CN115806599A

CN115806599A - Swine fever virus soluble Erns recombinant protein, manufacturing method thereof, kit and method for identifying swine fever virus infection

Info

Publication number: CN115806599A
Application number: CN202111478925.3A
Authority: CN
Inventors: 庄秀琪; 鍾文彬; 陈计志
Original assignee: Pingtung University Of Science And Technology Taiwan
Current assignee: Pingtung University Of Science And Technology Taiwan
Priority date: 2021-09-15
Filing date: 2021-11-24
Publication date: 2023-03-17
Also published as: TW202313657A; TWI811793B

Abstract

The invention discloses a soluble Erns recombinant protein of classical swine fever virus, a manufacturing method thereof, and a kit and a method for identifying classical swine fever virus infection by using the recombinant protein. The soluble recombinant protein of classical swine fever virus Erns can be applied to an identification kit and/or a method for distinguishing whether a swine subject from a biological sample is infected by classical swine fever virus or is immunized by an E2 subunit vaccine of classical swine fever virus.

Description

Swine fever virus soluble Erns recombinant protein, manufacturing method thereof, kit for identifying swine fever virus infection and method

Technical Field

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

Background

Classical swine fever (Classical swine fever virus) is a highly contagious and highly lethal disease caused by Classical Swine Fever Virus (CSFV). After first discovered in 1903, the swine flu vaccine has been popular in Asia, africa, europe and Central and south America, causing serious economic loss of the global swine industry and further causing great threat to agricultural safety of the countries without swine fever. For example: classical swine fever in the united states is a swine disease with a strong infectious virus, which is caused by classical swine fever virus. Countries where this virus is prevalent must use conventional vaccination to prevent and control the spread of swine fever. If used properly, vaccination can be an effective method to limit the spread of swine fever, prevent disease outbreaks, and establish protective immunity in uninfected herds.

Classical swine fever virus belongs to the genus pestivirus (pestivirus) in the family Flaviviridae (Flaviviridae), and a single strand of positive RNA, approximately 12.3kb in length, of the genome is translated into a polyprotein of 3898 amino acids. After protease processing, four structural proteins (C, erns, E1 and E2) and eight non-structural viral proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A and NS 5B) can be formed, wherein the E2 protein, the Erns recombinant protein and the NS3 protein can induce the antibody response of a host.

The current commercial swine fever vaccines commonly used include Live attenuated swine fever Vaccine (MLV) and swine fever virus envelope protein E2 subunit Vaccine. Although the swine fever MLV vaccine is cheap and effective, the positive immune response of the antibody in the serum antibody detection can only detect that the tested pig only has the endogenous antibody against the swine fever virus, but the endogenous antibody is not identified to be generated by the inoculation of the MLV vaccine or the infection of the swine fever virus.

Secondly, after the animals are inoculated with the swine fever MLV vaccine, the viruses existing in the environment can still continuously infect the animals, and the animals still have a certain low virus amount in the bodies, so that the viruses existing in the environment and the bodies of the animals are difficult to eliminate. In order to completely extinguish the source of classical swine fever virus infection, it is highly desirable to provide an identification agent to distinguish pigs in an animal farm from pigs that have been immunized or infected with classical swine fever virus, so as to facilitate subsequent extinguishment programs.

Disclosure of Invention

Therefore, one aspect of the present invention is to provide a method for producing a soluble Erns recombinant protein of a swine fever virus, wherein the soluble Erns recombinant protein of the swine fever virus can be obtained by performing homogenization treatment and solubilization treatment with an extraction buffer solution after expressing the recombinant protein of the swine fever virus (CSFV) Erns by using a prokaryotic transformed cell.

In another aspect, the present invention provides a soluble Erns recombinant protein of classical swine fever virus, which is prepared by the above method.

In another aspect, the present invention provides a kit for detecting classical swine fever virus infection, wherein the soluble Erns recombinant protein of classical swine fever virus is immobilized on the surface of a reaction area, and a secondary antibody is used to specifically recognize a first antibody against the Erns recombinant protein, but the secondary antibody does not interact with a second antibody generated by immunization with an E2 subunit vaccine of classical swine fever virus, thereby facilitating detection of classical swine fever virus infection or immunization with the E2 subunit vaccine of classical swine fever virus.

In addition, the invention further provides a method for identifying swine fever virus infection, which comprises identifying swine fever virus infection or receiving swine fever virus E2 subunit vaccine immunization by using the kit.

According to the above aspect of the present invention, a method for producing a soluble Erns recombinant protein of classical swine fever virus is provided. First, a protein expression step is performed on the prokaryotic transformed cells, which may comprise recombinant plasmids to express Classical Swine Fever Virus (CSFV) Erns recombinant protein having an amino acid sequence shown in SEQ ID NO: 1. Then, the prokaryotic transformed cells are homogenized to obtain a cell precipitate, wherein the cell precipitate comprises inclusion bodies of the CSFV Erns recombinant protein. Then, the cell pellet is solubilized by an extraction buffer solution to obtain soluble recombinant proteins of CSFV Erns, wherein the extraction buffer solution comprises 20 mM-30 mM Tris salt, 450 mM-550 mM NaCl, and 7M-9M Urea, and the pH of the extraction buffer solution is 7.0-8.0.

