TWI615401B - Soluble recombinant flic fusion protein in improving activities of porcine immune cells and prokaryotic transformant for over-expressing the same - Google Patents

Abstract

The present invention relates to a soluble recombinant FliC flagellin fusion protein which promotes the activity of porcine immune cells, which is a recombinant gene of Salmonella enteritidis wild strain flagellin FliC, which expresses soluble recombinant FliC flagella by a prokaryotic expression system. protein. The soluble recombinant FliC flagellin can promote the innate immune response of porcine immune cells, and not only increase the gene expression of toll-like receptor 7 (TLR7) and steroid receptor 8 (TLR8). At the same time, it promotes the secretion of interleukin-4 (IL-4) and IL-10 by porcine immune cells, so it can be used as a vaccine for pig vaccine.

Description

Soluble recombinant FliC whip that promotes porcine immune cell activity Hair fusion protein and its prokaryotic transformation strain

The invention relates to a soluble recombinant FliC flagellin fusion protein and a performance strain thereof for promoting the activity of porcine immune cells, and has the function of a porcine vaccine adjuvant, in particular to a sputum receptor 7 which promotes porcine immune cells ( Toll-Like Receptor 7; TLR7) and the gene expression of steroid receptor 8 (TLR8) and soluble recombinant FliC flagella fusion protein which promotes the secretion of Th2 cytokines IL-4 and IL-10 and their prokaryotic transformation strains It can be used as an adjuvant for pig vaccines.

At present, the main adjuvants for animal vaccines are aluminum gel adjuvants and oily adjuvants, but the disadvantage is that both are chemical adjuvants, which cannot enhance the immune response of specific Th1 cells, especially for inactivated vaccines and live poisons. Vaccines or attenuated vaccines do not produce adequate protection against the adaptive immune response.

The immune system includes an innate immune response and an acquired immune response. Innate immune response against viruses, bacteria and extracellular parasites, one of the current vaccine development strategies Adjuvant stimulates innate immune response, promotes acquired immune response by innate immune response, regulates the expression of cytokines, and promotes the reaction of antigen-presenting cells with T cells to make appropriate humoral and cellular immunity. reaction. For example, activated B cells produce antibodies, and antigen-presenting cells (APCs) secrete cytokines to promote the immune response of vaccines.

Toll-like receptor (TLR) is one of the important factors in the innate immune response. According to different types of TLR, different pathogenic factors such as bacteria and viruses can be identified, for example, steroid receptor 5 (TLR 5) can recognize bacterial flagella, steroid receptor 7 (TLR 7) and steroid receptor 8 (TLR 8) recognizes viral single strand RNA (ssRNA). Different types of TLRs can be used to adjust the immune response by signal transmission to achieve the effect of resisting different pathogens.

It has been reported in the literature that the binding of TLR agonists to TLRs can alter the microenvironment of antigen-presenting cells in contact with T cells, and promote the formation of stable immune synapses between antigen-presenting cells and T-cells ( Immune synapse). In addition, the microenvironment in which antigen-presenting cells are exposed to T cells requires the participation of specific cytokines to form a stable immune synapse.

The bacterial flagella is a TLR 5 agonist. The conventional bacterial flagella is a polymer formed from a plurality of flagellin monomers. Bacterial flagella, which is naturally isolated from the environment, can promote the production of antibodies, but it can cause excessively strong inflammatory reactions, and it does not help to identify mechanisms such as viral ssRNA, and is not suitable for vaccines. Agent.

In view of this, it is urgent to propose an ingredient which can be used as an adjuvant for pig vaccines to overcome the disadvantages of the conventional bacterial flagella used in adjuvants.

Therefore, one aspect of the present invention provides a soluble recombinant FliC flagellin fusion protein having the function of a porcine vaccine adjuvant, which not only activates the porcine immune cell activity in vivo, but also promotes the innate immune response of the pig, thereby enhancing its Immunocyte activity.

Another aspect of the invention provides a recombinant gene for a soluble recombinant FliC flagellin fusion protein encoding a soluble recombinant FliC flagellin fusion protein as described above.

A further aspect of the invention provides a transformed strain for the expression of a soluble recombinant FliC flagellin fusion protein deposited at the Food Industry Development Institute of Hsinchu, Republic of China, under the accession number BCRC 940651.

A further aspect of the present invention provides an adjuvant for a porcine vaccine comprising the above-described soluble recombinant FliC flagellin fusion protein.

