KR20090011215A - Transfer vector to express the single chain variable fragment of neutralizing antibody against porcine epidemic diarrhea virus on the bacterial outer membrane and the e.coli to inhibiting the propagation of porcine epidemic diarrhea virus - Google Patents

Abstract

A vector and Escherichia coli suppressing proliferation of PED virus are provided to neutralize specifically the porcine epidemic diarrhea virus by being transformed to a surface expression vector and to reveal short chain variable segments of a porcine epidemic diarrhea virus neutralizing antibody at a film surface of a bacterial body. A surface expression vector reveals short chain variable segments of porcine epidemic diarrhea virus neutralizing antibody consisting of a sequence 1 at a film surface of a bacterial body. The surface expression vector includes a base sequence consisting of a sequence 1 and coding the short chain variable cleavage site specifically neutralized to porcine epidemic diarrhea virus(PEDV), T7 RNA polymerase consisting of a base sequence of 21bp~40bp of a sequence 4 and revealing the short chain variable split gene at a film surface of a bacterial body, a base sequence consisting of A sequence 2 coding A signal peptide of IgA proteinase gene of a gonococcus and an autotransporter beta domain gene consisting of a sequence 3 of the IgA proteinase of a base sequence coding six histidines and a gonococcus inserted into a bacterial membrane.

Description

Vector to express the single chain variable fragment of neutralizing antibody against porcine epidemic diarrhea virus on the bacterial outer membrane and the E. coli to inhibiting the propagation of porcine epidemic diarrhea virus}

The present invention relates to a vector for expressing a short chain variable segment of a swine pandemic diarrheal virus neutralizing antibody and E. coli to inhibit the growth of swine pandemic diarrheal virus, and more particularly to a short chain variable of a swine pandemic diarrheal virus neutralizing antibody E. coli and pigs containing E. coli expressing a surface expression vector for expressing the fragment on the bacterial membrane surface, a short chain variable segment protein transformed with the surface expression vector to specifically neutralize the swine pandemic diarrheal virus It relates to a composition for preventing or treating a pandemic diarrheal virus.

Porcine endemic diarrhea (PED) is a major disease that causes diarrhea and mortality in young pigs and causes a lot of economic damage to pig farmers. It is caused by the porcine endemic diarrhea virus (PEDV).

A study on the technical preparation of single chain variable fragments (scFv) has shown that Ward et al. (1989) found that E. coli-expressing single variable domains of mouse-derived antibodies have the same specificity as the original IgG antibody. Since it has been reported to have antigen-binding ability, Holliger and Hudson (2005, Review) have reported various applications and industrialization status and prospects of biotherapeutics using various types of antibody molecules including scFv.

In the field of veterinary medicine, studies that demonstrate the effectiveness of prophylactic or therapeutic agents using neutralizing scFv against pathogens include not only the virus field, such as Gould et al. (2005), but also Almquist et al. Bacteria, such as the study on the neutralizing ability of tetanus toxin of 2006) has been actively reported until recently.

In particular, Veiga et al. (2003) have a neutralizing ability against the transmissible gastroenteritis virus (hereinafter referred to as "TGEV") belonging to the coronavirus (coronavirus) and causing diarrhea in pigs together with PEDV, the neutralizing agent of the present invention. We have reported a study using monoclonal antibody scFv as a therapeutic.

Pigs with PED have more severe symptoms at younger age, and most of them die after vomiting and severe watery diarrhea of suckling pigs less than 1 week old, and weaned piglets recover after 4-6 days of watery diarrhea. It is severely reduced, and hogs and tendons show diarrhea.

PEDs, like most viruses-associated diseases, rely solely on vaccine prevention, especially those who are directly affected by the PED outbreak, a very young piglet that can induce active immunity to piglets by vaccination. Since there is no time to spare, most of them rely on passive immunization to prevent PED as a maternal transfecting antibody that is vaccinated into sows and delivered via colostrum.

If the vaccine is not vaccinated or the sows are incompletely immunized to deliver sufficient maternal antibodies to their piglets, young pigs can easily get PEDs and cause severe damage such as diarrhea and mortality. There is no situation.

An object of the present invention is to provide a surface expression vector for expressing the short-chain variable segment of the swine epidemic diarrheal virus neutralizing antibody on the bacterial membrane surface.

Another object of the present invention is to provide E. coli, which is transformed with the surface expression vector to express a single chain variable segment protein that specifically neutralizes swine pandemic diarrheal virus.

Another object of the present invention to provide a composition for preventing or treating swine epidemic diarrhea virus containing E. coli.

The present invention for achieving the above object provides a surface expression vector for expressing the short chain variable segment of the swine epidemic diarrhea virus neutralizing antibody consisting of SEQ ID NO: 1 on the bacterial membrane surface.

The present invention provides E. coli, which is transformed with the surface expression vector to express a single chain variable segment protein that specifically neutralizes swine epidemic diarrheal virus.

The present invention provides a composition for preventing or treating swine epidemic diarrhea virus containing E. coli.

The present invention provides a short-chain variable segment protein that can neutralize the swine pancreatic diarrheal virus, a cause of swine pandemic diarrheal disease, which causes a great deal of economic damage to pigs. Oral administration to young pigs on farms can neutralize the swine pandemic diarrheal virus, which can be a therapeutic or prophylactic agent that can reduce the damage of young pigs by PED. The development of probiotics is expected to contribute greatly.