In the above examples, the host cell of the prokaryotic transformed cell may be, for example, escherichia coli BL21 (DE 3) strain.

In the above examples, the recombinant plasmid may comprise the nucleic acid sequence shown in SEQ ID NO. 2, encoding a classical swine fever virus Erns recombinant protein.

In the above examples, the extraction buffer solution is composed of, for example, 25mM Tris salt, 500mM sodium chloride (NaCl) and 8M Urea (Urea), and the pH of the extraction buffer solution is pH7.5.

In the above examples, the aforementioned solubilization treatment may be carried out, for example, at 0 ℃ to 4 ℃ for 20 minutes to 40 minutes.

In the above embodiments, after the solubilizing treatment, a column purification step can be optionally performed on the soluble recombinant protein of classical swine fever virus Erns to obtain a purified soluble recombinant protein of classical swine fever virus Erns.

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

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

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

According to another aspect of the present invention, a method for identifying a swine fever virus infection is provided. In one example, first, the classical swine fever virus soluble Erns recombinant protein having the amino acid sequence shown in SEQ ID NO:1 is immobilized on the surface of the reaction region. Then, the biological sample is added into the reaction area, so that the biological sample and the soluble Erns recombinant protein of the classical swine fever virus perform a bonding reaction. Unreacted biological sample is then removed. And then, introducing a secondary antibody into the reaction region, and carrying out identification reaction on the secondary antibody and the biological sample bonded with the soluble Erns recombinant protein of the classical swine fever virus, wherein the secondary antibody only specifically identifies the first antibody of the anti-Erns recombinant protein in the biological sample, but does not react with the second antibody generated by immunization of E2 subunit vaccine of the classical swine fever virus in the biological sample. Then, judging the result of the identification reaction, and when the identification reaction is positive, judging that the pig from the biological sample is infected by the classical swine fever virus; or when the identification reaction is negative, judging that the pig from the biological sample is immunized by the E2 subunit vaccine of the classical swine fever virus.

In the above embodiments, the biological sample may include an organ, tissue, cell, body fluid, lymph, urine, whole blood, plasma, serum, and/or cell culture supernatant isolated from a subject.

After the soluble Erns recombinant protein of the hog cholera virus and the preparation method thereof are applied, the cell sediment of the prokaryotic transformed cell is subjected to the solubilization treatment by using the extraction buffer solution after the prokaryotic transformed cell shows the soluble Erns recombinant protein of the hog cholera virus, so that the soluble Erns recombinant protein of the hog cholera virus can be obtained. When the soluble recombinant protein of classical swine fever virus Erns is applied to the identification kit and/or the method, whether a swine fever virus infected subject from a biological sample or not is infected by the classical swine fever virus or has received classical swine fever virus E2 subunit vaccine immunity can be effectively distinguished.

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

Drawings

The foregoing and other objects, features, and advantages of the invention will be apparent from the following more particular description of the embodiments of the invention, as illustrated in the accompanying drawings in which:

FIG. 1A is a colloidal image of a classical swine fever virus soluble Erns recombinant protein according to an embodiment of the present invention after solubilization treatment by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

FIG. 1B shows the image results of the Western transfer process of FIG. 1A.

FIG. 2A is a colloid image of SDS-PAGE showing soluble classical swine fever virus Erns recombinant protein after column purification according to one embodiment of the present invention.

Fig. 2B shows the image results of the western transfer process of fig. 2A.

FIG. 3 is a bar graph depicting the identification of different biological samples using classical swine fever virus Erns soluble recombinant protein, according to one embodiment of the present invention.

Wherein, the reference numbers:

101: step 1

103: step 2

105: insoluble proteins

107: soluble proteins

Detailed Description

To the extent that a term is defined or used in a reference, it is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term provided herein applies. Furthermore, unless the context dictates otherwise, singular terms may include the plural and plural terms may also include the singular.

As described above, the present invention provides a soluble Erns recombinant protein of a classical swine fever virus, a method for producing the same, and a kit and a method for identifying classical swine fever virus infection using the same. After the swine fever virus Erns recombinant protein is expressed by the prokaryotic transformed cell, the swine fever virus Erns recombinant protein is homogenized and solubilized by an extraction buffer solution to obtain the swine fever virus Erns soluble recombinant protein, and the swine fever virus Erns soluble recombinant protein can be applied to a kit and/or a method for identifying swine fever virus infection.

The terms "recombinant protein", "chimeric protein", "recombinant antigen", "protein", "peptide" and "polypeptide" as used herein are interchangeable and refer to a polymer of amino acids, typically joined together by peptide or disulfide bonds. "peptides" can also be used in 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 the naturally occurring amino acids. "peptide" also includes modified amino acid polymers, e.g., glycoproteins having 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, eukaryotic expression systems. In one embodiment, the recombinant protein referred to herein is a classical swine fever virus Erns recombinant protein.