Still another aspect of the present invention provides an adjuvant for a porcine vaccine comprising the above-described transformed strain for expressing a soluble recombinant FliC flagellin fusion protein.

According to the above aspect of the present invention, a recombinant protein which activates porcine immune cell activity has a function of a porcine vaccine adjuvant which is composed of an amino acid sequence such as the sequence of the sequence identification number (SEQ ID NO) 1. Composition, which promotes the activity of gene expression of sputum receptor 7 (TLR7) and steroid receptor 8 (TLR8), and promotes Th2 cytokines Secretion of IL-4 and IL-10.

According to an embodiment of the present invention, the amino acid sequence SEQ ID NO 1 is derived from Salmonella enteritidis SE-K40, deposited in the Hsinchu Food Industry Development Research Institute of the Republic of China, and the accession number is BCRC 940651. .

According to another aspect of the invention, a recombinant gene of a soluble recombinant FliC flagellin fusion protein comprising the DNA sequence of SEQ ID NO 2 is provided to encode the amino acid sequence described above.

According to still another aspect of the present invention, a transformant strain for expressing a soluble recombinant FliC flagellin fusion protein comprising a recombinant vector comprising a recombinant gene encoding the amino acid sequence described above is provided.

According to still another aspect of the present invention, an adjuvant for a porcine vaccine comprising the above-described soluble recombinant FliC flagellin fusion protein is provided.

According to still another aspect of the present invention, an adjuvant for a porcine vaccine comprising the above-described transformant strain for expressing a soluble recombinant FliC flagellin fusion protein is formed by removing an insoluble protein.

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; An electrophoretic analysis map of a nucleic acid fragment of a recombinant gene.

Figure 2 is a diagram showing SDS-PAGE electrophoresis analysis of soluble recombinant FliC flagellar fusion protein according to an embodiment of the present invention.

Figure 3A is a diagram showing the soluble recombinant FliC flagella fusion egg of Example 2. The amount of TLR7 gene expression after white treatment of blood mononuclear cells in pigs.

Figure 3B is a graph showing the expression of TLR8 gene after treatment of peripheral blood mononuclear cells of pigs by the soluble recombinant FliC flagellin fusion protein of Example 2.

Figure 4A is a graph showing the amount of IL-4 produced by treating the peripheral blood mononuclear cells of pigs by the soluble recombinant FliC flagellin fusion protein of Example 2.

Figure 4B is a graph showing the amount of IL-10 produced by treating the peripheral blood mononuclear cells of pigs by the soluble recombinant FliC flagellin fusion protein of Example 2.

As described above, the present invention provides a soluble recombinant FliC flagellin fusion protein which promotes the activity of porcine immune cells and a prokaryotic transformation strain thereof, which is a recombinant gene of the flagellin FliC of the wild strain of Salmonella, which is expressed by prokaryote. A large number of soluble recombinant FliC flagellin were systematically expressed. The obtained soluble recombinant FliC flagellin can promote the innate immune response of porcine immune cells, not only increase the gene expression of TLR7 and TLR8 in porcine immune cells, but also promote the secretion of IL-4 and IL-10 by porcine Th2 cells, so it can be used as a pig. Use a vaccine adjuvant.

The "soluble recombinant FliC flagellin fusion protein" as used herein refers to the amino acid sequence SEQ ID NO 1 obtained from the wild strain of S. enteritidis. In one embodiment, the above-mentioned S. enteritidis SE-K40 is provided by the laboratory of the Department of Veterinary Medicine of Pingtung University of Science and Technology, and is deposited in the Food Industry Development Research Institute of Hsinchu, Republic of China, on October 12, 2012. The registration number is BCRC 940651.

Method for producing soluble recombinant FliC flagella fusion protein

In one embodiment, the soluble recombinant FliC flagellin fusion protein described above can further utilize a prokaryotic expression system to express the DNA sequence SEQ ID NO 2 to obtain the amino acid sequence SEQ ID NO 1. In this embodiment, the DNA sequence SEQ ID NO 2 comprises an open reading frame of the start codon (ATG) to stop codon (TAA) of the Salmonella flagellin (FliC) (open reading) The frame; ORF) sequence is about 1515 base pairs in length. In an exemplary embodiment, a polymerase chain reaction (PCR) can be amplified using a specific primer pair to obtain a DNA sequence of SEQ ID NO 2.

