TW202313657A - Soluble erns recombinant protein of classical swine fever virus, method of making the same, kit and method of identifying classical swine fever virus infection using the same - Google Patents

Abstract

The present invention relates to a soluble Erns recombinant protein of classical swine fever virus (CSFV) and a method of making the same, a kit and a method of identifying classical swine fever virus infection using the same. After the Erns recombinant antigen is expressed by a prokaryotic transformed cell, the prokaryotic transformed cell is subjected to a homogenization treatment and a solubilization treatment by an extraction buffer, so as to obtain the soluble Erns recombinant protein of CSFV, which can be applied to a kit and/or a method of identifying a biological sample derived from a porcine subject that has been infected by CSFV infection or vaccinated by a CSFV E2 subunit vaccine.

Description

Soluble Erns recombinant protein of classical swine fever virus, its production method and its kit and method for identifying classical swine fever virus infection

The present invention relates to a recombinant protein of animal virus and its application, in particular to a soluble Erns recombinant protein of classical swine fever virus, its production method and its kit and method for identifying the infection of classical swine fever virus.

Classical swine fever (Classical swine fever) is a highly contagious and highly lethal disease caused by classical swine fever virus (CSFV). After it was first discovered in 1903, it has been popular in Asia, Africa, Europe, and Central and South America, causing serious economic losses to the global swine industry and posing a great threat to the agricultural safety of countries without swine fever. For example: swine fever in the United States is a pig disease with a highly contagious virus, which is caused by the classical swine fever virus. Countries where the virus is endemic must use routine vaccination to prevent and control the spread of swine fever. When used correctly, vaccination can be an effective method of limiting the spread of CSF, preventing disease outbreaks and establishing protective immunity in uninfected herds.

CSFV belongs to the genus Pestivius in the family Flaviviridae. Its genome has a single-stranded forward RNA with a total length of about 12.3 kb, which can be translated into a polyprotein with 3898 amino acids. Four structural proteins (C, Erns, E1 and E2) and eight non-structural viral proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) can be formed after protease processing, among which E2 protein, Erns recombinant Protein and NS3 protein can induce the host to produce antibody response.

The currently widely used commercialized CSF vaccines include Modified Live Vaccine (MLV) and CSFV envelope protein E2 subunit vaccine. Although the CSF MLV vaccine is cheap and effective, however, in the detection of serum antibodies, the positive immune reaction of the antibody can only detect the endogenous antibodies against CSFV in the tested pigs, but it is impossible to identify whether the endogenous antibodies are It is caused by vaccination with MLV or infection by classical swine fever virus.

Secondly, after animals are vaccinated against classical swine fever MLV vaccine, the virus existing in the environment may continue to infect the animal, and the animal still has a certain degree of low virus load, making it difficult to eliminate the virus existing in the environment and in the animal body. In order to completely eradicate the source of infection of CSFV, it is urgent to provide a differential reagent to distinguish between pigs that have been vaccinated or infected by CSFV in animal farms, so as to facilitate subsequent extermination plans.

Therefore, one aspect of the present invention is to provide a method for producing soluble Erns recombinant protein of CSFV, which uses prokaryotic transformed cells to express CSFV Erns recombinant protein, and then undergoes homogenization and extraction buffer solution After solubilization treatment, the soluble recombinant protein of swine fever virus Erns can be obtained.

Secondly, another aspect of the present invention is to provide a soluble Erns recombinant protein of classical swine fever virus, which is produced by the above method.

Furthermore, another aspect of the present invention is to provide a kit for identifying CSFV infection, which is to immobilize the above-mentioned CSFV soluble Erns recombinant protein on the surface of the reaction area, and use the secondary antibody to specifically recognize the anti-Erns The primary antibody of the recombinant protein, but the secondary antibody does not interact with the secondary antibody produced by the immunization of the classical swine fever virus E2 subunit vaccine, which is beneficial to the identification of CSFV infection or has received the immunization of the classical swine fever virus E2 subunit vaccine.

In addition, another aspect of the present invention is to provide a method for identifying CSFV infection, which comprises using the above-mentioned kit to identify CSFV infection or having received CSFV E2 subunit vaccine immunization.

According to the above aspect of the present invention, a method for producing the soluble Erns recombinant protein of classical swine fever virus is proposed. First, the prokaryotic transformed cells are subjected to a protein expression step, wherein the prokaryotic transformed cells may contain recombinant plastids to express the classical swine fever virus (CSFV) of the amino acid sequence shown in Sequence Identification Number (SEQ ID NO): 1 Erns recombinant protein. Next, the aforementioned prokaryotic transformed cells were homogenized to obtain a cell sediment, wherein the cell sediment contained the inclusion body of the Erns recombinant protein of classical swine fever virus. Then, use the extraction buffer solution to solubilize the aforementioned cell pellet to obtain the soluble recombinant protein of swine fever virus Erns, wherein the aforementioned extraction buffer solution can be composed of 20 mM to 30 mM Tris (Tris) salt , 450 mM to 550 mM sodium chloride (NaCl) and 7 M to 9 M urea (Urea), and its pH value is pH 7.0 to pH 8.0.