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

The virus strain of the "hog cholera virus" is not particularly limited, and may be, for example, the TW strain of hog cholera virus (genotype 2.1, NP_777496.1), although in other embodiments, other virus strains may be used, depending on the actual requirements.

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 SEQ ID No.: 1, or the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID No.: 2. In addition, for convenience of subsequent protein purification, a histidine (His) tag [ His tag; or polyhistidine) ], wherein the His-tag may comprise, but is not limited to, 6 to 10 histidine residues.

In some embodiments, the above-described soluble Erns recombinant proteins of hog cholera virus can be produced by conventional methods or by methods described below. First, a protein expression step is performed on a prokaryotic transformed cell, wherein the prokaryotic transformed cell may comprise a recombinant plasmid containing a recombinant gene having a nucleic acid sequence as shown in SEQ ID NO. 2 to express a Classical Swine Fever Virus (CSFV) Erns recombinant protein having an amino acid sequence as shown in SEQ ID NO. 1. For the subsequent purification of the recombinant protein, a nucleic acid sequence encoding a His tag may be optionally added to the 5' end of the recombinant gene. In some instances, the aforementioned nucleic acid sequence encoding the His-tag can be provided, for example, from a commercially available plastid and designed to be linked to the 5' end of the recombinant gene described above. The nucleic acid sequence related to the His tag is well known in the art to which the present invention pertains, and will not be described herein.

Then, the prokaryotic transformed cells are homogenized to obtain a cell pellet (also referred to as cell debris), wherein the cell pellet comprises inclusion bodies of the CSFV Erns recombinant protein.

Since the inclusion body of the recombinant protein of classical swine fever virus Erns is formed by accumulation of insoluble protein, the original biological function of the Erns recombinant protein can be influenced or even not exerted. Therefore, the present invention utilizes the extraction buffer solution to perform the solubilization treatment on the cell precipitate to obtain the soluble recombinant protein of CSFV Erns. In the above embodiment, the extraction buffer solution may be composed of 20mM to 30mM Tris salt, 450mM to 550mM sodium chloride (NaCl), and 7M to 9M Urea (Urea), and the pH of the extraction buffer solution is 7.0 to 8.0. In one example, the extraction buffer solution may be composed of, for example, 25mM Tris salt, 500mM sodium chloride (NaCl) and 8M Urea (Urea), and the pH of the extraction buffer solution may be pH7.5. In another example, the solubilization treatment may be performed, for example, at 0 ℃ to 4 ℃ for 20 minutes to 40 minutes. In other examples, the solubilization treatment may be performed at 0 ℃ for 30 minutes, for example, and the agitation treatment may be used in combination during the solubilization treatment.

In the above embodiments, after the above-mentioned solubilizing treatment, a conventional column purification step can be optionally performed on the soluble recombinant protein of classical swine fever virus Erns to obtain a purified soluble recombinant protein of classical swine fever virus Erns. It should be added that, compared to the conventional column purification step that needs to add β -mercaptoethanol (β -mercaptoethanol, b-ME or BME) to avoid the folding of the N-terminal His tag, the obtained soluble recombinant protein of classical swine fever virus Erns can be purified better without adding b-ME during the column purification step.

Generally, the production of the Erns recombinant protein can yield at least 21.8 milligrams (mg) of the soluble Erns recombinant protein per liter of bacterial fluid, which can be subsequently used in a detection kit and/or method to distinguish whether a swine subject from a biological sample is infected with a swine fever virus or has received an E2 subunit vaccine of a swine fever virus. The identification kits and/or methods may comprise immunoassay kits and/or methods, such as enzyme-linked immunosorbent assay (ELISA) kits and/or methods (e.g., indirect ELISA), for detecting antibodies contained in the biological sample.

The "biological sample" referred to herein is not particularly limited, and may be, for example, an isolated organ, tissue, cell, body fluid, lymph, urine, whole blood, plasma, serum, and/or cell culture supernatant, but the present invention is not limited thereto.

In some examples, the above-described identification methods can be performed using a conventional ELISA kit (and/or biochip). In general, the type of suitable ELISA kits and/or biochips is not particularly limited, and in some instances, such kits can include a classical swine fever virus soluble Erns recombinant protein having an amino acid sequence as set forth in SEQ ID NO. 1, and a secondary antibody. The above soluble Erns recombinant proteins of classical swine fever virus can be immobilized on the surface of the reaction area of the kit. The secondary antibody may be stored in solution in a reagent zone and communicated with the reaction zone via a microchannel, wherein the secondary antibody specifically recognizes the first antibody against the Erns recombinant protein, but does not interact with the second antibody generated by immunization with the classical swine fever virus E2 subunit vaccine, and does not have any antibody-antigen binding reaction with Specific Pathogen Free (SPF) swine serum. In other embodiments, the kit may optionally include a display area to present the result of the identification reaction. The kind of the kit is not particularly limited, and for example, when the kit is a quick-screening reagent, the display region may be, for example, a viewing window to visually observe the results of identifying the detection line and the control line of the color bar (strip) after the reaction. When the kit is a biochip, the display area may be, for example, a display for displaying an image or a signal of the reaction product detected by optical or electrochemical methods.