The content of the present invention will be described in more detail as follows.

The present invention is a porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, which is a cause of porcine epidemic diarrhea (hereinafter referred to as "PED"), which is a major disease that causes diarrhea and mortality in pigs and causes a lot of economic damage to pig farms; ScFv capable of neutralizing hereinafter referred to as "PEDV" was expressed on the surface of the bacterial membrane to obtain live bacteria that can be developed as a therapeutic agent.

The present invention clones the VH and VL genes of mouse monoclonal antibodies from hybridoma cells and links them with a linker to short-chain variable segments from hybridomas capable of neutralizing swine pandemic diarrheal virus (PEDV). Cloning the chain variable fragment antibody (scFv) gene and constructing a vector capable of expressing it on the surface of the bacterial body, and using this to express the surface of the scFv neutralizing ability on PEDV to the bacterial body to produce a bacterial body having a neutralizing ability on PEDV can do.

The present invention establishes a technique for cloning a single chain variable segment (scFv) of monoclonal antibody from mouse-derived hybridoma cells and uses the same to clone the short chain variable segment gene of 2C10 antibody which is neutralizing the swine epidemic diarrheal virus (PEDV). By sequencing, the amino acid composition of the VH and VL domains of the monoclonal antibody can be determined.

In addition, by demonstrating the neutralizing ability of the recombinant protein expressing the short-chain variable segment gene of the monoclonal antibody to PEDV, the swine epidemic diarrheal disease (PED) problem in the hog site of the short-chain variable segment gene of the monoclonal antibody of the present invention and the protein expressing it The possibility of direct treatment for) can be confirmed.

In other words, by economically mass-producing the protein of the present invention, which has been found to be neutralized against PEDV, and using it as a treatment for PED, it is possible to secure an effective means of controlling the PED, which is difficult to manage the disease only by the efficacy of the existing vaccine at the hog site. Can be. The technique for cloning and expressing the single-chain variable segment gene of the monoclonal antibody, which is the technique used in the present invention, is considered to be a highly available technique applicable to other diseases in the veterinary field.

According to a preferred embodiment of the present invention, a surface expression vector for expressing the short chain variable segment of the swine epidemic diarrhea virus neutralizing antibody consisting of SEQ ID NO: 1 on the bacterial membrane surface can be prepared.

In the above, the surface expression vector is a nucleotide sequence consisting of SEQ ID NO: 1 encoding a single chain variable fragment antibody site that specifically neutralizes the swine pancreatic diarrheal disease virus (PEDV), the short chain variable segment gene is a bacterial membrane T7 RNA polymerase consisting of the nucleotide sequences of 21bp to 404p of SEQ ID NO: 4 to be expressed on the surface, nucleotide sequence consisting of SEQ ID NO: 2 encoding a signal peptide of the IgA protease gene of Neisseria gonorrhoeae , 6 It is preferable to include an autotransforter β domain gene consisting of a nucleotide sequence encoding a dog histidine and SEQ ID NO: 3 of an IgA protease of Neisseria gonorrhoeae inserted into a bacterial membrane.

In the above, the surface expression vector preferably has the same characteristics as the nucleotide sequence of SEQ ID NO: 4 and the gene map of FIG.

The present invention can produce Escherichia coli expressing a short chain variable segment protein that is transformed with the surface expression vector to specifically neutralize the swine pandemic diarrheal disease virus.

The present invention can be used as a composition for preventing or treating swine epidemic diarrhea virus.

Like most viral diseases, PEDs rely solely on vaccines to prevent vaccination, so if they don't receive enough maternal antibodies, young pigs can easily get PEDs, causing severe damage to diarrhea and mortality.

Bacteria, such as non-pathogenic Escherichia coli, which expresses a short-chain variable segment protein having excellent neutralization ability in PEDV according to the present invention on the bacterial envelope, can be used as a composition for preventing or treating PED.

According to the present invention, a non-pathogenic E. coli bacterium expressing a scFv protein having excellent neutralization ability to PEDV in a bacterial envelope is orally administered to pigs of a farm in which PED is generated and neutralized PEDV while being present as part of the normal bacterial flora in the intestine. Can reduce pig damage

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

Example 1 Isolation of Total RNA from 2C10 Hybridoma Cells

The present invention is to select a 2C10 monoclonal antibody with excellent neutralization ability against PEDV, and to display the variable heavy chain (VH) and light chain variable light (VL) of the mouse monoclonal antibody from hybridoma cells The scFv gene was cloned from 2C10 hybridomas capable of neutralizing PEDV using a technique of cloning short-chain variable segment genes linked by a linker.

The present invention is to clone the VH and VL genes of the 2C10 antibody showing a neutralizing ability to PEDV among monoclonal antibodies prepared by fusing splenocytes and bone marrow cancer cells (myeloma) of Bolb / c mice immunized with PEDV antigen as shown in Figure 2 RT-PCR method was used, and total RNA was isolated from hybridoma cells producing 2C10 monoclonal antibody. Thus, cDNA was prepared as in Example 2. Total RNA was extracted according to the manufacturer's method using RNeasy mini kit (Qiagen) and the total RNA extracted was stored and used at -70 ℃.

Example 2 Amplification and Cloning of 2C10 VH and VL Genes from Total RNA

VH and VL genes in mice, referring to articles reported by Klebber et al. (1997) and Burmester and Pluckthun (2001) and in Chapter 2 of Kontermann and Dubel (antibody Engineering [Springer]; ISBN 3-540-41354-5). Primers for amplifying were designed.