The above suitable pair of primers can be based on, for example, the GenBank of the National Center for Biotechnology Information (NCBI) website (No. AM933172.1), using commercially available sequence analysis software or other functions equivalent. The software, such as the EditSeq 5.0 DNASTAR Expert Sequence Analysis Software (DNASTAR Inc.), redesigned the sequence of the specific primer pair and modified to contain restriction enzyme cleavage sites for alternative colonization. The above primer pairs include an upstream primer and a downstream primer, wherein the 5' end of the upstream primer is designed with a first restriction enzyme cleavage site, as shown in the DNA sequence SEQ ID NO 3; and the 5' end of the downstream primer is designed with a second restriction enzyme digestion. The position is as shown in the DNA sequence SEQ ID NO 4.

In an illustration, the first restriction enzyme may be, for example, NheI , and the second restriction enzyme may be, for example, XhoI . A recombinant gene of a soluble recombinant FliC flagellin fusion protein of about 1515 bp which can be amplified by the chromosomal DNA of Salmonella (BCRC 940651) using the above primer, is the DNA sequence SEQ ID NO 2. The cited pairs of the above-described examples are shown in Table 1 which will be described later.

Next, the recombinant gene of the soluble recombinant FliC flagellin fusion protein and the commercially available vector can be respectively cleaved by the first restriction enzyme and the second restriction enzyme, and then ligated by a ligase to be ligated into a soluble recombinant FliC flagellin fusion protein. Recombination of recombinant genes expresses plastids. Thereafter, the recombinant expression plastid is transformed into a prokaryotic expression system, and thereby the soluble recombinant FliC flagellin fusion protein is expressed (or translated).

It was confirmed in the following examples that the amino acid sequence of the soluble recombinant FliC flagellin fusion protein of the present invention differs from the known sequence, but does not affect its antigenicity. The transformed strain of the invention can not only increase the yield of soluble recombinant FliC flagellin fusion protein, but the soluble recombinant FliC flagellin fusion protein can increase the gene expression of TLR7 and TLR8 in porcine immune cells, and promote the secretion of IL-4 by porcine Th2 cells. IL-10, it can be used as a vaccine for pig vaccine.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Run Decoration.

Example 1: Construction of recombinant plastids of full-length gene of Salmonella flagellin (FliC) and large-scale culture

1. A nucleic acid fragment encoding a full-length gene of Salmonella flagellin (FliC)

This example is a recombinant plastid, a transformed strain, and a large number of cultures of the Salmonella flagellin (FliC) gene. The nucleic acid fragment of the full-length gene of Salmonella flagellin (FliC) contained in the gene was subjected to polymerase chain reaction using a specific primer pair from Salmonella enteritidis SE-K40 (Accession No. BCRC 940651) DNA. The increase is achieved.

In other words, after extracting the Salmonella DNA using a commercially available kit, the primer pair shown in Table 1 and the DNA are added to a commercially available PCR reaction reagent (Promega, Madison, WI, US). The polymerase chain reaction was carried out using a temperature cycle controller (Thermocycler; TaKaRa, Shiga, Japan) to obtain a nucleic acid fragment. The reagents for the above PCR reaction are as illustrated in Table 2:

In the case of performing polymerase chain reaction with DNA using a primer pair, the reaction conditions may include, but are not limited to, exemplified by about 1 minute at about 94 ° C, about 30 seconds at 94 ° C, and about 30 seconds at 52 ° C. The reaction was repeated for 30 minutes at 72 ° C for a total of 30 cycles to obtain a nucleic acid fragment of the FliC full-length gene, which was about 1515 base pairs in length.

Please refer to FIG. 1 , which is an electrophoresis analysis diagram of a nucleic acid fragment of a Salmonella flagellin gene according to an embodiment of the present invention, wherein the Mth channel represents a DNA ladder of 100 base pairs. "1500 bp" on the left side of the Mth channel indicates a DNA ladder mark of 1500 base pairs in size, and FliC indicates a nucleic acid fragment of the Salmonella flagellin gene (about 1515 base pairs, as indicated by arrow 101). The nucleic acid fragment of the obtained FliC full-length gene was confirmed by DNA sequencing to confirm the sequence was correct.

2. Construction of a recombinant plasmid containing Salmonella flagellin (FliC) and a transformed strain containing the same

After the above polymerase chain reaction, the nucleic acid fragment of the FliC full-length gene obtained by the primer is subjected to restriction enzyme action, for example, NheI and XhoI (both TaKaRa, Shiga, Japan), and the nucleic acid fragment of the FliC full-length gene is truncated and purified. Thereafter, it can be constructed and ligated into a vector which is treated and purified by the same restriction enzyme, for example, a mammalian cell expression vector pET21b vector (Promega, USA) to form a recombinant plastid to obtain a recombinant substance containing a nucleic acid fragment of the FliC full-length gene. body. The resulting recombinant plasmid was also confirmed by PCR and DNA sequencing. However, the restriction enzyme action, the recombinant plastid, and the ligation reaction relating to the recombinant plastid are well known to those of ordinary skill in the art, and therefore will not be further described herein.