It is clear that the above-mentioned recombinant ems proteins are used for immunoassays and not as vaccines, and therefore it is necessary to use full-length recombinant ems proteins to effectively distinguish between swine from biological samples whether the subject is infected with classical swine fever virus or has received classical swine fever virus E2-unit vaccine immunization. If the recombinant protein obtained by using the amino acid sequence other than SEQ ID NO. 1 is used, for example, by changing the length or by changing the sequence range selected, the above-mentioned effect cannot be exerted, i.e., whether the swine subject from which the biological sample is derived is infected with CSFV or has been immunized with CSFV E2 subunit vaccine.

In some embodiments, the specific manner for differentiating whether a swine subject from which a biological sample is derived is infected with a classical swine fever virus refers to that the Erns recombinant protein specifically recognizes an antibody against the Erns recombinant protein in the biological sample (also referred to as a first antibody), but does not react with an antibody against the biological sample that is produced by immunization with an E2 subunit vaccine of a classical swine fever virus (also referred to as a second antibody). If a recombinant protein obtained using an amino acid sequence other than SEQ ID No. 1 is used, for example, to change the length or to change the sequence range selected, the modified Erns recombinant protein may react with both the first antibody and the second antibody, and it is not possible to distinguish whether the swine fever virus-infected swine fever virus-derived swine subject or the swine fever virus-immunized E2-unit vaccine has been received.

It is to be understood that the specific recombinant protein sequences, specific formulations, specific dosages, specific assay formats, protocols, illustrations and examples set forth below are illustrative only and are not intended to be limiting. The principal features of this invention can be employed in various embodiments without departing from the spirit and scope of the invention. Therefore, those skilled in the art can easily ascertain the essential characteristics of this invention and various changes and modifications can be made to the invention to adapt it to various usages and conditions without departing from the spirit and scope of the invention.

Example 1: preparation of soluble Erns recombinant protein of classical swine fever virus

1.1 construction of recombinant plasmids of classical swine fever Virus Erns recombinant proteins

An Erns whole gene fragment derived from Classical swine fever virus (CSFV Egystrup strain, genotype1.1) was synthesized by whole gene synthesis (bio ltd). Via polymerase chain reaction [ Polymerase Chain Reaction (PCR), thermoFisher scientific; phusion high Fidelity DNA polymerase, phusion high-Fidelity DNA polymerase, advanced Biotechnology Ltd ] amplified Erns whole gene fragment (sequence shown in SEQ ID NO: 2). Then, 1. Mu.L of the Erns gene fragment (10 ng/. Mu.L, sequence shown In SEQ ID NO: 2), 1. Mu.L of pET21 plasmid (10 ng/. Mu.L) (Invitrogen), and 0.5. Mu.L of ligase (TaKaRa Bio, USA) were reacted at 50 ℃ for 20 minutes according to the method provided by the commercially available In-fusion construction kit (TaKaRa Bio, USA). Then, 25. Mu.L of ECOSTM X-competent cells [ DH5a ] (Probiotics development Co., ltd.) were added to the reaction product, and transformation reaction was carried out at 42 ℃ for 50 seconds. The transformed cell suspension was then spread on the surface of LB medium (Difco. TM. LB Broth, miller, BD) containing Ampicillin (Ampicillin, biobasic). Left at 37 ℃ for about 16 to 18 hours to await colony formation. Thereafter, single colonies were picked for gene sequencing.

1.2 expression of classical swine fever virus Erns recombinant protein

mu.L of pET-21-Erns plasmid (i.e., recombinant plasmid containing the nucleic acid sequence shown in SEQ ID NO: 2) confirmed by gene sequencing was taken and 25. Mu.L of ECOSTM 21 competent cells [ BL21 (DE 3) ] (Probiotics technology development Co., ltd.) were added, and the cells were transfected at 42 ℃ for 50 seconds. Then, the bacterial solution was applied to LB medium (Difco. TM. LB Broth, miller, BD) containing 100. Mu.g/. Mu.L of ampicillin (Biobasic). The mixture was left at 37 ℃ for about 16 to 18 hours to wait for colony formation. Selecting a single bacterial colony to 500 mu L of LB culture solution containing 100 mu g/mu L of ampicillin, culturing at 37 ℃ for 6-8 hours, taking 50 mu L of bacterial liquid to 50mL of LB culture solution containing 100 mu g/mu L of ampicillin, culturing at 37 ℃ for 16-18 hours, taking 10mL of bacterial liquid to 1L of LB culture solution containing 100 mu g/mu L of ampicillin, culturing at 37 ℃ for about 3 hours, adding 500 mu L of 1M Isopropyl-beta-D-thiogalactopyranoside (Isopropyl beta-D-1-thiogalactopyranoside, IPTG; youkou and trade company Limited) to perform protein induction expression, and culturing at 22 ℃ for about 16-18 hours.

