A GP5 protein, a gene encoding the same, a production vector, a preparation method and applications thereof

Abstract

The present invention provides a GP5 protein, a gene encoding the same, a production vector, a preparation method and an application thereof, wherein the GP5 protein has a GP5 protein active portion, and the GP5 protein active portion is shown in SEQ ID NO: 1. In the present invention, the GP5 protein is codon-optimized so that it depends only on its own gene for expression. At the same time, it has been experimentally verified that the recombinant protein of the present invention can have a good effect on inducing an immune response of an organism to porcine reproductive and respiratory syndrome virus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a GP5 protein, a gene encoding the GP5 protein, a production vector, a preparation method and applications thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-sense RNA enveloped virus that can cause severe reproductive disorders in boars and sows and respiratory diseases in piglets. In addition, the virus can cause severe immunosuppression in the host, leading to secondary infections with opportunistic pathogens, resulting in severe economic losses.

[0003] The genome of porcine reproductive and respiratory syndrome virus is about 15kb, and its ORF2 to ORF7 encode GP2a, GP3, GP4, GP5, M and N proteins respectively. In addition, there is a reading frame encoding E protein in ORF2, and ORF5 contains a reading frame encoding GP5a. On the viral envelope, GP5 and M proteins form heterodimers through disulfide bonds, and GP2a, GP3 and GP4 form heterotrimers. GP5-M dimers may be related to binding to the host receptor CD169, but GP5 monomer protein is difficult to prepare in vitro, and its specific pathogenic mechanism in porcine reproductive and respiratory syndrome symptoms is still unclear.

[0004] The in vitro preparation of GP5 protein has great application prospects for the production of porcine reproductive and respiratory syndrome virus gene vaccines, but GP5 is a membrane protein, which makes in vitro preparation very difficult. The GP5 protein prepared using conventional Escherichia coli as a production vector needs to be connected to trigger factors and other tags to promote its expression, making it difficult to obtain GP5 protein that relies on its own gene expression. Summary of the invention

[0005] The main purpose of the present invention is to provide a GP5 protein, a gene encoding the GP5 protein, a production vector, a preparation method and an application thereof, aiming to provide a protein that relies on its own gene expression.

[0006] To achieve the above object, the present invention provides a GP5 protein, wherein the GP5 protein has a GP5 protein active portion, and the sequence of the GP5 protein active portion is shown in SEQ ID NO:1.

[0007] Optionally, the GP5 protein further has a tag protein connected to the N-terminus and / or C-terminus of the active part of the GP5 protein.

[0008] Optionally, the tag protein is connected to the C-terminus of the active part of the GP5 protein, and the sequence of the GP5 protein is shown in SEQ ID NO:2.

[0009] In addition, the present invention also provides a coding gene sequence as shown in SEQ ID NO:3.

[0010] In addition, the present invention provides a vector containing the above encoding gene, wherein the vector is a plasmid or an engineered bacterium.

[0011] In addition, the present invention also provides a method for preparing the above-mentioned GP5 protein, and the method for preparing the GP5 protein comprises the following steps:

[0012] Constructing a recombinant plasmid containing a gene encoding the active part of the GP5 protein;

[0013] The recombinant plasmid is transferred into competent cells and induced for expression to obtain a GP5 expression strain;

[0014] The expressed protein is extracted from the GP5 expression strain to obtain the GP5 protein.

[0015] Optionally, the step of constructing a recombinant plasmid containing a gene encoding the active part of the GP5 protein comprises:

[0016] Using GP5 amplification primers to clone the coding gene of the GP5 protein as a template to obtain a GP5 gene amplification fragment containing a restriction site, wherein the sequence of the GP5 protein is shown in SEQ ID NO: 2;

[0017] The GP5 gene amplified fragment is connected to the vector plasmid to obtain the recombinant plasmid.

[0018] Optionally, the sequence of the GP5 amplification primer pair is as shown in SEQ ID NO: 4-5; and / or,

[0019] The sequence of the gene encoding the GP5 protein is shown in SEQ ID NO:3.

[0020] Optionally, the step of transferring the recombinant plasmid into competent cells and inducing expression to obtain a GP5 expression strain comprises:

[0021] After the recombinant plasmid is introduced into the competent cells derived from Escherichia coli, isopropylthiogalactoside is used to induce expression to obtain the GP5 expression strain.

[0022] Optionally, the step of extracting the expressed protein from the GP5 expression strain to obtain the GP5 protein comprises:

[0023] The GP5 expression strain was sequentially crushed, centrifuged and filtered, and then the bacterial liquid was collected.

[0024] The bacterial solution is subjected to column chromatography using a nickel column affinity chromatography method with an eluent containing imidazole;

[0025] The bacterial solution after the column chromatography is dialyzed and filtered in sequence to obtain the GP5 protein.

[0026] In addition, the present invention also provides the use of the above GP5 protein or the above method for preparing the GP5 protein in preparing a preparation for inducing an immune response of an organism to porcine reproductive and respiratory syndrome virus.