The VH sense primers designed the "VH1BACK" primer for the primary amplification of the VH gene and extended the SfiI, a restriction enzyme site for insertion into the expression vector after linker linkage, to the 5 'end and the translation initiation codon. The inserted "VH1sfiI" primer was designed. VH antisense primers designed "VH2FOR" primers for cDNA synthesis and VH gene primary amplification, and each primer was used to synthesize oligonucleotides (Bioneer, Korea).

The VL sense primers designed the "VL1BACK" primers for the primary amplification of the VL gene, and the VL antisense primers were designed for four primers, "VL2FOR1", due to the diversity of the VL gene 3 'end for cDNA synthesis and VH gene primary amplification. "VL2FOR2", "VL2FOR3" and "VL2FOR4" were designed and four types of NotI, restriction enzyme sites for insertion into the expression vector after linker linkage, were extended to the 5 'end of each VL antisense primer and a stop codon was inserted. "VL2NOT1", "VL2NOT2", "VL2NOT3" and "VL2NOT4" primers were designed and oligonucleic acid was used by order synthesis (Bioneer, Korea).

TABLE 1 Primer Design

primer Sequence VH1BACK 5'-AGGTSMARCTGCAGSAGTCWGG-3 ' VH1sfiI 5'-GCAACTGC GGCC CAGCC GGCC - ATG -GCCCAGGTSMARCTGCAGSAGTCWGG-3 ' VH2FOR 5'-TGAGGAGACGGTGACCGTGGTCCCTTGGC CCC-3 ' VL1BACK 5'-GACATTGAGCTCACCCAGTCTCCA-3 ' VL2FOR1 5'-CCGTTTGATTTCCAGCTTGG TGCC-3 ' VL2FOR2 5'-CCGTTTTATTTCCAGCTTGGTCCC-3 ' VL2FOR3 5'-CCGTTTTATTTCCAACTTTGTCCC-3 ' VL2FOR4 5'-CCGTTTCAGCT CCAGCTTGGTCCC-3 ' VL2NOT1 5'-GAG- TCA -TTCT- GCGGCCGC -CCGTTTGATTTCCAGCTTGGTGCC-3 ' VL2NOT2 5'-GAG- TCA -TTCT- GCGGCCGC -CCGTTTTATTTCCAGCTTGGTCCC-3 ' VL2NOT3 5'-GAG TCA -TTCT- GCGGCCGC -CCGTTTTATTTCCAACTTTGTCCC-3 ' VL2NOT4 5'-GAG- TCA -TTCT- GCGGCCGC -CCGTTTCAGCTCCAGCTTGGT CCC-3 '

To clone the VH gene, first, 2 μg of total RNA extracted in Example 1 and 2 pmole of the VH antisense primer “VH2FOR” were used. To clone the VL gene, 2 μg of total RNA extracted in Example 1 and the VL antisense primer were used. Four types of "VL2FOR1", "VL2FOR2", "VL2FOR3" and "VL2FOR4" were added at 0.5 pmole, respectively, to a final concentration of 2 pmole, and SuperScript ^TM II reverse transcriptase (Invitrogen) was used at 42 ° C according to the manufacturer's method. In response, the first-strand cDNAs of the VH and VL genes were prepared respectively.

For the amplification of the VH gene, 5 μl of cDNA of the prepared VH gene and 1 μl of “VH1BACK” and “VH2FOR” primers of 10 pmole / μl were used. For the amplification of the VL gene, 5 μl of the cDNA of the prepared VH gene and 10 pmole / μl. 1 μl of the same amount of “VL1BACK” and 10 μl / μl of each of the “VH2FOR1”, “VH2FOR2”, “VH2FOR3” and “VH2FOR4” primers were used to expand the Long Template PCR system (Roche, Germany). PCR was performed according to the manufacturer's method. PCR reaction conditions were 10 cycles at 94 ℃ 10 seconds, 54 ℃ 30 seconds, 68 ℃ 4 minutes after 94 ℃ 2 minutes reaction, 94 ℃ 15 seconds, 54 ℃ 30 seconds, 68 ℃ 4 minutes every cycle After 20 cycles were carried out under increasing conditions of 20 seconds per cycle, an elongation reaction was performed at 68 ° C. for 8 minutes.

After PCR, the amplified VH gene could identify a band corresponding to about 363 bp as in lane 1 of FIG. 4, and the VL gene could identify a band corresponding to about 325 bp as in lane 3.

Example 3 scFv generation and sequencing confirmation from 2C10 VH and VL genes

In order to connect the amplified VH and VL genes in Example 2 in a single chain form, as shown in FIG. 2, the 3 'end of the VH gene and the 3' end of the linker are the 5 'terminal base of the VL gene. _Three types of linkers (Gly ₄ Ser), each extended to overlap sequences, were designed and oligonucleic acid was used by order synthesis (Bioneer, Korea). Sense "LINKVH" and antisense "LINKVL" primers were designed to make this single stranded oligonucleotide linker gene double stranded and oligonucleic acids were used in order to synthesize them (Bioneer, Korea). The synthesized single chain linker gene was PCR amplified using the "LINKVH" and "LINKVL" primers, and the band of about 94 bp was identified as in lane 2 of B in FIG. 4.