1.3 Swine fever Virus Erns recombinant protein homogenization, solubilization and column purification

In this example, the recombinant protein of classical swine fever virus Erns is subjected to homogenization (hereinafter also referred to as step 1) and solubilization (hereinafter also referred to as step 2) sequentially, as follows.

Step 1: coli cells were homogenized by adding 100mL of a cell lysis buffer (containing 25mM Tris salt and 500mM NaCl, pH 7.5) using a commercially available homogenizer (IKA Han New International Co., ltd.). Then, 0.5g of lysozyme (lysozyme, cyrusbioscience, selukasseki technology Co., ltd.) was added to the above reaction product and mixed well, followed by standing on ice for 30 minutes. Next, 0.05g of DNase I (Cyrusbioscience Seoulson technology Co., ltd.) was added to the reaction product, and the mixture was shaken by a commercially available ultrasonic cell disrupter (Viagra Co., ltd.) for a total of 20 minutes with stopping the shaking for 5 seconds every 3 seconds to obtain a homogenized product. The homogenized product was then centrifuged at 12000 Xg for 40 minutes to obtain a first supernatant (1S, lane S of graph 101) and a first pellet (1P, lane P of graph 101), wherein the first pellet (1P) comprises inclusion bodies of classical swine fever virus Erns recombinant protein.

And 2, step: to the first precipitate (1P), an extraction buffer solution (composed of 25mM Tris salt, 500mM sodium chloride and 8M urea) was added, mixed well, and then placed on ice (0 ℃ C.) and stirred for 30 minutes for solubilization. Thereafter, the solubilized product was centrifuged at 12000 Xg for 40 minutes, thereby obtaining a second supernatant (abbreviated as 2S, lane S of diagram No. 103) and a second precipitate (abbreviated as 2P, lane P of diagram No. 103).

Please refer to fig. 1A, which shows the colloid image result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of step 1 (fig. 101) and step 2 (fig. 103) of the method for producing the recombinant protein of classical swine fever virus soluble Erns according to an embodiment of the present invention. From the results in FIG. 1A, it is shown that the Erns recombinant protein produced by the prokaryotic expression system is insoluble protein 105, the protein of which is present in the first precipitate (1P) (insoluble protein 105 indicated by the arrow in lane P of FIG. 1A, graph number 101), and the molecular weight of which is 28kDa.

However, insoluble protein 105 in the first pellet (1P) was solubilized using Erns extraction buffer (consisting of 25mM Tris salt, 500mM NaCl, 8M urea, pH 7.5) to obtain soluble protein 107 in the second supernatant (2S).

Specifically, 100mL of the extraction buffer solution was added to the first precipitate (1P), and after stirring on ice for 30 minutes and centrifugation at 12000 Xg for 40 minutes, the second supernatant (2S) and the second precipitate (2P) were obtained and analyzed by SDS-PAGE electrophoresis. Referring to FIG. 1A, insoluble protein 105 in the first precipitate (1P) has been converted to soluble protein 107 in the second supernatant (2S) (soluble protein 107 shown by the arrow in lane S of FIG. 1A, plot 103).

Referring to FIG. 1B, the image result of the Western transfer method of FIG. 1A is shown, and the flow of the Western transfer method is as follows. SDS-PAGE films containing Erns recombinant proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (GE healthcare, advanced Biotechnology GmbH) and reacted with 20mL of blocking buffer (Tris buffered saline with Tween20, TBST) containing polysorbate 20 (Tween 20) and 5% Bovine serum albumin (Bovine serum albumin, BSA) at room temperature (about 25 ℃) for 2 hours. Then, the blocking buffer solution was removed, and the PVDF membrane was reacted with 10mL of an anti-His-tag antibody solution [ antibody diluted by 1 fold with TBST buffer solution (1 × PBS with 0.1% tween-20 added) ], at 4 ℃ for 16 to 18 hours. Thereafter, the anti-His-tag antibody solution was removed, and the PVDF membrane was rinsed three times with 10mL of TBST buffer solution for 20 minutes each. Then, the PVDF membrane was reacted with a TBST buffer solution of an anti-mouse antibody (anti-mouse antibody, diluted by 1 fold to 10000) at room temperature for 1 hour. Thereafter, the antibody-containing TBST buffer solution was removed, and after washing the PVDF membrane three times with 10mL of TBST buffer solution, a coloring agent (ECL stain kit, advanced Biotechnology, inc.) was added for color development 20 minutes at a time, and the results are shown in FIG. 1B.

The results in fig. 1B show that the anti-His tag antibody reacted with polyhistidine (N-terminal) of Erns recombinant protein to confirm that insoluble protein 105 in the first precipitate (1P, lane P of fig. 101) had been solubilized and then converted to soluble protein 107 in the second supernatant (2S), as shown by the arrow in lane S of fig. 103 of fig. 1B, the soluble protein 107 having a molecular weight of 28kDa and containing an N-terminal His-tag.