[0027] In the present invention, the GP5 protein is codon-optimized so that it relies solely on its own gene for expression. At the same time, it has been experimentally verified that the recombinant protein of the present invention can have a good effect on inducing an immune response of an organism to porcine reproductive and respiratory syndrome virus. Specifically, in some embodiments, the preparation includes a compound preparation and the above-mentioned protein composition. In the vaccine, the mass of the GP5 protein is 25 to 300 μg / ml; the mass of the GP234 protein is 25 to 100 μg / ml, and the volume percentage of the compound preparation is 20 to 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 It is the detection result of the gene amplification fragment size;

[0030] Figure 2 Identification of GP5 protein PCR-positive transformants;

[0031] Figure 3 The results of induced expression of the GP5 protein recombinant protein in Example 2;

[0032] Figure 4 The immunoblotting of the purified GP5 protein of Example 3 was used to verify the reactivity;

[0033] Figure 5 This is the result of monitoring the body temperature of piglets after virus attack;

[0034] Figure 6 The results are shown in Figure 2. The fusion expression of Trigger factor and GP5 and the SDS-PAGE electrophoresis results before and after TEV enzyme digestion of the fusion protein.

[0035] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0036] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0037] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0038] Since the existing GP5 protein is difficult to express by its own gene, it is difficult to obtain GP5 protein monomer. The present invention provides a GP5 protein, wherein the GP5 protein has a GP5 protein active part, and the GP5 protein active part is shown in SEQ ID NO:1.

[0039] In the present invention, the amino acid sequence of GP5 protein is optimized so that it can be expressed in a soluble form only by its own gene. At the same time, experimental verification shows that the recombinant protein of the present invention can have a good effect on inducing an immune response of an organism to porcine reproductive and respiratory syndrome virus.

[0040] In some embodiments, the GP5 recombinant protein also has a tag protein connected to the N-terminus and / or C-terminus of the active part of the GP5 protein. By connecting the tag protein, it is convenient to purify the protein without affecting the activity and self-expression of the protein. Specifically, the tag protein is connected to the C-terminus of the active part of the GP5 protein, and the sequence of the GP5 recombinant protein containing the tag protein is shown in SEQ ID NO: 2.

[0041] In addition, the present invention also provides a coding gene sequence as shown in SEQ ID NO: 3. By optimizing the codons of the active part of the GP5 protein and the coding gene of the GP5 protein at the same time, the soluble expression amount of the GP5 protein in Escherichia coli can be increased.

[0042] In addition, the present invention provides a vector containing the above coding gene, wherein the vector is a plasmid or an engineered bacterium. The GP5 protein is prepared by using the vector containing the above coding gene as a raw material.

[0043] In addition, the present invention also provides a method for preparing the above-mentioned GP5 protein, and the method for preparing the GP5 recombinant protein comprises the following steps:

[0044] Step S10: constructing a recombinant plasmid containing a gene encoding the active part of the GP5 protein;

[0045] Step S20: transferring the recombinant plasmid into competent cells and inducing expression to obtain a GP5 expression strain;

[0046] Step S30: extracting the expressed protein from the GP5 expression strain to obtain the GP5 recombinant protein.

[0047] The process of preparing the GP5 protein of the present invention is simple, which is conducive to the scaled-up production of the GP5 protein and is conducive to industrial application.

[0048] Step S10 includes:

[0049] Step S101: using a GP5 amplification primer pair to clone the gene encoding the GP5 protein as a template to obtain a GP5 gene amplified fragment containing a restriction site, wherein the sequence of the GP5 protein is shown in SEQ ID NO: 2, and correspondingly, the sequence of the gene encoding the GP5 protein is shown in SEQ ID NO: 3;

[0050] Step S102: Connect the GP5 gene amplified fragment to the vector plasmid to obtain the recombinant plasmid.

[0051] It should be noted that the GP5 amplification primer pair can be selected according to the amplification effect. In the present invention, the sequence of the GP5 amplification primer pair is shown in SEQ ID NO: 4 to 5. In step S102, the vector plasmid is not limited as long as it can be connected to the GP5 connection product, and can be specifically a pET28a(+) plasmid.

[0052] In some embodiments, step S20 includes:

[0053] After the recombinant plasmid is introduced into the competent cells derived from Escherichia coli, isopropylthiogalactoside is used to induce expression to obtain the GP5 expression strain.

[0054] Escherichia coli is an important carrier for plasmid expansion production, which can well expand the yield of plasmid. However, the GP5 protein of the existing amino acid sequence is either unable to be expressed using the vector, or the expression is in the form of inclusion bodies, which greatly increases the subsequent renaturation process. The present invention optimizes the amino acid codons and the coding gene to enable soluble expression in the competent cells of Escherichia coli, which not only expands its yield, but also greatly shortens the preparation process. It should also be noted that the method in which expression can be induced is not limited. In the present invention, IPTG is used for expression.

[0055] Under the premise that extraction can be performed, the extraction method is not limited. Specifically, step S30 specifically includes the following steps:

[0056] Step S301: The GP5 expression strain is sequentially crushed, centrifuged and filtered, and the bacterial liquid is collected.

[0057] Step S302: using an eluent containing imidazole to perform column chromatography on the bacterial solution using a nickel column affinity chromatography method;

[0058] Step S303: dialyzing and filtering the bacterial solution after the column chromatography in sequence to obtain the GP5 recombinant protein.

[0059] In addition, the present invention also provides the use of the above GP5 protein or the above method for preparing the GP5 protein in preparing a preparation for inducing an immune response of an organism to porcine reproductive and respiratory syndrome virus.

[0060] In the present invention, an animal model was constructed using pigs to verify that the recombinant protein of the present invention can induce an immune response of an organism to porcine reproductive and respiratory syndrome virus.

[0061] Specifically, the application includes compounding GP234 protein and GP5 protein for use, wherein the sequence of the GP234 protein is shown in SEQ ID NO:6.