In the above embodiment, the host cell of the aforementioned prokaryotic transformed cell can be, for example, Escherichia coli BL21 (DE3) strain.

In the above embodiment, the aforementioned recombinant plasmid may comprise the nucleic acid sequence shown in SEQ ID NO: 2 to encode the Erns recombinant protein of classical swine fever virus.

In the above embodiment, the aforementioned extraction buffer solution is composed of, for example, 25 mM Tris salt, 500 mM sodium chloride (NaCl) and 8 M urea (Urea), and the pH value of the extraction buffer solution is pH 7.5 .

In the above embodiment, the aforementioned solubilization treatment may be performed at 0° C. to 4° C. for 20 minutes to 40 minutes, for example.

In the above embodiment, after the aforementioned solubilization treatment, the soluble recombinant protein of CSFV Erns can be optionally subjected to a column purification step to obtain purified soluble recombinant protein of CSFV Erns.

According to another aspect of the present invention, a soluble Erns recombinant protein of classical swine fever virus is proposed, which is prepared by the above-mentioned method.

According to another aspect of the present invention, a kit for identifying CSFV infection is provided. In one embodiment, the aforementioned kit may include soluble Erns recombinant protein of classical swine fever virus immobilized on the surface of the reaction area, and a secondary antibody, and the secondary antibody system communicates with the reaction area. In the above embodiment, the soluble Erns recombinant protein of CSFV can be, for example, the amino acid sequence shown in SEQ ID NO:1. The aforementioned secondary antibody only specifically recognizes the primary antibody against the Erns recombinant protein, but does not interact with the secondary antibody produced by the immunization of the classical swine fever virus E2 subunit vaccine.

In the above embodiment, the aforementioned set may optionally include a display area to present the result of the aforementioned identification reaction.

According to another aspect of the present invention, a method for identifying CSFV infection is provided. In one embodiment, firstly, the soluble Erns recombinant protein of classical swine fever virus with the amino acid sequence shown in SEQ ID NO:1 is immobilized on the surface of the reaction area. Next, adding a biological sample into the reaction area, so that the biological sample and the soluble Erns recombinant protein of CSFV are subjected to a bonding reaction. Then, remove the unreacted biological sample. Afterwards, the secondary antibody is introduced into the reaction area, and the biological sample bound to the soluble Erns recombinant protein of classical swine fever virus by the secondary antibody is identified and reacted, wherein the secondary antibody only specifically recognizes the first anti-Erns recombinant protein in the biological sample Antibodies, but do not react with the secondary antibodies produced by immunization with classical swine fever virus E2 subunit vaccine in biological samples. Then, the result of the differential reaction is judged. When the differential reaction is positive, it is judged that the pig subject from the biological sample is infected with classical swine fever virus; The CSFV E2 sub-unit vaccine immunization.

In the above embodiments, the aforementioned biological samples may include isolated organs, tissues, cells, body fluids, lymph fluid, urine, whole blood, plasma, serum and/or cell culture supernatant.

Applying the soluble Erns recombinant protein of swine fever virus of the present invention and the manufacturing method thereof, it can use prokaryotic transformed cells to express the recombinant protein of swine fever virus Erns, and then use the extraction buffer solution to dissolve the cell sediment of the prokaryotic transformed cells, Soluble recombinant protein of classical swine fever virus Erns can be obtained. When the soluble recombinant protein of CSFV Erns is applied to the identification kit and/or method, it can effectively distinguish whether the swine subject from the biological sample is infected with CSFV or has received the E2 subunit vaccine immunization of CSFV.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and intended to provide further explanation of the claimed invention.

If the definition or usage of a term in a cited document is inconsistent with or contrary to the definition of the term here, the definition of the term here applies instead of the definition of the term in the cited document. Secondly, unless otherwise defined by context, singular terms may include pluralities and plural terms may also include the singular.

As mentioned above, the present invention provides a soluble Erns recombinant protein of CSFV and its production method as well as its kit and method for identifying CSFV infection. After the aforementioned recombinant protein of CSFV Erns is expressed by prokaryotic transformed cells, it is homogenized and solubilized by extraction buffer solution to obtain the soluble recombinant protein of CSFV Erns, which can be applied to the kit for identifying CSFV infection group and/or method.