In addition, absorbance (OD) of the soluble protein 107 at 280nm was measured using a commercially available spectrometer (e.g., bioSpectrometer instrument, eppendorf) _280nm ) And calculating the protein concentration using the following formula (I):

(a 280 absorbance x protein extinction coefficient) x sample volume = protein concentration (I).

The concentration of soluble Erns recombinant protein was calculated to be (1.2/2.2) × 40ml =21.8mg, which means 21.8mg (mg) of soluble Erns recombinant protein per liter of bacterial suspension was obtained.

The second supernatant (graph No. 2S) containing the soluble Erns recombinant protein was optionally subjected to a column purification step by a nickel-nitrogen [ base ] triacetic acid agarose column (Ni-nitrilotriacetic acid agarose column, ni-NTA agarose column, cytiva, advanced Biotech Co., ltd.), and washed with a washing solution [ graph No. FT, containing 100mM imidazole (imidazole) ]. The protein thus obtained is a purified soluble Erns recombinant protein, such as soluble protein 107 shown by the arrow labeled Erns channel in fig. 2A, with a molecular weight of 28kDa.

FIG. 2B shows the western transfer image of FIG. 2A, wherein the purified soluble Erns recombinant protein is soluble protein 107 with a molecular weight of 28kDa and an N-terminal His tag as indicated by the arrow in the Erns lane of FIG. 2B.

Example 2: specificity identification combination reaction of Erns recombinant protein of hog cholera virus

This example establishes an immunological binding functional validation of antigen and antibody, and uses enzyme-linked immunosorbent assay (ELISA) to perform specific differential binding of the recombinant proteins of the hog cholera virus Erns.

1.4.1 biological samples

This example identifies the serum sources in the following three groups: (1) positive serogroup: in a certain pasture in the south of taiwan,serum of six-week-old piglets, to which live attenuated classical swine fever virus vaccine (originated from dry lapinized classical swine fever vaccine of high-market, farm-society biological pharmaceutical factory of taiwan high-male city, simulated classical swine fever virus infection) was applied, were sampled two weeks after the application of the live attenuated classical swine fever virus vaccine. (2) negative serogroup: serum of eight weeks old piglets without specific pathogen (from Taiwan animal science and technology institute). (3) vaccine group: specific Pathogen Free (SPF) four week old piglets (from Taiwan institute of animal science and technology), commercially available from Shidai

Serum collected four weeks after CSF-E2 subunit vaccine (Bayer). Each group is at least three repeats.

1.4.2 Experimental procedures

The purified Erns recombinant protein was adjusted to a protein concentration of 1 μ g/μ L using carbonate-bicarbonate buffer (Sigma, friend and trade gmbh). Next, 60 ng/. Mu.L of Erns recombinant protein was spread onto the bottom of a 96-well (well) flat-bottomed microtiter plate (Corning). The mixture was left at 4 ℃ for 16 to 18 hours. Then, each well was rinsed by adding 250. Mu.L of PBST (1 XPBS and 0.5% Tween-20), shaking at 125rpm for 10 minutes at room temperature, and repeating this step 5 times. Thereafter, 250. Mu.L of barrier buffer [ PBST and 5% BSA (Sigma, friend and trade Co., ltd.) was added to each well]At 37 ℃ for 2 hours. After removing the blocking buffer solution, 100. Mu.L (dilution ratio of 1 to 1500) of each serum was added to each well, and after 1 hour of reaction at 37 ℃, each serum was removed. Next, 250. Mu.L of PBST (1 XPBS and 0.5% Tween-20) was added to each well, reacted at room temperature for 10 minutes, shaken at 125rpm, and the procedure was repeated 5 times. Then, 100. Mu.L of a secondary antibody [ Goat-anti-pig horseradish peroxidase-labeled antibody, goat-anti pig Horseradish peroxidase (HRP) Ab, dilution ratio 1:10000, abcam (Ab 18184)]After 1 hour of reaction at 37 ℃, the secondary antibody was removed. Next, 250. Mu.L of PBST (1 XPBS and 0.5% Tween-20) was added to each well, reacted at room temperature for 10 minutes, shaken at 125rpm, and the procedure was repeated 5 times. Then, 100. Mu.L of a color-developing substance (e.g., tetramethylbiphenyl) was added to each wellAmine dihydrochloride, 3', 5' -tetramethylbenzidinihydrochloride, TMB), was reacted at room temperature for 15 to 20 minutes, 100. Mu.L of STOP (STOP) solution (ScyTek, yokoku, co., ltd.) was added, and absorbance (OD) at 450nm per well was measured _450nm ) The results are shown in FIG. 3.

FIG. 3 is a bar graph showing the identification of different biological samples using soluble recombinant proteins of classical swine fever virus Erns according to one embodiment of the present invention. As shown in the results of FIG. 3, the Erns soluble recombinant protein purified by the above example showed a binding reaction with serum antibodies of positive serogroup (piglet serum administered live attenuated vaccine) and a statistically significant difference (p <0.05 as shown in the figure) after ELISA test. However, the purified soluble recombinant protein of classical swine fever virus Erns has no immune binding reaction with negative serogroup (i.e. piglet serum of SPF piglets) and vaccine group (i.e. piglet serum administered with commercial E2 unit vaccine (Bayer)).