[0062] By combining with GP234 protein, it can have a good preventive and therapeutic effect on NADC30-like strains.

[0063] In the present invention, the compound adjuvant used in the present invention is a litchi-shaped nano zinc oxide cluster adjuvant (patent publication number: CN113616787A), and its preparation method is:

[0064] 1. Add 1.1 g zinc acetate into a round-bottom flask containing 50 mL diethylene glycol, and stir magnetically at 300 rpm for 30 min to fully dissolve the zinc acetate for later use;

[0065] 2. Place the round-bottom flask containing the above solution in silicone oil at 180°C and preheat for 10 minutes. During the heating process, stir the solution evenly by magnetic stirring at a speed of 500 rpm.

[0066] 3. Under continuous magnetic stirring at 500 rpm, deionized water was quickly added to the preheated solution at a volume ratio of water to diethylene glycol of 1:50 to initiate hydrolysis of the zinc salt. The solution became turbid after heating for 10 min, indicating that the lychee-shaped nano-zinc oxide clusters were successfully synthesized.

[0067] 4. Remove the heat source and continue magnetic stirring until the solution cools to room temperature. The product is diluted with an equal volume of ethanol, centrifuged at 10,000 rpm for 5 min to collect the zinc oxide nanocluster precipitate, add an appropriate amount of ethanol and disperse the precipitate under the assistance of ultrasound, then collect the zinc oxide clusters by centrifugation at 10,000 rpm for 5 min, and repeat the above steps 3 times to obtain a purified litchi-shaped nano zinc oxide cluster adjuvant. The protein prepared by the above adjuvant and the present invention has higher compatibility.

[0068] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0069] Example 1

[0070] This embodiment provides the construction of a recombinant plasmid and a recombinant Escherichia coli expression strain, which has the following steps:

[0071] 1) Codon optimization and gene synthesis

[0072] The GP5 coding sequence was optimized according to the codon preference of E. coli and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0073] 2) Cloning

[0074] The synthetic GP5 coding gene (SEQ ID NO: 3) was amplified by PCR using primers F1 / R1 (F1: SEQ ID NO: 4; R2: SEQ ID NO: 5) using Takara's Primestar@ high-fidelity enzyme. The amplified gene fragment was identified by 0.8% agarose gel electrophoresis to meet the target band size ( Figure 1), the amplified gene fragment was purified using Omega's Cyclepure kit, and the operation was carried out according to the instructions of the kit. Then, GP5 was double-digested with restriction endonucleases NcoI and XhoI in a 37°C water bath, and the digestion system was: 7μl of target gene, 1μl of NcoI and XhoI, and 1μl of 10×buffer. Then, the amplified gene fragment was purified using Omega's Cyclepure kit, and the operation was carried out according to the instructions of the kit. At the same time, the pET28a(+) plasmid was also double-digested and purified with restriction endonucleases NcoI and XhoI in a similar manner. Then, Takara's T4 DNA Ligase was used to connect the plasmid and gene after double digestion with NcoI and XhoI, and the connection system was: 7μl of target gene, 1μl of plasmid, 1μ of T4 DNA Ligase and 1μl of 10×buffer. The ligation system was connected in a 4°C refrigerator overnight, and then transformed into BL21 (DE3) competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.) according to the operating instructions, and then coated with LB agar medium containing 50 μg / ml kanamycin sulfate and cultured in a 37°C constant temperature incubator for 16 to 18 hours. Single colonies were picked for PCR identification, the primers were T7 / T7ter, and the DNA polymerase was Taq@ produced by Takara (results see Figure 2 ), Figure 2 In the figure, M is Marker, lane 1 is transformant 1, lane 2 is transformant 2, lane 3 is transformant 3, lane 4 is transformant 4, lane 5 is transformant 5, and the recombinant Escherichia coli expressing recombinant GP5 were named BL21(DE3)-GP5.

[0075] Example 2

[0076] The expression method of the recombinant GP5 protein in this example is as follows:

[0077] Pick a single colony of BL21(DE3)-GP5, inoculate 10 ml of LB medium containing 50 μg / ml kanamycin sulfate, and culture overnight at 37°C with shaking at 180 rpm. Then take 1% (v / v) of the overnight cultured bacterial solution and inoculate it into 200 ml of LB medium containing antibiotics. After culturing for 3.5 hours, add IPTG at a final concentration of 0.5 mM, lower the culture temperature to 25°C, and induce for 16 hours. Then centrifuge at 8000 rpm for 5 minutes to collect the bacterial sludge, resuspend the bacterial sludge with 1 / 10 volume of PBS, and perform SDS-PAGE analysis of recombinant protein expression after ultrasonic disruption (see Figure 3 ), Figure 31 is the supernatant of optimized GP5 induced expression, 2 is the precipitate of optimized GP5 induced expression, 3 is the supernatant of unoptimized GP5 (coding gene as shown in SEQ ID NO: 14), and 4 is the precipitate of unoptimized GP5 expression. By optimizing the coding gene, the yield of soluble expression is increased.