As used herein, "recombinant protein", "chimeric protein", "recombinant antigen", "protein", "peptide" and "polypeptide" are interchangeable and refer to a polymer of amino acids, usually by Peptide bonds or disulfide bonds link them together. "Peptide" also refers to amino acid polymers in which one or more amino acid residues are naturally occurring amino acids and polymers thereof, or analogs or mimetics corresponding to naturally occurring amino acids . "Peptide" also includes modified amino acid polymers, eg, glycoproteins with carbohydrate residues, or phosphorylated peptides. Peptides, polypeptides and proteins can be produced by liquid-phase synthesis, solid-phase synthesis, or by genetic engineering, recombinant cells, prokaryotic expression systems, and eukaryotic expression systems. In one embodiment, the recombinant protein referred to herein refers to the Erns recombinant protein of classical swine fever virus.

As used herein, "amino acid" and "residue" are interchangeable and, when used in conjunction with peptides, refer to naturally occurring and synthetic amino acids, amino acid analogs, amino acid mimetics and A non-naturally occurring amino acid that is chemically similar to a naturally occurring amino acid.

The "CSFV" referred to here is not particularly limited to the type of virus strain, such as the Taiwanese strain of CSFV (genotype 2.1, NP_777496.1), but in other embodiments, other virus strains can also be used, It depends on the actual needs.

The "Erns recombinant protein" referred to herein can be, for example, a full-length Erns recombinant protein, such as the amino acid sequence shown in Sequence Identification Number (SEQ ID NO): 1, or the nucleic acid sequence shown in SEQ ID NO: 2 The encoded amino acid sequence. In addition, in order to facilitate the subsequent protein purification, the N-terminus of the Erns recombinant protein can optionally add a histidine (His) tag [His tag; or polyhistidine (polyhistidine)], wherein the His tag can include but not limited to 6 to 10 histidine residues.

In some embodiments, the above-mentioned soluble Erns recombinant protein of CSFV can be produced by conventional methods or the following methods. First, the protein expression step is performed on the prokaryotic transformed cells, wherein the prokaryotic transformed cells may contain recombinant plastids, which contain the recombinant gene of the nucleic acid sequence shown in SEQ ID NO: 2, to express the sequence identification number (SEQ ID NO) : The recombinant protein of classical swine fever virus (CSFV) Erns of the amino acid sequence shown in 1. For the subsequent purification of the recombinant protein, the 5' end of the above-mentioned recombinant gene can selectively add the nucleic acid sequence for editing the His tag. In some examples, the aforementioned nucleic acid sequence for encoding the His tag can be provided, for example, by commercially available plastids, and designed to be connected to the 5' end of the aforementioned recombinant gene. The nucleic acid sequence of the His tag is common knowledge in the technical field of the present invention and will not be further described here.

Next, the aforementioned prokaryotic transformed cells were homogenized to obtain cell pellets (also called cell debris), wherein the cell pellets contained the inclusion bodies of the Erns recombinant protein of classical swine fever virus.

Since the inclusion body of the Erns recombinant protein of swine fever virus is formed by the accumulation of insoluble proteins, it will affect or even fail to exert the original biological function of the Erns recombinant protein. Therefore, the present invention uses the extraction buffer solution to solubilize the aforementioned cell sediment to obtain the soluble recombinant protein of swine fever virus Erns. In the above embodiment, the aforementioned extraction buffer solution can be composed of 20 mM to 30 mM Tris salt, 450 mM to 550 mM sodium chloride (NaCl) and 7 M to 9 M urea (Urea), and the extraction buffer The pH value of the solution is from pH 7.0 to pH 8.0. In one example, the aforementioned extraction buffer solution can be composed of, for example, 25 mM Tris salt, 500 mM sodium chloride (NaCl) and 8 M urea (Urea), and the pH value of the extraction buffer solution can be pH 7.5. In another example, the solubilization treatment may be performed at 0°C to 4°C for 20 minutes to 40 minutes. In other examples, the solubilization treatment may be performed at 0° C. for 30 minutes, and stirring may be used during the solubilization treatment.

In the above embodiment, after the aforementioned solubilization treatment, the conventional column purification step can be selectively performed on the soluble recombinant protein of CSFV Erns to obtain purified soluble recombinant protein of CSFV Erns. It is supplemented that, compared with the conventional column purification step, β-mercaptoethanol (β-mercaptoethanol, b-ME or BME) must be added to avoid the folding of the N-terminal His tag. When the protein is subjected to the column purification step, it is not necessary to add b-ME to obtain a better purification effect.

Generally speaking, the production capacity of the above-mentioned Erns recombinant protein can obtain at least 21.8 milligrams (mg) of soluble Erns recombinant protein per liter of bacterial liquid, which can be subsequently applied to identification kits and/or methods to distinguish pigs from biological samples Whether the subject is infected with CSFV, or has been immunized with CSFV E2 sub-unit vaccine. The above identification kits and/or methods may include immunoassay kits and/or methods, such as enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) kits and/or methods (such as indirect ELISA), to detect biological Antibodies contained in the sample.