(Gly ₄ Ser) ₃ Form Linker:

5'-GACCACGGTCACCGTCTCCTCA- GGTGGAGGCGGTTCA-GGCGGAGGTGGCTCT-GGCGGTGGCGGATCG -GACATTGAGCTCACCCAGTCTC-3 '

Sense "LINKVH": 5'-GGGACCACGGTCACCGTCTCCTCA -3 '

Antisense "LINKVL": 5'-TGGAGACTGGGTGAGCTCAATGTC-3 '

The double-linked linker gene and the VH and VL genes amplified in Example 2 were amplified by the Linker PCR as shown in FIG. The sense primers "VH1sfiI" extending the SfiI at the 5 'end and inserting the translation initiation codon and 4 antisense primers "VH2NOT1" extending the NotI at the 5' end and inserting the stop codon. , "VH2NOT2", "VH2NOT3" and "VH2NOT4" were used to amplify the 2C10 scFV gene with the SfiI at the 5 'end and the NotI site at the 3' end, and the result was about 760 as in lane 2 of c in FIG. The band of bp could be confirmed. The amplified gene was cloned into the pGemTeasy (Promega) plasmid to prepare a "pG2C10scFvF" plasmid and confirmed by sequencing. The nucleotide sequence of 2C10 scFv is shown in SEQ ID NO: 1. The framework regions of the VH and VL genes ("FRs") and complementarity determining regions (hereinafter referred to as "CDRs"), and linkers linking the two genes, etc. As shown in the following, a "pG2C10scFvF" plasmid was cloned. The entire process of constructing the "pG2C10scFvF" plasmid from the total RNA isolation in 2C10 hybridoma cells is shown in FIG. 2.

The cloned 5 2C10 scFv gene so as to insert in the E. coli expression vector, surface-to 'was 2C10FB extend the BamHI site at the terminal (5'-CGC GGATCC ATG GCCCAGGTCCAACTGCAG- 3') and 3 'KpnI end, 6x histidine (histidine), and 2C10RXHis (5'-CG GGTACC - ATGATGATGATGATGATG - CTCGAG -TTCTGCGGCCGCCCGTTT-3 ') primers with the extended XhoI site were raised and synthesized using Bioneer and PCR amplified using "pG2C10scFvF" as a template as shown in FIG. TA cloned the pGemT-easy (Promega) plasmid as in lane 2 of "pGscFvHisF" plasmid was prepared, and the cleavage map of the plasmid is shown in FIG.

[Example 4] Cloning of signal peptide gene of IgA protease of Neisseria gonorrhoeae for vector creation to express 2C10 scFv gene on bacterial surface

To clone the signal peptide gene of the vector for expressing the 2C10 scFv gene on the surface of the bacterial body, the report of Pohlner et al. (1987) and the sequence information of X04835 listed in their GenBank were referred to Neisseria gonorrhoeae . The signal peptide gene of IgA protease was selected and "NGSIGF"(5'-GC CATATG -AAAGCCAAACGTTTT-3 '), which is a sense primer extended at the 5' end to the NdeI site including the start codon (ATG) ) and 3 to synthesize a "extending a BamHI site for the gene associated with the following antisense primer at the terminal" NGSIGR "(5'-CG GGATCC -CGCTTCTGAGTATGGCGTAAG -3 ') (Bioneer) imjilgyun received pre-sale from Center for disease Control (hereinafter referred to as" PCR amplification from genomic DNA of NG "and TA cloning into pGemT-easy (Promega) plasmid as shown in A of FIG. 6 to prepare a" pGNGsigF "plasmid. It was. The base sequence of the signal peptide gene of the cloned NG IgA protease is shown in SEQ ID NO: 2 and FIG. 7 and a cleavage map of the "pGNGsigF" plasmid is shown in FIG.

[Example 5] Cloning of beta domain gene of IgA protease of Neisseria gonorrhoeae for vector creation for expressing 2C10 scFv gene on bacterial surface

In order to clone the beta domain gene of the IgA protease of Neisseria gonorrhoeae (NG) corresponding to the domain fixed to the bacterial membrane to express the 2C10 scFv gene on the bacterial surface, the same information as in Example 4 was used. "NGIgABF"(5'-GC GGTACC -CCAAGAGCCGCGCAGCCGCGA-3 '), which is a sense primer that extends the KpnI site for connection with the scFv gene region at the 5' end, and an antisense primer that extends the EcoRI site at the 3 'end. NGIgABR "(5'-CG GAATCC - TTA GAAACGAAGCTGTATTTT-3 ') (Bioneer) was also PCR amplified from genomic DNA of gonorrhoeae (Nisseria gonorrhoeae ), which was also distributed from the Center for Disease Control and pGemT as shown in lane 3 of FIG. TA cloning in the -easy (Promega) plasmid produced a "pGNGIgABF" plasmid and confirmed by sequencing. The nucleotide sequence of the beta domain gene of IgA protease of cloned NG is shown in SEQ ID NO: 3 and FIG. 10 and a cleavage map of the "pGNGIgABF" plasmid is shown in FIG.

Example 6 Preparation of the vector "pET5aSig-scFv-IgABeNE" plasmid for expressing the 2C10 scFv gene on the bacterial surface

3 using the "pGscFvHisF" shown in FIG. 9, the "pGNGsigF" shown in FIG. 8, and the "pGNGIgABF" plasmid shown in FIG. In order to express the 2C10 scFv gene on the bacterial surface, a plasmid of vector "pET5aSig-scFv-IgABeNE" having the characteristics as shown in the nucleotide sequence of SEQ ID NO: 4 (FIGS. 12A and 12B) and the cleavage map of FIG. 13 was prepared. .