[0078] Example 3

[0079] This example provides a method for purifying a recombinant protein and identifying its reactogenicity, and the operation is as follows:

[0080] The bacterial sludge collected by centrifugation was resuspended with PBS, and the ratio of bacterial sludge to PBS was 1:10 (W / V). It was broken with a high-pressure homogenizer, and the parameters were 1000bar and cyclically broken 3 times. After the broken bacterial solution was centrifuged at 10000rpm for 15min, the supernatant was taken and filtered with a 0.45μm filter membrane. After the nickel ion affinity chromatography column was equilibrated with PBS, the filtered bacterial solution was added at 1ml / min to allow the recombinant protein to bind to the nickel ions of the chromatography column through the histidine tag, and then the chromatography column was rinsed with PBS containing 100mM imidazole to remove non-target proteins, and finally the target protein was eluted with PBS containing 500mM imidazole. The purified protein was dialyzed to remove imidazole, and then sterilized by filtration with a 0.2μm filter membrane. The immunoblotting experiment was performed in accordance with "Molecular Cloning", in which the porcine reproductive and respiratory syndrome virus positive serum was diluted 500 times, and the horseradish peroxidase-labeled rabbit anti-pig secondary antibody was diluted 3000 times. The results showed that GP5 protein could react with porcine reproductive and respiratory syndrome virus positive serum ( Figure 4 ), indicating that the expressed proteins all have corresponding biological activities, Figure 4 To verify GP5 by immunoblotting, the GP5 protein shown in SEQ ID NO: 2 was obtained by sequencing.

[0081] Example 4

[0082] This embodiment provides a method for preparing GP234 protein, and its operation is generally consistent with that of GP5 protein, except that the amino acid sequence of GP234 protein is shown in SEQ ID NO: 6, its coding gene sequence is shown in SEQ ID NO: 7, and its amplification primer pair sequence is shown in SEQ ID NO: 8-9.

[0083] Comparative Example 1

[0084] The present comparative example provides a method for preparing a Trigger factor-GP5 protein, and the operation is substantially the same as that of Examples 1 to 3, except that the sequence of the Trigger factor-GP5 protein is as shown in SEQ ID NO: 10, the coding sequence thereof is as shown in SEQ ID NO: 11, the amplification primer pair is as shown in SEQ ID NO: 12 to 13, and TEV enzyme is added to the expression supernatant and digested at 30° C. for 16 h.

[0085] The results of SDS-PAGE electrophoresis of the fusion expression of trigger factor and GP5 and the fusion protein before and after TEV digestion are as follows: Figure 6 As shown, Figure 6 In the figure, M is a protein marker, 1 is the whole bacteria expressing the fusion of Trigger factor and GP5, 2 is the supernatant expressing the fusion of Triggerfactor and GP5, 3 is the supernatant expressing the fusion of Triggerfactor and GP5 without adding TEV enzyme, and 4 is the supernatant expressing the fusion of Triggerfactor and GP5 with TEV enzyme added and digested at 30℃ for 16h. The arrow indicates the position of the fusion protein of Triggerfactor and GP5, and the asterisk indicates the position of the TEV enzyme.

[0086] The results showed that after adding the Trigger factor for fusion expression, the Trigger factor could not be removed, and the pure GP5 protein could not be obtained in this comparative example.

[0087] From Examples 1 to 3 and Comparative Example 1, the GP5 amino acids were optimized for preparation, and the GP5 protein expressed only by GP5 itself was prepared.

[0088] Embodiments 5 to 8

[0089] This embodiment provides a vaccine, and the preparation method includes:

[0090] The GP5 recombinant protein of Example 3 and the GP234 recombinant protein of Example 4 were diluted to 1000 μg / ml with sterile PBS, mixed with the litchi-shaped nano zinc oxide cluster adjuvant, and prepared into a vaccine after high-speed stirring and shearing emulsification. The vaccine was stored at 4°C for later use. The vaccine was prepared according to the components and contents of Examples 5 to 8 in Table 1 to obtain a vaccine preparation.

[0091] Table 1 Components and contents of vaccines in Examples 5 to 8 Vaccine configuration

[0092]

[0093]

[0094] Example 9

[0095] This example provides a vaccine safety test, and the operating steps are as follows:

[0096] 1) Five mice weighing 18-22g were used, and each mouse was subcutaneously injected with 0.5ml of the vaccine. No deaths or obvious adverse reactions or systemic reactions caused by the vaccine were observed for 7 consecutive days.

[0097] 2) Five weaned piglets aged 21 to 28 days were injected with 2 ml of the vaccine into the neck muscle and observed daily. No local or systemic adverse reactions were caused by the injection of the vaccine.

[0098] Example 10

[0099] This embodiment provides a vaccine effectiveness test, and the operating steps are as follows:

[0100] 1) Immune challenge test

[0101] Marc145 cells were cultured in cell bottles, and the cells were subcultured to 96-well culture plates 48 hours before the determination of the virus content. They were cultured in a 37°C cell culture incubator with 5% CO2 for 2 days to form a monolayer of cells. The virus solution was diluted 10 times in a gradient, and 3 dilutions of 10-4, 10-5, and 10-6 were taken to inoculate the Marc145 cell monolayer of a 96-well culture plate. Each dilution was repeated for 6 wells, 0.2 ml / well; then placed in a constant temperature incubator for 5 days. Observe CPE and calculate TCID50 according to the Reed-Muench method. Dilute the virus solution to 105.0TCID50 / ml with sterile PBS for virus attack.

[0102] Five healthy piglets with negative PRRSV antibodies at 25 to 30 days of age were injected with 1 ml of the vaccine preparation of Example 5 into the neck muscle of each piglet, and 1 ml was boosted 21 days later; the other 8 healthy piglets with negative PRRSV antibodies were not immunized. 14 days after the second immunization, 5 immunized pigs and 5 non-immunized pigs were injected with 2 ml of porcine reproductive and respiratory syndrome virus NADC30-like strain DB strain (105.0TCID50 / ml) by intramuscular injection and observed for 3 consecutive weeks. Judgment was made based on clinical symptoms. The other 3 non-immunized pigs were not challenged with the virus and served as blank controls.