The "biological sample" referred to here is not particularly limited, and can be, for example, isolated organs, tissues, cells, body fluids, lymph, urine, whole blood, plasma, serum and/or cell culture supernatant, etc. However, The present invention is not limited to what is presented here.

In some examples, the above identification methods can be performed using conventional ELISA kits (and/or biochips). In general, the type of suitable ELISA set and/or biochip is not particularly limited. In some examples, the above set can include the soluble Erns of classical swine fever virus with the amino acid sequence shown in SEQ ID NO:1 Recombinant protein and secondary antibody. The above-mentioned soluble Erns recombinant protein of CSFV can be fixed on the surface of the reaction area of the kit. The above-mentioned secondary antibody can be stored in the reagent area in a solution state and communicated with the reaction area through a microfluidic channel, wherein the secondary antibody only specifically recognizes the primary antibody against Erns recombinant protein, but does not interact with the E2 subunit of classical swine fever virus The secondary antibodies produced by the vaccine immunization did not interact with each other, nor did they have any antibody-antigen binding reaction with the serum of specific pathogen-free (SPF) pigs. In other embodiments, the aforementioned kit may optionally include a display area to present the results of the identification responses. The type of the above-mentioned set is not particularly limited. For example, when the above-mentioned set is a rapid screening reagent, the display area can be such as an observation window, so as to utilize visual observation to identify the position of the detection line and the control line of the color strip (strip) after the reaction. result. When the above-mentioned set is a biochip, the display area can be, for example, a display to display images or signals of the reaction products by optical or electrochemical detection.

What is clarified here is that the Erns recombinant protein obtained above is used for immune analysis, not as a vaccine, so the full-length Erns recombinant protein must be used to effectively distinguish whether the pig subject from the biological sample is infected by CSFV , or have been immunized with classical swine fever virus E2 unit vaccine. If the recombinant protein obtained by using an amino acid sequence other than SEQ ID NO: 1, such as changing the length or changing the range of the selected sequence, will not be able to exert the above effect, that is, to distinguish whether the pig subject from the biological sample is infected by the classical swine fever virus , or have been immunized with classical swine fever virus E2 unit vaccine.

In some embodiments, the specific method for distinguishing whether the pig subject from the biological sample is infected by classical swine fever virus refers to that the above-mentioned Erns recombinant protein only specifically recognizes the antibody against the Erns recombinant protein in the biological sample (also known as the first Antibody), but does not react with the antibody (also known as the second antibody) produced by the immunization of the classical swine fever virus E2 sub-unit vaccine in the biological sample. If the recombinant protein obtained by using an amino acid sequence other than SEQ ID NO: 1, such as changing the length or changing the selected sequence range, the modified Erns recombinant protein may react with the first antibody and the second antibody at the same time, and cannot Distinguish whether the pig subject from which the biological sample is derived is infected with CSFV, or has been immunized with the E2 subunit vaccine against CSFV.

It can be understood that the following specific recombinant protein sequences, specific formulations, specific dosages, specific detection methods, viewpoints, illustrations and examples are for illustration only, and not as limitations of the present invention. The principal features of this invention can be employed in various embodiments without departing from the spirit and scope of the invention. Therefore, those with ordinary knowledge in the technical field of the present invention can easily determine the essential technical features of the present invention and make various changes and modifications to the present invention to apply to different purposes and conditions without departing from the spirit and scope of the present invention. Embodiment 1: Preparation of soluble Erns recombinant protein of classical swine fever virus 1.1 Construction of recombinant plastids of CSFV Erns recombinant protein

The whole gene fragment of Erns from Classical swine fever virus (CSFV Egystrup strain, Genotype1.1) was synthesized by total gene synthesis (Shenggong Co., Ltd.). Through the polymerase chain reaction [polymerase chain reaction (PCR), ThermoFisher scientific; Phusion high-fidelity DNA polymerase, Phusion High-Fidelity DNA polymerase, Advanced Company, Taiwan] amplified Erns whole gene fragment (as shown in SEQ ID NO: 2 display sequence). Then, according to the method provided by the commercially available In-fusion construction kit (TaKaRa Bio, USA), take 1 μL of Erns gene fragment (10 ng/μL, sequence shown in SEQ ID NO:2), 1 μL of pET21 plasmid (10 ng/μL) (Invitrogen) and 0.5 μL of ligase (TaKaRa Bio, USA) were reacted at 50°C for 20 minutes. Afterwards, the above reaction product was added to 25 μL of ECOSTM X Competent Cells [DH5a] (Yishengsheng Technology, Taiwan), and the transformation reaction was carried out at 42°C for 50 seconds. Then, the bacterial liquid of transformed cells was spread on the surface of LB medium (DifcoTM LB Broth, Miller, BD) containing ampicillin (Ampicillin, Biobasic, Taiwan). Leave at 37°C for about 16 to 18 hours to allow colonies to form. Afterwards, single colonies were picked for gene sequencing. 1.2 Expression of CSFV Erns recombinant protein