The recombinant plastid can be further transformed into a suitable host, such as a competent cell of an E. coli BL21 (DE3) strain, as a host for the transfer and preservation of the recombinant plastid. .

Thereafter, antibiotic screening was carried out by using a Luria-Bertani culture solution (LB culture solution) containing ampicillin (MD Bio, Germany) in a 37 ° C incubator, and after 16 hours of culture, the successfully transformed strain was selected. A large number of cultures can be carried out by PCR and DNA sequencing to determine that the sequence of the constructed sequence is backward.

The above PCR product was subjected to DNA sequencing (the sequencing of the DNA was performed by Kelonex Biotech Co., Ltd.), and the result was the nucleotide sequence shown in SEQ ID NO 2, wherein Salmonella The gene sequence of Bacillus flagellin (FliC) has 1515 nucleotides, and is linked to the National Center for Biotechnology Information (NCBI) website, GenBank accession number AM933172.1 The sequence alignment of Salmonella flagellin (FliC) has a 99% similarity.

Please refer to Table 3, which is the DNA sequence of the Salmonella flagellin (FliC) of the present invention, SEQ ID NO 2, amino acid sequence SEQ ID NO 1 and Salmonella standard strain (No. AM933172.1) The nucleotide sequence is aligned with a portion of the amino acid sequence.

From the sequence alignment result of the third table, it is known that the DNA sequence of the Salmonella flagellin (FliC) of the present invention is SEQ ID NO 2 at 1406 as compared with the standard strain of Salmonella (No.: AM933172.1). The nucleotide is mutated from A to G, resulting in the amino acid sequence of SEQ ID NO 1 of the Salmonella flagellin (FliC) of the present invention being mutated from cysteine (Cysteine; Cys) to tyramine at the 469th amino acid. Acid (Tyrosine; Tyr).

It is described herein that the above-mentioned construction of recombinant plastids, transformation to competent cells, mass culture, extraction of plastid DNA, optical density (OD) values, establishment of a standard curve, and DNA sequencing are in the technical field. Anyone with ordinary knowledge is well known, so it will not be repeated here.

Example 2: Preparation of soluble recombinant FliC flagellin fusion protein

1. Using a large number of transformed strains to express soluble recombinant FliC flagella fusion protein

In this embodiment, first, the transformed strain containing the above recombinant expression vector is cultured in 100 mL LB culture solution (containing amsillin, ampicillin), and cultured at 37 ° C until the OD _{600 nm} value is about 0.8, to 1 The expression of recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). After 5 hours of induction, the bacterial solution was collected and centrifuged at 8000 xg for 5 min at 4 °C. After centrifugation, the cells were suspended in 5 mL of phosphate buffer saline (PBS) (pH 7.8) for washing, and centrifuged at 8000 x g for 5 min at 4 °C. Next, 5 mL of PBS was added to suspend the cells, and then the cells were disrupted by ultrasonic waves, and then 1 mM 1 mM of phenylmethanesulfonyl fluoride (PMSF) (Roche Applied Scienc, Germany) was added to obtain coarse particles. Extract protein solution.

Next, the crude extract protein solution was centrifuged at 25000 xg for 10 min at 4 ° C to remove insoluble protein, and the resulting supernatant contained soluble recombinant FliC flagellin fusion protein.

Subsequently, the supernatant containing the soluble recombinant FliC flagellin fusion protein was replaced with a lyophilization buffer containing 0.03 M NH ₄ HCO ₃ (Sigma, USA), and subjected to a lyophilization step to obtain a soluble recombinant FliC flagella fusion. Protein powder. In an example, a freeze drying step can be performed using a multifunctional medium dryer (FDU-2100, EYELA, Tokyo).

2. Sodium dodecyl sulfate-polyacrylamide gel e1ectrophoresis; SDS-PAGE

The above-mentioned induced strain-expressing transformed strain (containing soluble recombinant FliC flagellin fusion protein) and 2-fold sample buffer (2X sample buffer; for example, containing 100 mM Tris-HCl pH 6.8, 200 mM dithiothreitol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol), mixed in an equal volume, boiled at 100 ° C for 4 minutes and cooled, placed on ice and prepared for analysis on 8% SDS-PAGE, at about 100 volts for about 90 minutes. Electrophoretic analysis, and then stained and decolorized according to standard procedures. Since SDS-PAGE can be carried out using commercially available products, there is no further explanation here.