[0103] 2) Body temperature, clinical symptoms, and autopsy

[0104] Before the virus attack, the body temperature was measured once in the morning and once in the afternoon, and the average value was taken as the basal body temperature. After the virus attack, the body temperature was measured once every morning for 21 consecutive days. At the same time, the clinical symptoms of each pig were recorded, including respiratory symptoms, mental state, appetite, etc. The results showed that there was no obvious change in the body temperature of the unimmunized and unchallenged piglets. The body temperature of the unimmunized piglets that were challenged with the virus began to rise after the virus attack and continued until the end of the experiment. Clinical symptoms included shortness of breath, loss of appetite, and mental depression. The body temperature of the immunized group of piglets increased by ≤1°C 1 to 4 days after the virus attack, then decreased, and returned to normal on the 7th day ( Figure 5 ). During the stage of elevated body temperature, appetite decreased to a certain extent, and only two piglets showed mild respiratory symptoms on the 4th day. After autopsy, typical lesions appeared in the lungs of non-immune piglets after challenge, including congestion and necrosis of the apical lobe of almost all piglets, and swelling of other parts of the lungs. However, shrimp-like lesions appeared in the lungs of only one piglet after challenge, indicating that the vaccine of the present invention has a good immune response effect.

[0105] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention. Sequence Listing <110> Sinopharm Animal Health Co., Ltd. <120> A GP5 protein, a gene encoding the same, a production vector, a preparation method and applications thereof <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 176 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Ser Ser Gly Asn Ser Asn Ser Ser Ser His Leu Gln Leu Ile 1 5 10 15 Tyr Asn Leu Thr Ile Cys Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys 20 25 30 Lys Phe Asp Trp Ala Val Glu Thr Phe Val Ile Phe Pro Ala Leu Thr 35 40 45 His Ile Val Ser Tyr Gly Ala Leu Thr Thr Ser His Phe Leu Asp Thr 50 55 60 Val Gly Leu Ile Thr Val Ser Thr Ala Gly Tyr Leu His Gly Arg Tyr 65 70 75 80 Val Leu Ser Ser Val Tyr Ala Val Cys Ala Leu Ala Ala Leu Ile Cys 85 90 95 Phe Thr Ile Arg Leu Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys 100 105 110 Thr Arg Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg 115 120 125 Trp Arg Ser Pro Val Val Ile Glu Lys Gly Gly Lys Val Asp Val Glu 130 135 140 Gly His Leu Ile Asp Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala 145 150 155 160 Thr Pro Val Thr Lys Ile Ser Ala Glu Gln Trp Gly Arg Pro Leu Glu 165 170 175 <210> 2 <211> 182 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Ser Ser Gly Asn Ser Asn Ser Ser Ser His Leu Gln Leu Ile 1 5 10 15 Tyr Asn Leu Thr Ile Cys Glu Leu Asn Gly Thr Asp Trp Leu Asn Lys 20 25 30 Lys Phe Asp Trp Ala Val Glu Thr Phe Val Ile Phe Pro Ala Leu Thr 35 40 45 His Ile Val Ser Tyr Gly Ala Leu Thr Thr Ser His Phe Leu Asp Thr 50 55 60 Val Gly Leu Ile Thr Val Ser Thr Ala Gly Tyr Leu His Gly Arg Tyr 65 70 75 80 Val Leu Ser Ser Val Tyr Ala Val Cys Ala Leu Ala Ala Leu Ile Cys 85 90 95 Phe Thr Ile Arg Leu Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys 100 105 110 Thr Arg Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly Lys Leu Tyr Arg 115 120 125 Trp Arg Ser Pro Val Val Ile Glu Lys Gly Gly Lys Val Asp Val Glu 130 135 140 Gly His Leu Ile Asp Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala 145 150 155 160 Thr Pro Val Thr Lys Ile Ser Ala Glu Gln Trp Gly Arg Pro Leu Glu 165 170 175 His His His His His His 180 <210> 3 <211> 549 <212> DNA <213> Artificial Sequence <400> 3 atgggttcca gcggtaactc taacagctct agccacctgc agctgattta taatctgacc 60 atctgcgaac tgaacggcac cgactggctg aacaaaaaat tcgactgggc agttgaaact 120 ttcgtgatct ttccggcgct gacccatatc gtgagctacg gcgcgctgac cacctcccac 180 ttcctggata ccgttggcct gattaccgtt agcaccgcgg gttatctgca cggccgttat 240 gttctgtcct ctgtatatgc cgtgtgcgcg ctggcggcgc tgatctgctt taccatccgc 300 ctggcaaaaa actgtatgtc ttggcgctac tcttgcactc gttataccaa ctttctgctg 360 gataccaaag gtaaactgta ccgttggcgc agccctgtag tgattgaaaa aggcggcaaa 420 gtggatgtag aaggccacct gattgacctg aaacgcgtag tcctggacgg ttctgctgct 480 actccggtca ctaaaatctc cgcggaacag tggggtcgtc cgctcgagca ccaccaccac 540 caccactaa 567 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <400> 4 gactccatgg gttccagcgg taactc 