First, a 796 bp fragment obtained by treating the "pGscFvHisF" plasmid prepared in Example 3 with BamHI and SpeI and a 3.0kp fragment obtained by treating the "pGNGsigF" plasmid prepared in Example 4 with BamHI and SpeI were ligated to "pGNGsig-scFvHis". Plasmid was prepared. A 3.8 kb fragment obtained by treating the prepared "pGNGsig-scFvHis" plasmid with KpnI and SpeI and a 932 bp fragment obtained by treating the "pGNGIgABF" plasmid created in Example 5 with KpnI and SpeI were ligated to "pGsig-scFv-IgAB". Plasmids were prepared. Finally, Nde I and EcoR I treatments were performed from the "pGsig-scFv-IgAB" plasmid, which signaled the sequence of the signal peptide of gonorrhea corresponding to 1.8 kb, the scFv of the 2C10 antibody, the six histidine sequences and the IgA protease of the gonorrhea. Gene fragments arranged in the beta domain of (protease) were ligated to E. coli expression plasmid pET5a (Promega) into a 4.1 kb fragment treated with NdeI and EcoRI and scFv of 2C10 in the form of the schematic diagram of FIG. The "pET5aSig-scFv-IgABeNE" which can express to was created.

Example 7 Identification of Escherichia Coli Expressing 2C10 scFv by Western Blot Analysis

Normal Escherichia coli transformed with "pET5aSig-scFv-IgABeNE" was used as a control, and SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis according to the manual of pRSET A, B, and C kit (Invitrogen) as a control. ) And anti-6x histidine monoclonal antibody and alkaline phosphotase (hereinafter referred to as "AP") conjugated anti-mouse IgG antibody (alkaline phosphotase conjugated anti-mouse IgG; Western blot analysis was performed using the mouse IgG AP ".

The transformed Escherichia coli was not induced with isopropyl-beta-D-thiogalactopyranoside (hereinafter referred to as "IPTG") and 0.1 mM, 0.2 mM, 0.4 mM , Inoculated in 25 ml SOB medium under the condition of inducing with IPTG of 0.5mM and 0.7mM concentration, respectively, multiply by shaking culture at 37 ° C for 4 hours, and take 1ml each and centrifuge the bacteria at 3,000 rpm for 10 minutes. After removing the supernatant, only the harvested bacterial bodies were stored and used at -20 ° C. The sample was dissolved in 100 μl of 20mM phosphate solution, repeated freezing and thawing three times using liquid nitrogen and a constant temperature water bath at 42 ° C., followed by centrifugation, and 1X SDS-PAGE was added to the precipitate from which the supernatant was removed. 100 μl was added and boiled for 5 minutes, and 10 μl each was subjected to electrophoresis at 190 volt for about 4 hours with a protein size marker (Bench Mark ^TM prestained protein ladder) on a 10% SDS-PAGE gel. The polyvinylidene difluoride membrane (hereinafter referred to as "PVDF membrane", Millipore, MA) was transferred to electricity using electricity and a blocking solution, i.e., PBS- containing 5% nonfat dried milk. Blocking was performed at room temperature with T (0.1% Tween 20 in PBS) solution for 1 hour. The anti 5x histidine antibody (Penta.His Antibody, Qiagene) was then diluted in a 1: 2,000-fold blocking solution and allowed to react at room temperature for 1 hour, washed three times with PBS-T solution, followed by alkaline phosphotase (alkaline phosphotase) conjugation ( The reaction solution prepared by diluting the conjugated anti-mouse IgG AP (BETHYL, Texas) in a blocking solution 1: 1,000 times was washed with PBS-T solution three times after the PVDF membrane was reacted at room temperature for 1 hour. The washed PVDF membrane was developed with 1-STEP ^™ NBT-BCIP (Pierce, IL) solution. As shown in FIG. 14, only 60 kDa protein bands were identified in E. coli transformed with "pET5aSig-scFv-IgABeNE", and it was confirmed that there was no significant difference in expression level according to IPTG concentration conditions.

[Example 8] Confirmation of PEDV neutralization ability of E. coli expressing 2C10 scFv on the surface of bacterial body

The "pET5aSig-scFv-IgABeNE" plasmid prepared to express the 2C10 scFv prepared in Example 6 was transformed into E. coli DH5α strain to contain 100 μg / ml of ampicillin. Colonies were selected from LB agar medium and cultured in LB liquid medium for neutralization assay for PEDV.

PEDV SM98P Note: PEDV's 98 domestic outdoor isolates, SM98 strains (Ref .; Veterinary Science and Technology Development Project 1999 Research Report p273-81), were subcultured in swine testicle (ST) cell lines and purified to tissue culture. The cell line was named SM98P strain (Ref. 2004 Veterinary Science and Technology Development Project, p681-703).

0.5 ml of PEDV SM98P strains of 2 × 10 ⁴ PFU / mL titer were mixed with E. coli bacteria expressing 10 ⁸ 2C10 scFv, followed by neutralization at 37 ° C. for 1 hour, followed by centrifugation to remove the bacteria. 12 well plates were inoculated in 10-fold dilutions each. The control group was inoculated by reacting with normal E. coli bacteria of the same number of cells instead of 2C10 scFv-expressing E. coli, or by reacting with PBS alone and diluting. When cell degeneration was observed after 72 hours of inoculation, it was fixed at room temperature for 5 minutes using a fixed solution of acetone and methanol 1: 1 mixed solution stored at −20 ° C. and dried.