Please refer to FIG. 2, which is a SDS-PAGE electrophoresis analysis diagram of a soluble recombinant FliC flagellin fusion protein according to an embodiment of the present invention, wherein the Mth lane represents a standard protein molecular weight marker, and represents “26 kDa, 34 kDa, 43 respectively. Proteins of molecular weight such as kDa, 55 kDa, 72 kDa, 95 kDa, 130a, 170a". Lanes 1 to 2 represent the protein ladder and pET21b vector, respectively, and lane 3 indicates that the soluble recombinant FliC flagellin fusion protein expressed by the transformant of Example 1 was induced by IPTG.

From the results of Fig. 2, it was revealed that the transformant strain of Example 1 was induced by IPTG to obtain a soluble recombinant FliC flagellin fusion protein having a molecular weight of about 55 kDa (as indicated by arrow 201) as compared with the standard protein molecular weight marker.

Example 3: Evaluation of the immune promoting effect of soluble recombinant FliC flagellin fusion protein in vitro

1. Extraction of peripheral blood mononuclear cells

This example is a peripheral blood mononuclear cell of 12 6-week-old Specific Pathogen-Free (SPF) Large White Pigs (Source: Taiwan Institute of Animal Science and Technology, Zhunan, Taiwan). ;PBMC).

First, whole blood of healthy pigs was taken, and heparin anticoagulant was added, and then centrifuged at 1300 × g for about 30 minutes at 4 ° C to collect a buffy coat. Mixing the aforementioned white blood cell layer with an equal volume of RPMI-1640 medium (for example, containing 2.05 mM L-glutamine, 25 mM HEPES buffer, 2 g of sodium bicarbonate) (GIBCO, NY, USA) at 37 ° C, and then It is added to an equal volume density gradient solution, such as the lymphocyte separation solution Ficoll-Paque (GE Healthcare, Stgiles, Sweden), and centrifuged at 200 x g for 25 minutes at about 4 °C.

Next, the cell layer was aspirated, washed once with 1 × PBS, centrifuged at 200 × g for 10 minutes at 4 ° C, and then washed twice with RPMI-1640 medium to obtain purified lymphocytes. After counting the cells, a cell culture medium such as RPMI-1640 (GIBCO, NY, USA) containing 10% fetal bovine serum, penicillin (40 μg/mL), and 5×10 ⁻⁵ M β-mercaptoethanol (GIBCO, NY, USA) is used. After the cell concentration was adjusted to 5.4 × 10 ⁶ cells/mL, 500 μL of the cell liquid (2.7 × 10 ⁶ cells/well) was added to a 24-well cell culture plate.

2. Evaluation of immunostimulatory activity of soluble recombinant FliC flagellar fusion protein

In the above-mentioned isolated peripheral blood mononuclear cells, 50 μL of 0.2 μg/μL of flagellin FliC, negative control group (PBS) and positive control group (containing 0.2 μg/μL of concanavalin A, Con) were added. A) After incubating for 6 hours in a 37 ° C incubator at 5% CO ₂ , centrifuge with a centrifuge (BECKMAN Cs-6R, USA) at 400 × g for 20 minutes at 4 ° C to collect the cells and supernatant respectively. Subsequently, real-time polymerase chain reaction (Real-Time PCR) and ELISA kits can be used to analyze the TLR gene expression and cytokine content of cells treated with soluble recombinant FliC flagellin fusion protein.

2.1 Evaluation of the expression of TLR7 and TLR8 genes by soluble recombinant FliC flagellin fusion protein

2.1.1 Synthesis of nucleic acid fragments of TLR7 and TLR8 genes

This example was evaluated by reverse transcription polymerase chain reaction (RT-PCR) using the reverse transcription polymerase chain reaction (RT-PCR) to evaluate the expression of the TLR7 and TLR8 genes in the animal model of Example 3. First, the above-mentioned peripheral blood mononuclear cells treated with flagellin FliC, negative control group (PBS), and positive control group (containing 0.2 μg/μL of concanavalin (Con A), respectively, were centrifuged at 4 ° C. The supernatant was centrifuged at 800 × g for 10 minutes, and the supernatant was collected by repeating the centrifugation step, followed by storage at -70 ° C for use.