Take 1 μL of the pET-21-Erns plasmid confirmed by gene sequencing (that is, the recombinant plasmid containing the nucleic acid sequence shown in SEQ ID NO: 2) plus 25 μL of ECOSTM 21 competent cells [BL21(DE3)] (prebiotic Biotechnology, Taiwan)] at 42°C for 50 seconds. Then, the bacterial solution was spread on LB medium (DifcoTM LB Broth, Miller, BD) containing 100 μg/μL of ampicillin (Biobasic, Taiwan). Leave at 37°C for about 16 to 18 hours to allow colonies to form. Pick a single colony into 500 μL of LB culture medium containing 100 μg/μL of ampicillin, culture at 37°C for 6 to 8 hours, transfer 50 μL of bacterial culture to 50 mL of LB culture medium containing 100 μg/μL of ampicillin cultured at 37°C for 16 to 18 hours, take 10mL of the bacterial liquid into 1L of LB culture medium containing 100 μg/μL of ampicillin, cultivate at 37°C for about 3 hours, then add 500 μL of 1M Isopropyl β-d-1-thiogalactopyranoside [Isopropyl β-d-1-thiogalactopyranoside, IPTG; Youhe Trading, Taiwan) was used for protein induction and cultured at 22°C for about 16 to 18 hours. 1.3 Homogenization treatment, solubilization treatment and column purification steps of CSFV Erns recombinant protein

In this example, the CSFV Erns recombinant protein was subjected to homogenization treatment (hereinafter also referred to as step 1) and solubilization treatment (hereinafter also referred to as step 2) in sequence, as described below.

Step 1: Take E. coli cells and add 100mL of cell lysis buffer solution (containing 25mM Tris salt and 500mM NaCl, pH 7.5), and use a commercially available homogenizer (IKA, Taiwan) for homogenization. Then, 0.5 g of lysozyme (lysozyme, Cyrusbioscience, Taiwan) was added to the above reaction product, mixed evenly, and left standing on ice for 30 minutes. Next, 0.05g DNase I (Cyrusbioscience, Taiwan) was added to the above reaction product, and a commercially available ultrasonic cell disruptor (Shangwei, Taiwan) was used to vibrate, oscillating for 3 seconds and stopping for 5 seconds, and the whole process took 20 minutes to obtain Homogenized product. Afterwards, the homogenized product was centrifuged at a speed of 12000×g for 40 minutes to obtain the first supernatant (1S, track S of figure number 101) and the first precipitate (1P, track P of figure number 101), wherein The first pellet (1P) contained inclusion bodies of the CSFV Erns recombinant protein.

Step 2: Add extraction buffer solution (composed of 25 mM Tris salt, 500 mM sodium chloride and 8 M urea) to the first precipitate (1P), mix well, and place on ice (0° C) stirring for 30 minutes for solubilization treatment. Afterwards, the solubilized product was centrifuged at a speed of 12000×g for 40 minutes to obtain the second supernatant (abbreviated as 2S, track S of figure number 103) and the second precipitate (abbreviated as 2P, track P of figure number 103) road).

Please refer to Fig. 1A, which shows the sodium dodecyl sulfate polypropylene of step 1 (figure number 101) and step 2 (figure number 103) of the manufacturing method of the swine fever virus soluble Erns recombinant protein according to an embodiment of the present invention Colloid image results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results in Figure 1A show that the Erns recombinant protein produced by the prokaryotic expression system is an insoluble protein 105, and its protein appears in the first precipitate (1P) (insoluble as shown by the arrow in the P channel of Figure 101 in Figure 1A protein 105), and its molecular weight is 28 kDa.

However, using Erns extraction buffer solution (composed of 25 mM Tris salt, 500 mM NaCl, 8 M urea, and pH 7.5), the insoluble Protein 105 was solubilized to obtain soluble protein 107 in the second supernatant (abbreviated as 2S).

Specifically, 100 mL of extraction buffer solution was added to the first precipitate (1P), placed on ice and stirred for 30 minutes, and centrifuged at 12000×g for 40 minutes to obtain the second supernatant (2S) and the second The second precipitate (2P) was analyzed by SDS-PAGE electrophoresis. Please refer to Figure 1A, the insoluble protein 105 in the first precipitate (1P) has been converted into soluble protein 107 in the second supernatant (2S) (as shown by the arrow in the S track of Figure 103 in Figure 1A Soluble protein 107).