 The color was then stained by virus plaques in the immobilized cell monolayer using the VECTASTAIN ABC (VECTOR, PK-4002, CA) kit according to the manufacturer's method. After immobilizing the fixed cells with PBS solution, the cells were blocked with 2% normal horse serum at 37 ° C for 30 minutes to reduce non-specific reactions, and anti-PEDV (anti-PEDV) monoclonal antibody was primary. The reaction was carried out at 37 ° C. for 30 minutes using an antibody (primary antibody). After the reaction, the first reaction antibody solution was removed, washed with PBS solution for 5 minutes, and then, the biotinylated anti mouse antibody was reacted with a secondary antibody for 30 minutes, washed, and then the biotinylated hose. The reaction solution was prepared by reacting horseradish peroxidase and avidin (avidine) in 10 ml of 50 µl each in the same amount, and then reacted and washed for 30 minutes. Finally, diaminobenzidine tetrahydrochioride (hereinafter referred to as "DAB") was finally colored gray / black by adding nickel ions as a substrate.

As shown in Fig. 15, the final dilution wells showing the cytopathic effect (CPE) showed a large difference in the number of DAB-plated plaques between the wells that reacted with normal E. coli bacteria or only PBS. Only the virus solution reacted with E. coli expressing 2C10 scFv was neutralized by more than 10-fold dilution step, and even at 10 ² PFU / mL, E. coli expressing 2C10 scFv protein on the surface showed a significant difference in the number of plaques. It was found that the PEDV neutralized about 95% or more.

Figure 1 shows the schematic diagram showing the composition of the gene expressing the neutralizing scFv against the swine epidemic diarrheal virus and the state of the protein expressed ("OM" represents "outer membrane", "AT" is "autotransforter").

Figure 2 is a schematic diagram showing the process of cloning the 2C10 scFv gene and individual amplification of the VH and VL genes of the 2C10 antibody with neutralizing ability in PEDV.

Figure 3 is a schematic diagram showing the manufacturing process of the expression vector expressing the 2C10 scFv gene on the bacterial outer membrane.

Figure 4 is a PCR amplification and electrophoresis of the VH and VL genes and linker genes of 2C10 antibody, respectively.

(A, PCR amplification of VH and VL genes of 2C10 antibody (lane 1: VH gene of PCR-amplified 2C10 antibody; lane 2: 1 kb size marker; lane 3: VL gene of PCR-amplified 2C10 antibody); B, 2C10 antibody VH and VL genes confirmed by PCR amplification (lane 1: 1 kb size marker; lane 2: PCR amplified linker gene); C, 2C10 antibody scFv gene confirmed by PCR amplification (lane 1) : 1 kb size marker; lane 2: PCR amplified linker gene))

Figure 5 shows the components of the scFV gene cloned by inserting the SfiI site and start codon (ATG) at the 5 'end and the NotI site and stop codon (TGA) at the 3' end for 2C10 scFv protein expression. Base and amino acid sequences. "FR" stands for "framework region" and "CDR" stands for "complementarity determining region".

6 is a photogram of the NG signal gene, 6C histidine-extended 2C10 scFv at the 3 'end, and PCR amplification of the beta domains of NG IgA protease, respectively, and electrophoresis by restriction enzyme treatment.

(Picture showing signal genes of A and NG (lane 1: 1 kb size marker; lane 2: “pGNGsigF” plasmid / EcoR I); B, 2C10 scFv gene with 6x histidine extended at the 3 'end and beta domain of IgA protease Photo (lane 1: 1 kb size marker; lane 2: "pGscFvHisF" plasmid / EcoR I; lane 3: "pGNGIgABF" plasmid / EcoR I))

Figure 7 shows the nucleotide sequence of the signal peptide gene of cloned gonorrhea (Nisseria gonorrhoeae ).

Figure 8 is a cleavage map of the "pGNGsigF" plasmid with TA cloning of the signal peptide of Neisseria gonorrhoeae .

9 is a cleavage map of the "pGscFvHisF" plasmid with TA cloning of the 2C10 scFv gene with 6x histidine extended at the 3 'end.

Figure 10 shows the base and amino acid sequence of the gene of the IgA protease beta-domain of the cloned NG.

Figure 11 is a cleavage map of the "pGNGIgABF" plasmid with TA cloning of the IgA protease β-domain of Neisseria gonorrhoeae .

12A and 12B show the entire sequence of the "pET5asig-sFv-igABeNE" plasmid, which is a bacterial envelope expression vector cloned into the pET5a plasmid in order of signal peptide-2C10 scFv-IgA protease β-domain of Neisseria gonorrhoeae .

Figure 13 is a cleavage map of the "pET5aSig-sFv-IgABeNE" plasmid, a bacterial envelope expression vector prepared by cloning genes to the pET5a plasmid in order of signal peptide-2C10 scFv-IgA protease β-domain of gonorrhea (Nisseria gonorrhoeae ).