Subsequently, each well was added in approximately 1 mL of a commercially available RNA extraction reagent (e.g. TriSolution ^TM Reagent; GeneMark, Canada) destruction of the cell membrane, and then chloroform was added approximately 200 μL of a commercially available (chloroform; Sigma, USA) to make DNA, proteins It is layered with RNA, while RNA is present in the water layer. After centrifuging the supernatant, first use about 200 μL of isopropanol (Sigma, USA) to precipitate total RNA at about -20 ° C, and then use about 1 mL of 100% ethanol, about 1 mL of 75% pyrocarbonic acid. Deuterated water (DEPC-ddH ₂ O) treated with diethyl pyrocarbonate (DEPC; Sigma, USA) to remove impurities from total RNA. Thereafter, after removing the supernatant by centrifugation and evaporating residual ethanol, about 25 μL of DEPC-ddH ₂ O was added to reconstitute the total RNA. The total RNA extracted by the above extraction was confirmed by RNA electrophoresis and measured by optical density (OD) ₂₆₀ / OD ₂₈₀ (OD ₂₆₀ / OD ₂₈₀ > 1.8) at wavelengths of 260 nm and 280 nm. Subsequent RT-PCR was performed to detect the concentration of total RNA. However, the above-mentioned extraction of total RNA and optical density (OD ₂₆₀ ) values are well known to those of ordinary skill in the art and will not be described herein.

The reverse transcription reaction is carried out by using the extracted total RNA as a template to obtain cDNA, and the reagent for reverse transcription reaction is exemplified in Table 4-1:

When performing a reverse transcription reaction, the reaction conditions may include, but are not limited to, As exemplified below, first, the reaction was carried out at 42 ° C for 60 minutes and then at 95 ° C for 5 minutes. After completion of the reverse transcription reaction, the resulting cDNA was stored at 0 °C.

Next, using the cDNA obtained above as a template, the polymerase chain reaction was carried out by using a primer pair to increase the nucleic acid fragment of TLR7, TLR8 and cyclophilin (as a control group for internal control), wherein the polymerase The reagent for the chain reaction is exemplified in Table 5:

This example is referred to the National Center for Biotechnology Information. (National Center for Biotechnology Information; NCBI) website, gene bank (GenBank) accession number (accession number) AF245702, AB092975 and AY266299 designed TLR7, TLR8, and cyclophilin primer, wherein the TLR7 gene upstream primer system 6 is the sequence shown in SEQ ID NO: 5; the downstream primer of the TLR7 gene is the sequence shown in SEQ ID NO 6. The upstream primer of the TLR8 gene is the sequence shown in SEQ ID NO: 7; the downstream primer of the TLR8 gene is the sequence shown in SEQ ID NO 8. The control group for internal control was a cyclophilin gene in which the upstream primer of the cyclophilin gene was sequenced as shown in SEQ ID NO: 9 and the downstream primer of the cyclophilin gene was listed in the sequence shown in SEQ ID NO 10.

Polymerase chain-locking with the primers described above using the primer pair The reaction conditions may include, but are not limited to, exemplified below: First, the double-stranded template DNA is subjected to a pre-separation reaction by performing at 94 ° C for 5 minutes to 10 minutes. Next, the reaction is carried out at 94 ° C for 30 seconds to 1 minute, and the reaction is carried out at 50 ° C to 60 ° C for 30 seconds to 1 minute to carry out the primer pair-template DNA binding reaction, and the DNA extension reaction is carried out at 72 ° C for 30 seconds to 2 minutes. In this cycle, a total of 30 to 40 cycles of the reaction are repeated, but in another embodiment, the primer-template DNA bonding reaction can be carried out, for example, at 55 ° C to 60 ° C for 30 seconds, and then at 72 ° C. The reaction was extended for 1 minute to 1.5 minutes, and a total of 30 cycles of the reaction were repeated. The PCR product was sent to Kelonex Biotech Co., Ltd. for sequencing to confirm its nucleic acid sequence.

2.1.2 Evaluation of the effect of soluble recombinant FliC flagellin fusion protein on the expression of TLR7 and TLR8 genes

This example utilizes commercially available commercially available gene expression assays, such as the LightCycler-Faststart DNA Master SYBR green I reagent set (Roche, Germany) or other functionally equivalent commodity to assess soluble recombinant FliC flagellin fusion protein for TLR7 and TLR8 genes. The impact of performance.