Please refer to FIG. 1B , which shows the image result of the western transfer method in FIG. 1A , and the process of the western transfer method is as follows. Transfer the SDS-PAGE film containing Erns recombinant protein to polyvinylidene difluoride (polyvinylidene difluoride, PVDF) membrane (GE healthcare, Taiwan advanced), and 20 mL of blocking buffer solution [blocking buffer, including adding polysorbate Tris Buffered Saline with Tween 20 (Tris Buffered Saline with Tween 20, TBST) and 5% bovine serum albumin (Bovine serum albumin, BSA)] reacted at room temperature (about 25°C) for 2 hours . Then, the blocking buffer solution was removed, and the PVDF membrane was reacted with 10 mL of anti-His tag antibody solution [diluted 1:1000 antibody and TBST buffer solution (1x PBS with 0.1% Tween-20 added)] at 4°C for 16 to 18 hours. Afterwards, the anti-His tag antibody solution was removed, and the PVDF membrane was rinsed with 10 mL of TBST buffer solution three times for 20 minutes each time. Then, the PVDF membrane was reacted with TBST buffer solution of anti-mouse antibody (anti-mouse antibody, dilution factor 1:10000) at room temperature for 1 hour. Afterwards, remove the TBST buffer solution containing the antibody, add 10 mL of TBST buffer solution to wash the PVDF membrane three times, 20 minutes each time, add a coloring agent (ECL stain kit, Taiwan Advanced) for color development, and the result is shown in Figure 1B shown.

The results in Figure 1B show that the N-terminal polyhistidine (polyhistidine) of the Erns recombinant protein was reacted with the antibody against the His tag, and it was confirmed that the insoluble protein 105 in the first precipitate (1P, track P of Figure No. 101) passed through soluble After the melting treatment, it has been converted into soluble protein 107 in the second supernatant (2S). The soluble protein 107 shown by the arrow in the S track of Figure 103 in Figure 1B has a molecular weight of 28 kDa and contains N-terminal His Label.

In addition, use a commercially available spectrometer (such as BioSpectrometer instrument, Eppendorf, Taiwan) to measure the absorbance value of soluble protein 107 at 280 nm (OD _280nm ), and use the following formula (I) to calculate the protein concentration: (A280 absorbance value × protein extinction coefficient) x sample volume = protein concentration (I). The calculated concentration of soluble Erns recombinant protein is (1.2/2.2)×40 mL=21.8 mg, which means that 21.8 milligrams (mg) of soluble Erns recombinant protein can be obtained per liter of bacterial liquid.

The second supernatant (Figure No. 2S) containing soluble Erns recombinant protein can be selectively passed through nickel-nitrogen [base] triacetic acid agarose column (Ni-nitrolotriacetic acid agarose column, Ni-NTA agarose column, Cytiva, Taiwan) stage), carry out the column purification step, and wash with the eluent [Figure No. FT, containing 100 mM imidazole (imidazole)]. The protein thus obtained is the purified soluble Erns recombinant protein, such as soluble protein 107 shown by the arrow in the Erns lane of Figure 2A, with a molecular weight of 28 kDa.

Please refer to Fig. 2B, which shows the image result of the western blotting method in Fig. 2A, wherein the purified soluble Erns recombinant protein is soluble protein 107 as shown by the arrow in Fig. N-terminal His tag. Example 2: Specific identification binding reaction of Erns recombinant protein of classical swine fever virus

This example establishes the verification of the immune binding function of the antigen and the antibody, and uses the enzyme binding immunosorbent assay (ELISA) to carry out the specific identification binding reaction of the Erns recombinant protein of the classical swine fever virus. 1.4.1 Biological samples

In this embodiment, the sources of serum are divided into the following three groups: (1) Positive serum group: a pasture in southern Taiwan, administered with live virus attenuated swine fever virus vaccine (the source is Gaoshi Nong dry rabbitized swine fever vaccine, Kaohsiung City Farm The serum of six-week-old piglets from a biopharmaceutical factory simulated classical swine fever virus infection was collected after two weeks of vaccination. (2) Negative serum group: the serum of eight-week-old piglets without specific pathogens (sourced from the Institute of Animal Science and Technology, Zhunan). (3) Vaccine group: Specific pathogen-free (SPF) four-week-old piglets (source: Institute of Animal Science and Technology, Zhunan), blood serum collected four weeks after the commercially available Bayovac® CSF-E 2-unit vaccine (Bayer). Each of the above groups was repeated at least three times. 1.4.2 Experimental steps