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <400> 5 cgcctcgagc ggacgacccc actgttc 27 <210> 6 <211> 305 <212> PRT <213> Artificial Sequence <400> 6 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Glu Met Val Ser Arg Arg Met Tyr Arg 20 25 30 Val Met Glu Lys Ala Gly Gln Ala Ala Trp Lys Gln Val Val Ser Glu 35 40 45 Ala Thr Gly Pro Gly Pro Gly Gln Ala Ala Ala Glu Ile Leu Glu Pro 50 55 60 Gly Lys Ser Phe Trp Cys Lys Ile Gly Asn Asp Arg Cys Ser Glu Asn 65 70 75 80 Asp His Asp Glu Leu Gly Pro Gly Pro Gly Asp Ile Ser Cys Arg Arg 85 90 95 His Gly Asp Ser Ser Ser Pro Thr Ile Arg Lys Ser Ser Gln Cys Arg 100 105 110 Thr Ala Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Met Leu Ser Lys Asp Ile Ile Lys Leu Leu Asn Glu Gln Val 130 135 140 Asn Lys Glu Met Asn Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp 145 150 155 160 Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp His 165 170 175 Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn 180 185 190 Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His 195 200 205 Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu 210 215 220 Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys 225 230 235 240 Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala Glu 245 250 255 Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile Glu 260 265 270 Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr Val 275 280 285 Lys Gly Ile Ala Lys Ser Arg Lys Ser Leu Glu His His His His His 290 295 300 His 305 <210> 7 <211> 918 <212> DNA <213> Artificial Sequence <400> 7 atgggttcta gccaccacca tcaccaccat tcttctggcc tggttccgcg tggttctcac 60 atggcgtccg aaatggtttc tcgtcgtatg taccgcgtta tggaaaaagc gggtcaggct 120 gcctggaagc aggttgtctc tgaagcaact ggtccaggtc cgggtcaggc tgctgcagaa 180 atcctggaac cgggtaaaag cttttggtgc aagatcggta atgaccgttg ctctgagaat 240 gaccacgacg aactgggccc aggtccaggt gatatctctt gtcgtcgtca cggtgattct 300 tcttctccta ccattcgcaa atcttcccaa tgtcgtactg ctattggtgg tggcggttct 360 ggtggtggtg gttctggtgg tggtggttct atgctgtcta aggacatcat taaactgctg 420 aacgagcagg tgaacaaaga aatgaactct agcaacctgt acatgtctat gtctagctgg 480 tgctataccc actctctgga tggtgccggc ctgtttctgt ttgatcacgc tgcagaggaa 540 tatgaacatg ctaaaaaact gatcatcttc ctgaacgaga acaacgttcc ggttcagctg 600 actagcattt ctgctccaga acacaagttc gagggtctga cgcagatctt ccagaaagcg 660 tatgaacacg aacagcacat ttctgaaagc atcaacaaca ttgttgatca cgcgatcaaa 720 tctaaagacc acgctacctt taactttctg cagtggtacg tcgcagagca gcatgaggaa 780 gaagttctgt tcaaggacat cctggataaa atcgagctga tcggtaacga aaaccacggc 840 ctgtacctgg cggatcagta cgttaaaggt attgctaaat ctcgtaaatc cctcgagcac 900 caccaccacc accactaa 948 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <400> 8 gactccatgg gttctagcca ccacc 25 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <400> 9 cgcctcgagg gatttacgag atttagcaat ac 32 <210> 10 <211> 634 <212> PRT <213> Artificial Sequence <400> 10 Met Gly Met Gln Val Ser Val Glu Thr Thr Gln Gly Leu Gly Arg Arg 1 5 10 15 Val Thr Ile Thr Ile Ala Ala Asp Ser Ile Glu Thr Ala Val Lys Ser 20 25 30 Glu Leu Val Asn Val Ala Lys Lys Val Arg Ile Asp Gly Phe Arg Lys 35 40 45 Gly Lys Val Pro Met Asn Ile Val Ala Gln Arg Tyr Gly Ala Ser Val 50 55 60 Arg Gln Asp Val Leu Gly Asp Leu Met Ser Arg Asn Phe Ile Asp Ala 65 70 75 80 Ile Ile Lys Glu Lys Ile Asn Pro Ala Gly Ala Pro Thr Tyr Val Pro 85 90 95 Gly Glu Tyr Lys Leu Gly Glu Asp Phe Thr Tyr Ser Val Glu Phe Glu 100 105 110 Val Tyr Pro Glu Val Glu Leu Gln Gly Leu Glu Ala Ile Glu Val Glu 115 120 125 Lys Pro Ile Val Glu Val Thr Asp Ala Asp Val Asp Gly Met Leu Asp 130 135 140 Thr Leu Arg Lys Gln Gln Ala Thr Trp Lys Glu Lys Asp Gly Ala Val 145 150 155 160 Glu Ala Glu Asp Arg Val Thr Ile Asp Phe Thr Gly Ser Val Asp Gly 165 170 175 Glu Glu Phe Glu Gly Gly Lys Ala Ser Asp Phe Val Leu Ala Met Gly 180 185 190 Gln Gly Arg Met Ile Pro Gly Phe Glu Asp Gly Ile Lys Gly His Lys 195 200 205 Ala Gly Glu Glu Phe Thr Ile Asp Val Thr Phe Pro Glu Glu Tyr His 210 215 220 Ala Glu Asn Leu Lys Gly Lys Ala Ala Lys Phe Ala Ile Asn Leu Lys 225 230 235 240 Lys Val Glu Glu Arg Glu Leu Pro Glu Leu Thr Ala Glu Phe Ile Lys 245 250 255 Arg Phe Gly Val Glu Asp Gly Ser Val Glu Gly Leu Arg Ala Glu Val 260 265 270 Arg Lys Asn Met Glu Arg Glu Leu Lys Ser Ala Ile Arg Asn Arg Val 275 280 285 Lys Ser Gln Ala Ile Glu Gly Leu Val Lys Ala Asn Asp Ile Asp Val 290 295 300 Pro Ala Ala Leu Ile Asp Ser Glu Ile Asp Val Leu Arg Arg Gln Ala 305 310 315 320 Ala Gln Arg Phe Gly Gly Asn Glu Lys Gln Ala Leu Glu Leu Pro Arg 325 330 335 Glu Leu Phe Glu Glu Gln Ala Lys Arg Arg Val Val Val