Figure 14 shows the anti-mouse IgG antibody (AP) conjugated with alkaline phosphotase after SDS-PAGE of lysate of E. coli cells expressing 2C10 scFv on the surface of the envelope and reacted with an anti 6x histidine antibody. The photograph was confirmed by Western blot analysis using conjugated anti-porcine IgG.

(M 1 and M 2: pre-stained protein size markers; lane 1: normal E. coli cell lysates; lane 2: for E. coli cells transformed with “pET5aSig-scFv-IgABeNE” and not IPTG-induced) Seafood; Lane 3-7: E. coli cell lysates transformed with "pET5aSig-scFv-IgABeNE" and induced with IPTG of 0.1 mM, 0.2 mM, 0.4 mM, 0.5 mM, 0.7 mM, respectively)

Figure 15 is a photograph staining the plaques of tissue culture cells confirmed that the PEDV plaques were reduced by the neutralizing ability of E. coli expressing 2C10 scFv on the outer envelope surface (No Bacteria: bacterial non-treated group; Normal E. coli : Escherichia coli treated group; E. coli expressing 2C10 scFv: scFv envelope surface expression E. coli treated group)

<110> National Veterinary Research Quarantine Service <120> Transfer vector to express the scFv of neutralizing antibody          against porcine epidemic diarrhea virus on the bacterial outer          membrane and the E.coli to inhibiting the propagation of porcine          epidemic diarrhea virus <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 777 <212> DNA <213> Artificial Sequence <220> <223> single chain variable fragment antibody <400> 1 gcaactgcgg cccagccggc catggcccag gtccaactgc agcagtctgg agctgagctg 60 gtaaggcctg ggacttcagt gaaggtgtcc tgcaaggctt ctggatacgc cttcacaaat 120 tacttgatag agtggataaa gcagaggcct ggacaggtcc ttgagtggat tggagtgatt 180 aatcctggga gtggtggtac taactacaat gagaaattca agggcaaggc aacactgact 240 gcagacaaat cctccagcac tgcctacatg cagctcagca gcctgacatc tgatgactct 300 gcggtctatt tctgtgcaag aaggggttac tacgtggggg gttatgctat ggactactgg 360 ggccaaggga ccacggtcac cgtctcctca ggtggaggcg gttcaggcgg aggtggctct 420 ggcggtggcg gatcagacat tgagctcacc cagtctccag cctccctatc tgcatctgtg 480 ggagaaactg tcaccatcac atgtcgagca agtgagaata tttacagtta tttagcatgg 540 tatcagcaga aacagggaaa atctcctcag ctcctggtct ataatggaaa aacctttccg 600 gaaggtgtgc cgtcaaggtt cactggcagt ggatcaggca cacagttttc tctgaagatc 660 aacagcctgc agcctgaaga ttttgggagt tattactgtc aacatcatta tggtagtccg 720 ctcacgttcg gtgctgggac caagctggaa ataaaacggg cggccgcaga atgactc 777 <210> 2 <211> 90 <212> DNA <213> Neisseria gonorrhoeae <400> 2 catatgaaag ccaaacgttt taaaattaac gccatatcct tatccatctt tcttgcctat 60 gcccttacgc catactcaga agcgggatcc 90 <210> 3 <211> 930 <212> DNA <213> Neisseria gonorrhoeae <400> 3 ggtaccccaa gagccgcgca gccgcgaacc caagccgccg cgcaagccga tgcagtcagc 60 accaatacta actcggcttt atctgacgca atggcaagca cgcaatctat cttgttggat 120 acaggtgctt acttaacacg gcacattgca caaaaatcac gcgctgatgc cgaaaaaaac 180 agtgtttgga tgtcaaacac cggttatggc cgtgattatg cttccgcaca atatcgccgg 240 tttagttcga aacgcacgca aacacaaatc ggcattgacc gcagcttgtc cgaaaatatg 300 cagataggcg gagtattgac ttactctgac agtcagcata cttttgatca ggcgagcggc 360 aaaaatactt ttgtgcaagc caacctttat ggtaagtatt atttaaatga tgcttggtat 420 gtggccggcg atattggtgc gggcagcttg agaagccggt tacaaacgca gcaaaaagca 480 aactttaacc gaacaagcat ccaaaccggc cttactttgg gcaatacgct gaaaatcaat 540 caattcgaga ttgtccctag tgcgggtatc cgttacagcc gcctgtcatc tgcagattac 600 aagttgggtg acgacagtgt taaagtaagt tctatggcag tgaaaacact aacggccgga 660 ctggattttg cttatcggtt taaagtcggc aaccttaccg taaaaccctt gttatctgca 720 gcttactttg ccaattatgg caaaggcggc gtgaatgtgg gcggtaaatc cttcgcctat 780 aaagcagata atcaacagca atattcagca ggcgccgcgt tactgtaccg taatgttaca 840 ttaaacgtaa atggcagtat tacaaaagga aaacaattgg aaaaacaaaa atccggacaa 900 attaaaatac agattcgttt ctaagaattc 930 <210> 4 <211> 5874 <212> DNA <213> Neisseria gonorrhoeae <400> 4 agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc 60 tagaaataat tttgtttaac tttaagaagg agatatacat atgaaagcca aacgttttaa 120 aattaacgcc atatccttat ccatctttct tgcctatgcc cttacgccat actcagaagc 180 gggatccatg gcccaggtcc aactgcagca gtctggagct gagctggtaa ggcctgggac 240 ttcagtgaag gtgtcctgca aggcttctgg atacgccttc acaaattact tgatagagtg 300 gataaagcag aggcctggac aggtccttga gtggattgga gtgattaatc ctgggagtgg 360 tggtactaac tacaatgaga aattcaaggg caaggcaaca ctgactgcag acaaatcctc 420 cagcactgcc tacatgcagc tcagcagcct gacatctgat gactctgcgg tctatttctg 480 tgcaagaagg ggttactacg tggggggtta tgctatggac tactggggcc aagggaccac 540 ggtcaccgtc tcctcaggtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc 600 ggacattgag ctcacccagt ctccagcctc cctatctgca tctgtgggag aaactgtcac 660 catcacatgt cgagcaagtg agaatattta cagttattta gcatggtatc agcagaaaca 720 gggaaaatct cctcagctcc tggtctataa tggaaaaacc tttccggaag gtgtgccgtc 780 aaggttcact ggcagtggat caggcacaca gttttctctg aagatcaaca gcctgcagcc 840 tgaagatttt gggagttatt actgtcaaca tcattatggt agtccgctca cgttcggtgc 900 tgggaccaag ctggaaataa aacgggcggc cgcagaactc gagcatcatc atcatcatca 960 tggtacccca agagccgcgc agccgcgaac ccaagccgcc gcgcaagccg atgcagtcag 1020 caccaatact aactcggctt tatctgacgc aatggcaagc acgcaatcta tcttgttgga 1080 tacaggtgct tacttaacac ggcacattgc acaaaaatca cgcgctgatg ccgaaaaaaa 1140 cagtgtttgg atgtcaaaca ccggttatgg ccgtgattat gcttccgcac aatatcgccg 1200 gtttagttcg aaacgcacgc aaacacaaat cggcattgac cgcagcttgt ccgaaaatat 1260 gcagataggc ggagtattga cttactctga cagtcagcat acttttgatc aggcgagcgg 1320 caaaaatact tttgtgcaag ccaaccttta tggtaagtat tatttaaatg atgcttggta 1380 tgtggccggc gatattggtg cgggcagctt gagaagccgg ttacaaacgc agcaaaaagc 1440 aaactttaac cgaacaagca tccaaaccgg ccttactttg ggcaatacgc tgaaaatcaa 1500 tcaattcgag attgtcccta gtgcgggtat ccgttacagc cgcctgtcat ctgcagatta 1560 caagttgggt gacgacagtg ttaaagtaag ttctatggca gtgaaaacac taacggccgg 1620 actggatttt gcttatcggt ttaaagtcgg caaccttacc gtaaaaccct tgttatctgc 1680 agcttacttt gccaattatg gcaaaggcgg cgtgaatgtg ggcggtaaat ccttcgccta 1740 taaagcagat aatcaacagc aatattcagc aggcgccgcg ttactgtacc gtaatgttac 1800 attaaacgta aatggcagta ttacaaaagg aaaacaattg gaaaaacaaa aatccggaca 1860 aattaaaata cagattcgtt tctaagaatt cttgaagacg aaagggcctc gtgatacgcc 1920 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 1980 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 2040 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 2100 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 2160 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 2220 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 2280 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg 2340 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 2400 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 2460 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 2520 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 2580 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 2640 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 2700 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 2760 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 2820 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 2880 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 2940 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 3000 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 3060 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 3120 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 3180 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 3240 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 3300 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 3360 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 3420 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 3480 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 3540 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 3600 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 3660 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 3720 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 3780 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 3840 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 3900 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac 3960 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 4020 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 4080 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 4140 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca 4200 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt 4260 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt 4320 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata 4380 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta 4440 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc 4500 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag 4560 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc 4620 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt 4680 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta 4740 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt 4800 ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg 4860 atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg 4920 gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg 4980 aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg 5040 cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga 5100 cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct 5160 gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga 5220 tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg 5280 ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc 5340 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga 5400 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa 5460 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa 5520 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc 5580 tcaagggcat cggtcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta 5640 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc 5700 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga 5760 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa 5820 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcg 5874  