The purified Erns recombinant protein was adjusted to a protein concentration of 1 μg/μL using carbonate-bicarbonate buffer solution (carbonate-bicarbonate buffer, Sigma, Youhe, Taiwan). Next, 60 ng/μL Erns recombinant protein was coated on the bottom of a 96-well (well) flat bottom microplate (Corning, Taiwan). Place at 4°C for 16 to 18 hours. Then, add 250 μL of PBST (1x PBS and 0.5% Tween-20) to each well to rinse, and carry out at room temperature for 10 minutes while shaking at 125 rpm, and repeat this step 5 times. Afterwards, 250 μL of blocking buffer solution [PBST and 5% BSA (Sigma, Youhe)] was added to each well and kept at 37°C for 2 hours. After removing the blocking buffer solution, add 100 μL (1:1500 dilution ratio) of various sera to each well, react at 37°C for 1 hour, and remove the various sera. Then, 250 μL of PBST (1x PBS and 0.5% Tween-20) was added to each well, reacted at room temperature for 10 minutes, and was shaken at 125 rpm, and this step was repeated 5 times. Then, add 100 μL of secondary antibody [goat anti-pig horseradish peroxidase-labeled antibody, Goat-anti pig horseradish peroxidase (HRP) Ab, dilution ratio 1:10000, abcam (ab18184), Taiwan], after reacting at 37°C for 1 hour, remove the secondary antibody. Then, 250 μL of PBST (1x PBS and 0.5% Tween-20) was added to each well, reacted at room temperature for 10 minutes, and was shaken at 125 rpm, and this step was repeated 5 times. Then, add 100 μL of chromogenic substrate (such as tetramethylbenzidine dihydrochloride, 3,3',5,5'-tetramethyl benzidine dihydrochloride, TMB) to each well, and react at room temperature for 15 to 20 minutes After that, 100 μL of stop (STOP) solution (ScyTek, Youhe) was added, and the absorbance value at 450 nm (OD _{450 nm} ) of each well was detected. The results are shown in Figure 3.

Please refer to FIG. 3 , which is a histogram showing the identification of different biological samples by using the soluble recombinant protein of CSFV Erns according to an embodiment of the present invention. As shown in the results in Figure 3, the Erns soluble recombinant protein of swine fever virus purified by the above-mentioned embodiment, after the ELISA test, the Erns soluble recombinant protein will react with the serum antibody of the positive serogroup (the serum of piglets injected with live attenuated vaccine) There is a binding reaction, and there is a statistically significant difference (as shown in the figure number *, * p <0.05). However, the above-mentioned purified soluble recombinant protein of CSFV Erns had no immunological binding reaction with the negative serum group (ie piglet serum of SPF piglets) and the vaccine group (ie piglet serum administered with commercially available E2 subunit vaccine (Bayer)).

The soluble recombinant protein of swine fever virus Erns purified in the above examples can indeed produce a specific binding reaction with the serum antibodies produced by the whole virus, but there is no binding reaction with the antibodies produced by the immune reaction of the E2 sub-unit vaccination, which represents the purification of the above examples. The soluble recombinant protein of CSFV Erns can indeed effectively distinguish whether the pig subjects from biological samples are infected with CSFV, or have been immunized with CSFV E2 sub-unit vaccine, and it is expected to be used as a differential diagnostic reagent for CSFV in the future , and then achieve the goal of eliminating swine fever virus.

In summary, the specific amino acid sequence, specific manufacturing method, specific composition, specific analysis mode or specific evaluation method of the present invention are only used to illustrate the soluble Erns recombinant protein of classical swine fever virus and its manufacturing method and its Kits and methods for identifying classical swine fever virus infection. However, those skilled in the art to which the present invention pertains should understand that without departing from the spirit and scope of the present invention, other amino acid sequences, other production methods, other compositions, other analysis modes or other The evaluation method can also be used for the soluble Erns recombinant protein of CSFV and its production method and its kit and method for identifying CSFV infection, and is not limited to the above.

For example, the soluble Erns recombinant protein of CSFV can be produced using other expression systems (such as eukaryotic expression systems) to optimize the process and mass production. The obtained soluble Erns recombinant protein of classical swine fever virus can be applied to rapid screening reagents or biochips or other commercially available inspection products according to actual needs. In addition, in addition to serum, the biological samples used to identify CSFV infection can also use, for example, isolated organs, tissues, cells, body fluids, lymph fluid, urine, whole blood, plasma and/or cell culture supernatant liquid etc.

According to the above-mentioned embodiments, the soluble Erns recombinant protein of swine fever virus of the present invention and the manufacturing method thereof have the advantage that after utilizing prokaryotic transformed cells to express the recombinant protein of swine fever virus Erns, the cell sediment of the prokaryotic transformed cells is extracted using an extraction buffer. The solution is solubilized to obtain the soluble recombinant protein of swine fever virus Erns. When the soluble recombinant protein of CSFV Erns can be applied to the identification kit and/or method in the future, it can effectively distinguish whether the pig subjects from the biological sample are infected with CSFV or have received the E2 sub-unit vaccine immunization of CSFV.

While the invention has been disclosed above in terms of a few specific embodiments, other embodiments are possible. Therefore, the spirit and scope of the invention appended hereto should not be limited to the description of the examples contained herein.