The real-time polymerase chain reaction (Real-Time PCR) was carried out by using the extracted cDNA as a template, wherein the reagent of the polymerase is as illustrated in Table 7:

Table 7

For details of the remaining operating steps, refer to the manufacturer's manual, which is not described here.

Thereafter, the reaction was carried out in Roche Molecular Biochemicals Lightcycle Software, and after completion of the reaction, the expression amounts of TLR7 and TLR8 genes were analyzed. The mode of operation and judgment are in accordance with the manufacturer's manual, which is not here.

Please refer to Figures 3A and 3B, wherein Figure 3A shows the TLR7 gene expression after treatment of the peripheral blood mononuclear cells of pigs by the soluble recombinant FliC flagellin fusion protein of Example 2, and Figure 3B depicts The amount of TLR8 gene expression after treatment of peripheral blood mononuclear cells of pigs by the soluble recombinant FliC flagellin fusion protein of Example 2 is shown.

The vertical axis of Fig. 3A and Fig. 3B shows the relative expression of the gene of TLR7 or TLR8 gene (2 ^{- △ △ Ct} ), each group of samples has at least three repeats, and the relative gene expression (2 ^{- △ △ Ct} ) -△△Ct represents the change in fluorescence intensity (ΔCt) of TLR7 or TLR8 gene in peripheral blood mononuclear cells treated with soluble recombinant FliC flagellin fusion protein of Example 2, minus the solubility of Example 2 Recombinant FliC flagellin fusion protein was used to treat the fluorescence intensity of TLR7 or TLR8 gene in peripheral blood mononuclear cells of pigs. The same figure number (eg, between a and a) indicates a non-significant difference between the groups. Conversely, different numbers between different numbers (eg, a and b) indicate significant differences between groups.

From Fig. 3A and Fig. 3B, it can be seen that compared with other groups (positive control group and negative control group), peripheral blood mononuclear cells were treated with soluble recombinant FliC flagellin fusion protein after treatment of peripheral blood mononuclear cells of pigs. The TLR7 or TLR8 gene expression was significantly higher than the other groups.

2.1.3 Assessing the effect of soluble recombinant FliC flagellin fusion protein on the production of cytokines (IL-4 and IL-10)

This example utilizes commercially available products for evaluating the production of interleukin IL-4 and IL-10 stimulated by soluble recombinant FliC flagellin fusion protein of pigs in peripheral blood mononuclear cells of the second embodiment, such as porcine IL-4. ELISA kits (R&D Systems, USA) and porcine IL-10 ELISA kits (R&D Systems, USA) were used to evaluate peripheral blood mononuclear cells stimulated by the soluble recombinant FliC flagellar fusion protein of Example 2 in Example 3. The amount of IL-4 and IL-10 produced by the cells.

The procedure for the porcine IL-4 ELISA kit and the porcine IL-10 ELISA kit is exemplified below. First, add analytical dilution (Assay Diluent RD1-63) and capture antibody (goat anti-porcine IL-4 IgG/goat anti-porcine) to each well in a 96-well microplate. After the mixture of IL-10 IgG; R&D Systems, U.S.A.), 100 μL of the standard solution of different dilution ratios and the sample to be tested were separately added and allowed to act at room temperature for about 1 hour. Next, it was washed three times by adding 200 μL of PBST (1× phosphate buffer saline, 0.5% Tween 20).

Then, 200 μL of detection antibody (detecting antibody goat anti-porcine IL-4 IgG/goat anti-porcine IL-10 IgG; R&D Systems, U.S.A.) was added, and after washing at room temperature for about 1 hour, it was washed three times with PBST.

Next, 100 μL of streptavidin-HRP (R&D Systems, U.S.A.) was added, and after washing at room temperature for about 30 min, it was washed three times with PBST.

Then, add an equal volume of cold light coloring agent, such as TMB perosidase substrate (KPL, Inc., MD, USA), and wait for 30 minutes at room temperature in the dark, add an equal amount of stop solution ( Stop solution; KPL, Inc., MD, USA) to stop the color reaction. Subsequently, a cold light fluorescence analyzer, such as an Anthos 2020 ELISA reader (Anthos 2020 ELISA reader; Anthos 2020, Cambridge, UK), was used to detect the absorbance at 450 nm (OD _{450 nm} ) to analyze the peripheral blood mononucleus of the pig. The amount of IL-4 and IL-10 produced in the globule cells is shown in Fig. 4A and Fig. 4B.

