-MBP vector (SEQ ID NO: 4) digested with the restriction enzyme. At this time, the MBP fusion protein was linked to the attachment factor and the toxin factor in order to enhance the water-soluble expression of the protein.

The plasmid thus constructed was finally named pET28 MBP-F4-F18-LT, and its cleavage map is shown in Fig.

≪ Example 2 > (Protein Expression of Recombinant Gene)

1. Recombinant vector pET28 MBP -F4-F18- LT  Induction of protein expression through inserted E. coli

Expression of the protein was induced through the transformed E. coli into which the recombinant vector pET28 MBP-F4-F18-LT prepared in Example 1 was inserted into the host E. coli.

At this time, as the host cells for expression of the enzyme gene, B-type E. coli BL21 (DE3) (E. coli BF ^- dcm opmT hsdS ( rB - mB-a ) gal [ lambda] novagen, USA) was used. To insert the recombinant vector, 100 μl of competent E. coli BL21 cells were thawed on ice, 5 μl of the recombinant plasmid was mixed, For 30 minutes.

Then, heat shock was applied at 42 캜 for 45 seconds, and after standing for 2 minutes on ice, 1 ml of LB medium was added and incubated at 37 캜 for 1 hour with stirring. The transformed bacteria were plated on a culture plate containing kanamycin and allowed to stand overnight at 37 占 폚. A bacterial clone having kanamycin resistance was selected to produce the transformed E. coli.

The transformed E. coli has been deposited on July 20, 2016 Date of the Biological Resource Center (KCTC) of Korea Research Institute of Bioscience and Biotechnology, Escherichia coli (Eschrerichia coli ) pET28 MBP F4-F18-LT (KCTC18478P).

The transformed Escherichia coli cells were cultured at 25 DEG C for 16 hours using 50 mu g / ml of kanamycin and auto induction medium composed of the following ingredients and contents as shown in Table 1 below.

The expression system generally induces overexpression of the protein by using an expensive isopropyl β-D-1-thiogalactopyranoside (IPTG) by activating the T7 promoter. However, lactose is used instead of the IPTG using the self- Inducing substance to induce the cell concentration of the recombinant strain and overexpression of the protein.

Collecting the expressed cells and a buffer solution in an amount of 15ml per cell 1g was suspended in _{1 (20mM NaH 2 PO 4,} 0.3M NaCl, pH 7.4). The suspension was sonicated and centrifuged at 12000 rpm for 30 min at 4 ° C to remove any remaining bacterial debris and the supernatant was then used for immunoblotting analysis.

The expressed recombinant protein was purified by MBP-trap column (GE healthcare) as follows.

The expressed recombinant protein samples pre-was added to the equilibrated MBP- Trap HP 1ml column, washing the bound protein in the buffer 1 of 10ml, and then elution buffer _{2 (20mM NaH 2 PO 4,} 0.3M NaCl, 10mM ≪ / RTI > maltose, pH 7.4) was used to elute the protein from the column.

The eluted protein was used for immunoblotting analysis.

≪ Example 3 > (Confirmation of Expression of Recombinant Protein and Immunoblotting Analysis)

One. Attachment factor Toxin factor

Expression of the recombinant antigen protein produced in Example 2 was confirmed by SDS-PAGE gel electrophoresis. The SDS-PAGE results confirmed that the molecular weight of the recombinant antigen protein was consistent with the expected and expressed water-soluble (Fig. 2).

Expression of the target protein was measured by immunoblotting.

Specifically, the recombinant antigen protein produced in Example 2 was subjected to SDS-PAGE, and the obtained gel was transferred to a nitrocellulose (NC) membrane.

The nitrocellulose membrane was blocked with 5% skim milk in PBS for 1 hour at room temperature to remove the nonspecific reaction and washed three times with PBST (PBS, 0.05% Tween 20) for 15 minutes.

Then, 1) mouse anti-E primary antibody for identification of each antigen protein in 0.5% skim milk-dissolved PBST. coli heat labile toxin (Abcam), 2) sheep anti-E. E. coli O149: K91: K88ac (F4; Abcam), 3) mouse anti-His (Santacruze), 4) mouse anti-E. coli F18 hybridoma unconcentration were added, respectively, and primary reaction was performed at room temperature for 1 hour.