Claims (5)

A surface expression vector for expressing a short chain variable segment of the swine epidemic diarrhea virus neutralizing antibody consisting of SEQ ID NO: 1 on the bacterial membrane surface. The method of claim 1, wherein the surface expression vector is a nucleotide sequence consisting of SEQ ID NO: 1 encoding a single chain variable fragment antibody site that specifically neutralizes Swine pandemic diarrheal virus (PEDV), the short chain variable segment gene A base consisting of T7 RNA polymerase consisting of the nucleotide sequences of 21bp to 40bp of the sequence 4 to be expressed on the bacterial membrane surface, and the sequence 2 encoding the signal peptide of the IgA protease gene of Neisseria gonorrhoeae . A surface expression vector comprising an autotransforter β domain gene consisting of a sequence, a base sequence encoding six histidines, and sequence 3 of an IgA protease of Neisseria gonorrhoeae inserted into a bacterial membrane. The surface expression vector of claim 1, wherein the surface expression vector has characteristics such as a nucleotide sequence of SEQ ID NO: 4 and a genetic map of FIG. 13. E. coli expressing on the surface of the bacterial membrane a short chain variable segment protein transformed with the surface expression vector of any one of claims 1 to 3 specifically neutralizing the swine pandemic diarrheal virus. Composition for preventing or treating swine pandemic diarrhea virus containing E. coli of claim 4.

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