The soluble recombinant protein of classical swine fever virus Erns purified in the above embodiment can actually generate specific binding reaction with serum antibody generated by whole virus, but has no binding reaction with antibody generated by immune reaction of E2 times unit vaccination, which means that the soluble recombinant protein of classical swine fever virus Erns purified in the above embodiment can actually distinguish whether a swine subject from a biological sample is infected by classical swine fever virus or has received immune reaction of E2 times unit vaccine of classical swine fever virus, and is expected to be applied to diagnostic reagent for differential swine fever virus in the future, thereby achieving the goal of removing classical swine fever virus.

In summary, the present invention is only exemplified by the soluble Erns recombinant protein of classical swine fever virus, the method for producing the same, and the kit and method for identifying classical swine fever virus infection, with a specific amino acid sequence, a specific manufacturing method, a specific composition, a specific analytical model or a specific evaluation method. However, it is understood by those skilled in the art that other amino acid sequences, other manufacturing methods, other compositions, other analysis modes or other evaluation methods can be used for the soluble Erns recombinant protein of classical swine fever virus and the manufacturing method thereof, and the kit and method for identifying classical swine fever virus infection without departing from the spirit and scope of the present invention.

For example, the soluble Erns recombinant proteins of classical swine fever virus can be produced using other expression systems (e.g., eukaryotic expression systems) to optimize process and mass production. The obtained soluble Erns recombinant protein of the classical swine fever virus can be applied to a quick screening reagent or a biochip or other test products sold in the market according to actual requirements. In addition, in addition to serum, the biological sample for identifying the swine fever virus infection may be, for example, an isolated organ, tissue, cell, body fluid, lymph fluid, urine, whole blood, plasma, and/or cell culture supernatant.

According to the embodiments, the soluble Erns recombinant protein of swine fever virus and the method for producing the same of the present invention have the advantages that after the original nuclear transformed cells express the soluble Erns recombinant protein of swine fever virus, the cell precipitates of the original nuclear transformed cells are solubilized by the extraction buffer solution, so as to obtain the soluble Erns recombinant protein of swine fever virus. When the soluble recombinant protein of classical swine fever virus Erns can be applied to an identification kit and/or method in the future, whether a swine subject from a biological sample is infected by classical swine fever virus or not or whether the swine subject is immunized by a classical swine fever virus E2 subunit vaccine can be effectively distinguished.

While the invention has been described with reference to specific embodiments, other embodiments are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the embodiments contained herein.

Sequence listing

<110> Taiwan east-screener science and technology university

<120> soluble Erns recombinant protein of classical swine fever virus, method for producing the same, kit and method for identifying classical swine fever virus infection

<130> do not

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<212> PRT

<213> Artificial sequence

<220>

<223> Erns recombinant protein of CSFV

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Met Glu Asn Ile Thr Gln Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly

5 10 15

Ile Gln His Ala Met Tyr Leu Arg Gly Val Ser Arg Ser Leu His Gly

20 25 30

Ile Trp Pro Glu Lys Ile Cys Lys Gly Val Pro Thr Tyr Leu Ala Thr

35 40 45

Asp Thr Glu Leu Lys Glu Ile Gln Gly Met Met Asp Ala Ser Glu Gly

50 55 60

Thr Asn Tyr Thr Cys Cys Lys Leu Gln Arg His Glu Trp Asn Lys His

65 70 75 80

Gly Trp Cys Asn Trp Tyr Asn Ile Asp Pro Trp Ile Gln Leu Met Asn

85 90 95

Arg Thr Gln Ala Asn Leu Ala Glu Gly Pro Pro Ala Lys Glu Cys Ala

100 105 110

Val Thr Cys Arg Tyr Asp Lys Asp Ala Asp Ile Asn Val Val Thr Gln

115 120 125

Ala Arg Asn Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn

130 135 140

Phe Ser Phe Ala Gly Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val

145 150 155 160

Ser Val Glu Asp Ile Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu

165 170 175

Gln Asp Thr Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu

180 185 190

Asn Ala Arg Gln Gly Ala Ala Arg Val Thr Ser Trp Leu Gly Arg Gln

195 200 205

Leu Ser Thr Ala Gly Lys Arg Leu Glu Gly Arg Ser Lys Thr Trp Phe

210 215 220

Gly Ala Tyr Ala

225

<210> 2

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<212> DNA

<213> Artificial sequence

<220>

<223> Erns recombinant protein of CSFV

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ATGGAAAATA TAACTCAATG GAACCTGAGT GACAACGGCA CTAATGGTAT CCAGCATGCT 60