The nitrocellulose membrane was washed three times for 15 minutes with PBST, and then reacted with anti-mouse IgG AP-linked antibody (Sigma) and anti-sheep IgG AP-linked antibody (Santacruze) for 1 hour at room temperature.

The nitrocellulose membrane was washed with PBST three times for 15 minutes, and the antigen protein was identified using the AP Conjugate Substrate Kit (bio-rad). As a result, it was confirmed that the adhesion factor F4 and the toxin factor LT protein were antigenic (Fig. 3).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, many variations and modifications may be made without departing from the scope of the present invention as defined by the following claims. It will be understood that the present invention can be changed.

The present invention uses F4 ciliate and LT toxin protein produced by the above strain as a preventive vaccine antigen using transformed E. coli that mass produce F4 ciliate protein, F18 cilioprotein and LT toxin protein, Can be effectively prevented and the mortality rate of pigs can be reduced. Therefore, the present invention can be effectively applied to the technical field to which the present invention belongs.

Korea Biotechnology Research Institute KCTC18475P 20160713 Korea Biotechnology Research Institute KCTC18476P 20160713 Korea Biotechnology Research Institute KCTC18478P 20160720

<110> Animal and Plant Quarantine Agency <120> Transformed E. coli producing pili (F4, F18) and heat labile          toxin (LT) for postweaing diarrhea vaccine in pigs and vaccine          composition comprising the pili and LT produced by the same <130> 10522 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 197 <212> PRT <213> Escherichia coli <400> 1 Trp Met Thr Gly Asp Phe Asn Gly Ser Val Asp Ile Gly Gly Thr Ile   1 5 10 15 Thr Ala Asp Asp Tyr Arg Gln Lys Trp Glu Trp Glu Val Gly Thr Gly              20 25 30 Leu Asn Gly Phe Gly Asn Val Leu Asn Asp Leu Thr Asn Gly Gly Thr          35 40 45 Lys Leu Thr Ile Thr Val Thr Gly Asn Lys Pro Ile Leu Leu Gly Arg      50 55 60 Thr Lys Glu Ala Phe Ala Thr Pro Val Ile Gly Gly Val Asp Gly Ile  65 70 75 80 Pro Gln Ile Ala Phe Thr Asp Tyr Glu Gly Ala Ser Val Glu Leu Arg                  85 90 95 Lys Pro Asp Gly Gly Thr Asn Lys Gly Leu Ala Tyr Phe Val Leu Pro             100 105 110 Met Lys Asn Ala Gly Gly Thr Lys Val Gly Ser Val Lys Val Asn Ala         115 120 125 Ser Tyr Ala Gly Val Leu Gly Arg Gly Gly Val Thr Ser Ala Asp Gly     130 135 140 Glu Leu Leu Ser Leu Phe Ala Asp Gly Leu Ser Ser Ile Phe Tyr Gly 145 150 155 160 Gly Leu Pro Arg Gly Ser Glu Leu Ser Ala Gly Ser Ala Ala Ala Ala                 165 170 175 Arg Thr Lys Leu Phe Gly Ser Leu Ser Arg Asp Asp Ile Leu Gly Gln             180 185 190 Ile Gln Arg Val Asn         195 <210> 2 <211> 50 <212> PRT <213> Escherichia coli <400> 2 Thr Leu Thr Cys Gln Ala Gly Thr Ile Leu Val Trp Lys Asn Gly Arg   1 5 10 15 Glu Thr Gln Tyr Ala Leu Glu Cys Arg Val Ser Ile His Ser Ser              20 25 30 Gly Ser Ile Asn Glu Ser Gln Trp Gly Gln Gln Ser Gln Val Gly Phe          35 40 45 Gly Thr      50 <210> 3 <211> 175 <212> PRT <213> Escherichia coli <400> 3 Ile His His Ala Pro Gln Gly Cys Gly Asn Leu Ser Arg Thr Ile Thr   1 5 10 15 Asp Asp Thr Cys Asn Glu Glu Thr Gln Asn Leu Ser Thr Ile Tyr Leu              20 25 30 Arg Glu Tyr Gln Ser Lys Val Lys Arg Gln Ile Phe Thr Asp Tyr Gln          35 40 45 Ser Glu Val Asp Ile Tyr Asn Arg Ile Arg Asp Glu Gly Pro Gly Pro      50 55 60 Gly Pro Gly Pro Gly Pro Gly Pro Ala Pro Gln Ser Ile Thr Glu Leu  65 70 75 80 Cys Ser Glu Tyr Arg Asn Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile                  85 90 95 Leu Ser Tyr Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile             100 105 110 Thr Phe Lys Ser Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln         115 120 125 His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu     130 135 140 Arg Ile Ala Tyr Leu Thr Glu Thr Lys Ile Asp Lys Leu Cys Val Trp 145 150 155 160 Asn Asn Lys Thr Pro Asn Ser Ile Ala Ala Ile Ser Met Glu Asn                 165 170 175 <210> 4 <211> 6470 <212> DNA <213> Artificial Sequence <220> <223> gene of pET28 MBP-F4-F18-LT <400> 4 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520 gtttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100 agcagcggcc tggtgccgcg cggcagccat atgaaaatcg aagaaggtaa actggtaatc 5160 tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa 5220 gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag 5280 gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc 5340 tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg 5400 tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct 5460 gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg 5520 gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc 5580 aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc 5640 aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa 5700 gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat 5760 tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg 5820 tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc 5880 ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc 5940 agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt 6000 ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa 6060 gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc 6120 atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac 6180 gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc 6240 tcgaacaaca acaacaataa caataacaac aacgaattcg agctccgtcg acaagcttgc 6300 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa 6360 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc 6420 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat 6470