Please refer to Figures 4A and 4B, wherein Figure 4A shows the production of IL-4 after treatment of peripheral blood mononuclear cells of pigs by the soluble recombinant FliC flagellin fusion protein of Example 2, and Figure 4B The amount of IL-10 produced by treating the peripheral blood mononuclear cells of pigs by the soluble recombinant FliC flagellin fusion protein of Example 2 is shown. Vertical axis of Figure 4A and Figure 4B The amount of IL-4 and IL-10 produced (pg/mL) is not indicated here. The same figure number (eg, between a and a) indicates a non-significant difference between the groups. Conversely, different numbers between different numbers (eg, a and b) indicate significant differences between groups. As for the figure ab, this value is not significantly different from a and b.

From Fig. 4A and Fig. 4B, it can be seen that compared with other groups (positive control group and negative control group), peripheral blood mononuclear cells were treated with soluble recombinant FliC flagellin fusion protein after treatment of peripheral blood mononuclear cells. The production of IL-4 or IL-10 was significantly higher than other groups.

It can be seen from the above examples of the present invention that the soluble recombinant FliC flagellin fusion protein of the present invention can promote an innate immune response, such as the gene expression of TLR7 and TLR8 in peripheral blood mononuclear cells of pigs, and the promotion of IL-4 and IL-10. Secretion, which in turn can be applied to medicines, foods, vaccines or other compositions.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

【Biomaterial Storage】

Domestic deposit information

[Hosting Agency: Food Industry Development Research Institute, the date of deposit: October 12, 2012, registration number: BCRC 940651]

<110> National Pingtung University of Science and Technology

<120> Soluble recombinant FliC flagellin fusion protein with porcine immune cell activity and its prokaryotic transformation strain

<130> None

<160> 10

<210> 1

<211> 503

<212> Protein

<213> Salmonella enteritidis flagellin

<400> 1

<210> 2

<211> 1515

<212> DNA

<213> Salmonella enteritidis flagellin cDNA

<400> 2

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence

<400> 3

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<400> 4

<210> 5

<211> 17

<212> DNA

<213> Artificial sequence

<400> 5

<210> 6

<211> 16

<212> DNA

<213> Artificial sequence

<400> 6

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence

<400> 7

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence

<400> 8

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence

<400> 9

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence

<400> 10

Claims (7)

A soluble recombinant FliC flagellin fusion protein consisting of the amino acid sequence SEQ ID NO 1 having a purine receptor 7 (Tol7) which promotes porcine immune cells (Toll-Like Receptor 7; TLR7) And the activity of the gene expression of the steroid receptor 8 (TLR8), and at the same time promote the secretion of interleukin-4 (IL-4) and IL-10 by the porcine Th2 cells. The soluble recombinant FliC flagellin fusion protein according to claim 1, wherein the amino acid sequence SEQ ID NO 1 is derived from Salmonella enteritidis SE-K40 and is deposited in the Hsinchu Food Industry Development Institute of the Republic of China. And the registration number is BCRC 940651. A recombinant gene of a soluble recombinant FliC flagellin fusion protein comprising the DNA sequence of SEQ ID NO 2 to encode the amino acid sequence of claim 1. A transformant strain for expressing a soluble recombinant FliC flagellin fusion protein comprising a recombinant vector, and the recombinant vector comprises the DNA sequence of SEQ ID NO 2 according to claim 3. An adjuvant for porcine vaccine consisting of the soluble recombinant FliC flagellin fusion protein of claim 1, and the soluble recombinant FliC flagellin fusion protein is one of the transformed strains as described in claim 4 The protein solution consists of removing one of the insoluble proteins. Use of a soluble recombinant FliC flagellin fusion protein for preparing a composition for promoting porcine immune activity, characterized in that the soluble recombinant FliC flagellin fusion protein is composed of the amino acid sequence SEQ ID NO 1 to promote a pig immune cell An immunological activity, wherein the porcine immune cell comprises a peripheral blood mononuclear cell of pig and a porcine Th2 cell, and the soluble recombinant FliC flagellin fusion protein promotes sputum receptor 7 such as peripheral blood mononuclear cells of the pig (Toll-Like) Gene expression of Receptor 7; TLR7) and steroid receptor 8 (TLR8). The use of the soluble recombinant FliC flagellin fusion protein according to claim 6 for the preparation of a composition for promoting immune activity of porcine immune cells, wherein the soluble recombinant FliC flagellin fusion protein promotes secretion of interleukin-4 from the porcine Th2 cell (interleukin- 4; IL-4) and IL-10 secretion.