ATGTACCTTA GAGGGGTTAG CAGGAGCTTG CATGGGATCT GGCCGGAAAA AATATGCAAA 120

GGAGTCCCCA CCTACCTGGC CACAGACACG GAACTGAAAG AAATACAGGG AATGATGGAT 180

GCCAGCGAGG GGACAAACTA TACGTGCTGT AAGTTACAGA GACATGAATG GAACAAACAT 240

GGATGGTGTA ACTGGTACAA TATAGACCCC TGGATACAGT TGATGAATAG AACCCAAGCA 300

AACTTGGCAG AAGGCCCTCC GGCCAAGGAG TGCGCTGTGA CTTGCAGGTA CGATAAAGAT 360

GCTGACATCA ACGTGGTCAC CCAGGCCAGA AACAGGCCAA CAACCCTGAC CGGTTGCAAG 420

AAAGGAAAAA ATTTTTCTTT TGCGGGTACA GTTATAGAGG GCCCATGTAA TTTCAATGTT 480

TCCGTGGAGG ATATCTTGTA TGGGGATCAT GAGTGCGGCA GTTTGCTTCA GGACACGGCT 540

CTGTACCTAG TGGATGGAAT GACCAACACT ATAGAGAATG CCAGACAGGG AGCAGCGAGG 600

GTAACATCTT GGCTCGGGAG GCAACTCAGC ACTGCCGGGA AGAGGTTGGA GGGTAGAAGC 660

AAAACCTGGT TTGGTGCCTA TGCC 684

Claims

1. A method for preparing soluble Erns recombinant protein of hog cholera virus, which is characterized by comprising the following steps:

performing a protein expression step on a prokaryotic transformed cell, wherein the prokaryotic transformed cell comprises a recombinant plasmid to express a classical swine fever virus Erns recombinant protein having an amino acid sequence as shown in SEQ ID NO: 1;

homogenizing the prokaryotic transformed cell to obtain a cell precipitate, wherein the cell precipitate comprises an inclusion body of the CSFV Erns recombinant protein; and

performing a solubilization process on the cell precipitate by using an extraction buffer solution to obtain the CSFV Erns soluble recombinant protein, wherein the extraction buffer solution is composed of 20mM to 30mM of tris, 450mM to 550mM of sodium chloride and 7M to 9M of urea, and the pH value of the extraction buffer solution is pH 7.0 to pH 8.0.

2. The method of claim 1, wherein a host cell of the prokaryotic transformant cell is an E.coli strain BL21 (DE 3).

3. The method of claim 1, wherein the recombinant plasmid comprises the nucleic acid sequence as set forth in SEQ ID NO. 2, encoding the classical swine fever virus Erns recombinant protein.

4. The method of claim 1, wherein the extraction buffer solution comprises tris (25 mM) salt, 500mM sodium chloride and 8M urea, and the pH of the extraction buffer solution is pH7.5.

5. The method of claim 1, wherein the solubilization is performed at 0 ℃ to 4 ℃ for 20 minutes to 40 minutes.

6. The method of claim 1, further comprising performing a column purification step on the CSFV Erns soluble recombinant protein after the solubilization process to obtain a purified CSFV Erns soluble recombinant protein.

7. A classical swine fever virus soluble Erns recombinant protein produced by the method of any one of claims 1 to 6.

8. A kit for identifying a swine fever virus infection, comprising:

a soluble Erns recombinant protein of classical swine fever virus is fixed on a surface of a reaction area, wherein the soluble Erns recombinant protein of classical swine fever virus is an amino acid sequence shown as SEQ ID NO. 1; and

a secondary antibody in communication with the reaction region, wherein the secondary antibody specifically recognizes a first antibody directed against an Erns recombinant protein, but does not interact with a second antibody produced by immunization with an E2 subunit vaccine of classical swine fever virus.

9. The kit for identifying classical swine fever virus infection according to claim 8, further comprising a display area for displaying a result of the identification reaction.

10. A method of identifying a classical swine fever virus infection, comprising:

fixing 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 an amino acid sequence shown as SEQ ID NO. 1;

adding a biological sample into the reaction area, and enabling the biological sample and the soluble Erns recombinant protein of the classical swine fever virus to perform a bonding reaction;

removing the unreacted biological sample;

introducing a secondary antibody into the reaction region, and performing an identification reaction between the secondary antibody and the biological sample bonded with the soluble Erns recombinant protein of the classical swine fever virus, wherein the secondary antibody only specifically identifies a first antibody against the Erns recombinant protein in the biological sample, but does not react with a second antibody generated by immunization with an E2 subunit vaccine of the classical swine fever virus in the biological sample; and

judging the result of the identification reaction, and if the result is positive,

wherein when the identification reaction is positive, it is judged that one of the swine subjects from which the biological sample is derived is infected with CSFV, or

And when the identification reaction is negative, judging that the pig subject from which the biological sample is derived is immunized by the classical swine fever virus E2 subunit vaccine.

11. The method according to claim 10, wherein the biological sample comprises ex vivo organs, tissues, cells, body fluids, lymph fluids, urine, whole blood, plasma, serum and/or cell culture supernatant.