Gly Leu Leu 340 345 350 Leu Gly Glu Val Ile Arg Thr Asn Glu Leu Lys Ala Asp Glu Glu Arg 355 360 365 Val Lys Gly Leu Ile Glu Glu Met Ala Ser Ala Tyr Glu Asp Pro Lys 370 375 380 Glu Val Ile Glu Phe Tyr Ser Lys Asn Lys Glu Leu Met Asp Asn Met 385 390 395 400 Arg Asn Val Ala Leu Glu Glu Gln Ala Val Glu Ala Val Leu Ala Lys 405 410 415 Ala Lys Val Thr Glu Lys Glu Thr Thr Phe Asn Glu Leu Met Asn Gln 420 425 430 Gln Ala Glu Asn Leu Tyr Phe Gln Ser Ala His Ile Val Met Val Asp 435 440 445 Ala Tyr Lys Pro Thr Lys Ser Ser Gly Asn Ser Asn Ser Ser Ser His 450 455 460 Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys Glu Leu Asn Gly Thr Asp 465 470 475 480 Trp Leu Asn Lys Lys Phe Asp Trp Ala Val Glu Thr Phe Val Ile Phe 485 490 495 Pro Ala Leu Thr His Ile Val Ser Tyr Gly Ala Leu Thr Thr Ser His 500 505 510 Phe Leu Asp Thr Val Gly Leu Ile Thr Val Ser Thr Ala Gly Tyr Leu 515 520 525 His Gly Arg Tyr Val Leu Ser Ser Val Tyr Ala Val Cys Ala Leu Ala 530 535 540 Ala Leu Ile Cys Phe Thr Ile Arg Leu Ala Lys Asn Cys Met Ser Trp 545 550 555 560 Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly 565 570 575 Lys Leu Tyr Arg Trp Arg Ser Pro Val Val Ile Glu Lys Gly Gly Lys 580 585 590 Val Asp Val Glu Gly His Leu Ile Asp Leu Lys Arg Val Val Leu Asp 595 600 605 Gly Ser Ala Ala Thr Pro Val Thr Lys Ile Ser Ala Glu Gln Trp Gly 610 615 620 Arg Pro Leu Glu His His His His His His 625 630 <210> 11 <211> 1905 <212> DNA <213> Artificial Sequence <400> 11 atgggcatgc aagtttcagt tgaaaccact caaggccttg gccgccgtgt aacgattact 60 atcgctgctg acagcatcga gaccgctgtt aaaagcgagc tggtcaacgt tgcgaaaaaa 120 gtacgtattg acggcttccg caagggcaaa gtgccaatga atatcgttgc tcagcgttat 180 ggcgcgtctg tacgccagga cgttctgggt gacctgatga gccgtaactt cattgacgcc 240 atcattaaag aaaaaatcaa tccggctggc gcaccgactt atgttccggg cgaatacaag 300 ctgggtgaag acttcactta ctctgtagag tttgaagttt atccggagagt tgaactgcaa 360 ggtctggaag cgatcgaagt tgaaaaaccg atcgttgaag tgaccgacgc tgacgttgac 420 ggcatgctgg atactctgcg taaacagcag gcgacctgga aagaaaaaga cggcgctgtt 480 540 ggcggtaaag cgtctgattt cgtactggcg atgggccagg gtcgtatgat cccgggcttt 600 gaacggta tcaaaggcca caaagctggc gaagagttca ccatcgacgt gaccttcccg 660 gaagaatacc acgcagaaaa cctgaaaggt aaagcagcga aattcgctat caacctgaag 720 aaagttgaag agcgtgaact gccggaactg accgcagagt tcatcaaacg tttcggcgtt 780 gaagatggtt ccgtagaagg tctgcgcgct gaagtgcgta aaaacatgga gcgcgagctg 840 aagagcgcca tccgtaaccg cgttaagtct caggcgatcg aaggtctggt aaaagctaac 900 gacatcgacg taccggctgc gctgatcgac agcgaaatcg acgttctgcg tcgccaggct 960 gcacagcgtt tcggtggcaa cgaaaaacaa gctctggaac tgccgcgcga actgttcgaa 1020 gaacaggcta aacgccgcgt agttgttggc ctgctgctgg gcgaagttat ccgcaccaac 1080 gagctgaaag ctgacgaaga gcgcgtgaaa ggcctgatcg aagagatggc ttctgcgtac 1140 gaagatccga aagaagttat cgagttctac agcaaaaaca aagaactgat ggacaacatg 1200 cgcaatgttg ctctggaaga acaggctgtt gaagctgtac tggcgaaagc gaaagtgact 1260 gaaaaagaaa ccactttcaa cgagctgatg aaccagcagg cggaaaacct gtacttccaa 1320 tccgctcaca tcgtgatggt ggacgcctac aagcccacca agagcagcgg caacagcaat 1380 agcagcagcc atctgcagct gatttataat ctgaccattt gtgaactgaa cggtaccgat 1440 tggctgaata aaaagtttga ttgggccgta gaaacatttg ttatttttcc ggcactgaca 1500 catattgttt cctatggtgc actgacaacc tcacattttc tggatacagt tggcctgatt 1560 accgtgagca ccgccggtta tctgcatggt cgttatgttc tgtcttcagt ttatgcagtt 1620 tgtgccctgg ccgcgctgat ttgttttacc attcgtctgg caaaaaattg tatgagctgg 1680 cgttatagct gtacccgtta taccaatttt ctgctggata ccaaaggtaa actgtatcgt 1740 tggcgtagcc cggttgttat tgaaaaaggt ggtaaagttg atgttgaagg tcatctgatt 1800 gatctgaaac gtgttgttct ggatggtagc gcagcaaccc cggttaccaa aattagcgca 1860 gaacagtggg gtcgtccgct cgagcaccac caccaccacc actga 1967 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <400> 12 cgatccatgg gcatgcaagt ttcagtt 27 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <400> 13 gtgctcgagc ggacgacccc actgttctgc g 31 <210> 14 <211> 549 <212> DNA <213> Artificial Sequence <400> 14 atgggttcca gcggtaacag caacagcagc tcccatttac agttgattta taacctgacg 60 atatgtgagc tgaatggcac agattggctg aacaaaaaat ttgactgggc agtggagact 120 ttcgttatct ttcctgcgtt gactcatatt gtctcctatg gcgcccttac cactagccat 180 ttccttgaca cggttggcct tatcactgta tctaccgccg gttatcttca cgggcggtat 240 gtgttgagta gcgtctatgc tgtctgtgcc ctggctgcgc tgatctgctt caccattagg 300 ctggcgaaaa attgcatgtc ctggcgttac tcatgcacta ggtataccaa ttttcttctt 360 gataccaagg gcaaactcta ccgctggcgg tcacccgtcg tcatagagaa agggggtaaa 420 gttgatgttg aggggcattt aatcgacctc aagagagttg tgcttgacgg ctccgcggca 480 acccctgtaa ccaagatttc agcggaacaa tggggtcgtc cactcgagca ccaccatcat 540 catcactaa 567