101: Step 1 103: Step 2 105: Insoluble protein 107: Soluble protein

In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, the detailed description of the accompanying drawings is as follows: [Fig. 1A] shows the sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) of the soluble Erns recombinant protein of classical swine fever virus according to an embodiment of the present invention after solubilization treatment ) colloid image. [Fig. 1B] shows the image result of the Western transfer method in Fig. 1A. [ FIG. 2A ] is a colloidal image showing the SDS-PAGE of the soluble Erns recombinant protein of CSFV after the column purification step according to an embodiment of the present invention. [Fig. 2B] shows the image result of the Western transfer method in Fig. 2A. [ Fig. 3 ] is a histogram showing the identification of different biological samples by using the soluble recombinant protein of classical swine fever virus Erns according to an embodiment of the present invention.

Domestic deposit information (please note in order of depositor, date, and number) none Overseas storage information (please note in order of storage country, institution, date, and number) none

Claims (11)

A kind of manufacture method of swine fever virus soluble Erns recombinant protein, comprising: A protein expression step is performed on a prokaryotic transformed cell, wherein the prokaryotic transformed cell comprises a recombinant plastid to express one of the classical swine fever virus ( CSFV) Erns recombinant protein; performing a homogenization process on the prokaryotic transformed cells to obtain a cell pellet, wherein the cell pellet contains an inclusion body of the CSFV Erns recombinant protein; and Utilize an extraction buffer solution to carry out a solubilization treatment to the cell pellet to obtain the soluble recombinant protein of swine fever virus Erns, wherein the extraction buffer solution is tris (Tris) of 20 mM to 30 mM Salt, 450 mM to 550 mM sodium chloride (NaCl) and 7 M to 9 M urea (Urea), and the pH value of the extraction buffer solution is pH 7.0 to pH 8.0. The method for producing the soluble Erns recombinant protein of classical swine fever virus as described in claim 1, wherein a host cell of the prokaryotic transformed cell is Escherichia coli BL21 (DE3) strain. The method for producing the soluble Erns recombinant protein of CSFV according to claim 1, wherein the recombinant plasmid comprises a nucleic acid sequence as shown in SEQ ID NO: 2 to encode the Erns recombinant protein of CSFV. The manufacture method of the swine fever virus soluble Erns recombinant protein as described in claim item 1, wherein the extraction buffer solution is made up of the urea of the Tris salt of 25 mM, the sodium chloride (NaCl) of 500 mM and 8 M, and the The pH value of the extraction buffer solution is pH 7.5. The method for producing the soluble Erns recombinant protein of classical swine fever virus as claimed in claim 1, wherein the solubilization treatment is carried out at 0°C to 4°C for 20 minutes to 40 minutes. The method for producing the soluble Erns recombinant protein of classical swine fever virus as described in claim 1, after the solubilization treatment, further comprises a column purification step for the soluble recombinant protein of classical swine fever virus Erns, to obtain the purified classical swine fever virus Viral Erns soluble recombinant protein. A soluble Erns recombinant protein of classical swine fever virus is prepared by the method described in any one of claims 1 to 6. A kit for identifying classical swine fever virus infection, comprising: A soluble Erns recombinant protein of classical swine fever virus is immobilized on a surface of a reaction region, wherein the soluble Erns recombinant protein of classical swine fever virus is the amino acid sequence shown in SEQ ID NO:1; and Secondary antibody, and the secondary antibody system is connected with the reaction area, wherein the secondary antibody only specifically recognizes a primary antibody against Erns recombinant protein, but does not immunize with the antibody produced by a classical swine fever virus E2 subunit vaccine A secondary antibody produces an interaction. The kit for identifying classical swine fever virus infection as described in Claim 8 further includes a display area for presenting a result of the identification reaction. A method of identifying classical swine fever virus infection, comprising: Immobilizing a soluble Erns recombinant protein of classical swine fever virus on a surface of a reaction area, wherein the soluble Erns recombinant protein of classical swine fever virus is the amino acid sequence shown in SEQ ID NO:1; adding a biological sample into the reaction area, so that the biological sample and the soluble Erns recombinant protein of classical swine fever virus undergo a bond reaction; remove unreacted biological samples; Introduce the secondary antibody into the reaction area, and make the secondary antibody and the biological sample bonded with the soluble Erns recombinant protein of the swine fever virus carry out a discrimination reaction, wherein the secondary antibody only specifically recognizes the anti-Erns in the biological sample a primary antibody to the recombinant protein, but does not react with a secondary antibody produced by immunization with a classical swine fever virus E2 subunit vaccine in the biological sample; and judge the outcome of one of the differential responses, Wherein when the differential reaction is positive, it is determined that a pig subject from which the biological sample is derived is infected with classical swine fever virus, or When the differential reaction is negative, it is judged that the pig subject from which the biological sample is derived has been immunized with the CSFV E2 subunit vaccine. The method for identifying classical swine fever virus infection as described in claim 10, wherein the biological sample includes isolated organs, tissues, cells, body fluids, lymph, urine, whole blood, plasma, serum and/or cell culture Serum.

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