Claims (10)

A gene encoding the F4 ciliate protein comprising the amino acid sequence of SEQ ID NO: 1, a gene encoding the F18 cilioprotein consisting of the amino acid sequence of SEQ ID NO: 2, and a LT encoding the amino acid sequence of SEQ ID NO: 3 A recombinant vector operably linked to a gene encoding a toxin protein,
Wherein said vector is a recombinant vector pET28 MBP-F4-F18-LT having a cleaved map of Fig. 1 below.
[Figure 1]

delete The transformed Escherichia coli in which the recombinant vector of claim 1 is inserted is Escherichia coli pET28 MBP F4-F18-LT (KCTC18478P). delete 4. The method of claim 3, wherein the transformed E. coli is selected from the group consisting of F4 cilioprotein consisting of the amino acid sequence of SEQ ID NO: 1, an adhesion factor F18 cilioprotein consisting of the amino acid sequence of SEQ ID NO: 2 and an LT toxin protein comprising the amino acid sequence of SEQ ID NO: Wherein the transformed E. coli is mass produced. A method for mass production of the F4 cili protein and LT toxin protein, comprising culturing the transformed Escherichia coli of claim 3 or 5 in a medium containing lactose as an inducer. The culture medium according to claim 6, wherein the medium is selected from the group consisting of kanamycin 50 mg / L, glucose 0.5 g / L, glycerol 3 g / L, lactose 2 g / L, MgSO ₄ .7H ₂ O 0.15 g / tryptone _{16 g / L, (NH 4} ) 2 SO 4 3.3 g / L, sodium chloride 0.5 g / L, trace elements _{1 ㎖ / L, KH 2 PO} 4 6.8 g / L and Na ₂ HPO ₄ and 12H ₂ O 7.1 g / L. &lt; / RTI &gt; A vaccine composition for preventing diarrheal diseases in pigs comprising the LT toxin protein comprising the F4 ciliate protein consisting of the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3 produced by the transgenic E. coli of claim 3 or 5. A vaccine composition for preventing diarrheal diseases in pigs, which comprises the LT toxin protein produced by the method of claim 6 and consisting of the F4 ciliate protein comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3. A vaccine composition for preventing diarrheal diseases in pigs, comprising the F4 ciliate protein consisting of the amino acid sequence of SEQ ID NO: 1 and the LT toxin protein consisting of the amino acid sequence of SEQ ID NO: 3 produced by the method of claim 7.

Images