Claims

1. A GP5 protein, characterized in that The GP5 protein consists of the GP5 protein active part, and the sequence of the GP5 protein active part is shown in SEQ ID NO:

1.

2. A GP5 protein, characterized in that The GP5 protein consists of a GP5 protein active portion and a tag protein, wherein the tag protein is connected to the N-terminus and / or C-terminus of the GP5 protein active portion, and the sequence of the GP5 protein active portion is shown in SEQ ID NO:

1.

3. The GP5 protein according to claim 2, characterized in that The tag protein is connected to the C-terminus of the active part of the GP5 protein, and the sequence of the GP5 protein is shown in SEQ ID NO:

2.

4. A gene encoding the GP5 protein according to any one of claims 1 to 3, characterized in that: The coding gene sequence is shown in SEQ ID NO:

3.

5. A vector containing the coding gene according to claim 4, characterized in that: The vector is a plasmid or an engineered bacterium.

6. A method for preparing the GP5 protein according to any one of claims 1 to 3, characterized in that: The method for preparing GP5 protein comprises the following steps: Constructing a recombinant plasmid containing a gene encoding the active part of the GP5 protein; The recombinant plasmid is transferred into competent cells and induced for expression to obtain a GP5 expression strain; The expressed protein is extracted from the GP5 expression strain to obtain the GP5 protein.

7. The method for preparing GP5 protein according to claim 6, characterized in that: The step of constructing a recombinant plasmid containing a gene encoding the active part of the GP5 protein comprises: Using GP5 amplification primers to clone the coding gene of the GP5 protein as a template to obtain a GP5 gene amplification fragment containing a restriction site, wherein the sequence of the GP5 protein is shown in SEQ ID NO: 2; The GP5 gene amplified fragment is connected to the vector plasmid to obtain the recombinant plasmid.

8. The method for preparing GP5 protein according to claim 7, characterized in that: The sequence of the GP5 amplification primer pair is shown in SEQ ID NO: 4-5; and / or, The coding gene sequence of the GP5 protein is shown in SEQ ID NO:

3.

9. The method for preparing GP5 protein according to claim 6, characterized in that: The step of transferring the recombinant plasmid into competent cells and inducing expression to obtain a GP5 expression strain comprises: After the recombinant plasmid is introduced into the competent cells derived from Escherichia coli, isopropylthiogalactoside is used to induce expression to obtain the GP5 expression strain; and / or, The step of extracting the expressed protein from the GP5 expression strain to obtain the GP5 protein comprises: The GP5 expression strain is sequentially crushed, centrifuged and filtered to collect the bacterial solution; The bacterial solution is subjected to column chromatography using a nickel column affinity chromatography method with an eluent containing imidazole; The bacterial solution after the column chromatography is dialyzed and filtered in sequence to obtain the GP5 protein.

10. Use of the GP5 protein according to any one of claims 1 to 3 in the preparation of a preparation for eliciting an immune response of pigs to porcine reproductive and respiratory syndrome virus.

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