A multi-epitope fusion protein containing an intramolecular adjuvant and its application in the preparation of porcine reproductive and respiratory syndrome virus vaccine

Abstract

The present invention relates to the field of biopharmaceutical technology. The present invention provides a PRRSV recombinant multi-epitope fusion protein, and a multi-epitope fusion protein containing an intramolecular adjuvant comprising the PRRSV recombinant multi-epitope fusion protein. The present invention also provides the use of the multi-epitope fusion protein containing an intramolecular adjuvant in the preparation of a porcine reproductive and respiratory syndrome virus vaccine. With GP5 protein as the core of PRRSV, bioinformatics analysis methods are used to assist protein design, screen epitopes, intramolecular adjuvants and tandem methods, and the obtained PRRSV recombinant multi-epitope fusion protein is then introduced with an intramolecular adjuvant to improve the immune level. The neutralizing epitope antibody level is enhanced through reasonable truncation, tandem connection, etc., the ADE effect is reduced, and humoral immunity and cellular immunity are further strengthened.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to a multi-epitope fusion protein containing an intramolecular adjuvant and application thereof in the preparation of a porcine reproductive and respiratory syndrome virus vaccine. Background Art

[0002] Animal husbandry is related to the national economy and people's livelihood. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most common swine pathogen in China's pig herds and a major disease that affects the healthy and sustainable development of the pig industry. Although several vaccines have been used for more than ten years, these vaccines have problems such as rapid virus mutation, low intramuscular cellular / mucosal immunity, and potential ADE to varying degrees, and cannot meet the needs.

[0003] The development of vaccines for PRRSV not only needs to deal with the impact of high mutation and potential ADE effects, but also needs to consider the economic feasibility of industrial applications. Based on the current technology and status quo, subunit vaccines have become the preferred direction of research and development because they do not have biosafety issues, have clear proteins, clear structures, precise dosages, do not waste animal immune potential, and can minimize the ADE effect. These vaccines can induce stronger cellular or humoral immunity in clinical practice, activate Th cell responses and germinal center responses, and form related memory cells, greatly improving the protection efficiency, and providing a good reference for the development of PRRSV vaccines. Therefore, the technical route of subunit vaccine development will surely become one of the effective means to solve the problems of existing PRRSV vaccines. Summary of the invention

[0004] The purpose of the present invention is to provide a multi-epitope fusion protein containing an intramolecular adjuvant and its application in the preparation of porcine reproductive and respiratory syndrome virus vaccine. With PRRSV as the core, GP5 protein is used to assist protein design, screen epitopes, intramolecular adjuvants and series connection methods, and the obtained PRRSV recombinant multi-epitope fusion protein is then introduced with an intramolecular adjuvant to improve the immune level. The neutralizing epitope antibody level is enhanced through reasonable truncation, series connection, etc., the ADE effect is reduced, and the humoral immunity and cellular immunity are further strengthened.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a PRRSV recombinant multi-epitope fusion protein, including PRRSV recombinant multi-epitope fusion protein 1, PRRSV recombinant multi-epitope fusion protein 2, PRRSV recombinant multi-epitope fusion protein 3, PRRSV recombinant multi-epitope fusion protein 4, PRRSV recombinant multi-epitope fusion protein 5 or PRRSV recombinant multi-epitope fusion protein 6.

[0007] Preferably, the amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 1 is shown in SEQ ID NO:1; the amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 2 is shown in SEQ ID NO:2; the amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 3 is shown in SEQ ID NO:3; the amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 4 is shown in SEQ ID NO:4; the amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 5 is shown in SEQ ID NO:5; the amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 6 is shown in SEQ ID NO:6.

[0008] The present invention provides a multi-epitope fusion protein containing an intramolecular adjuvant, comprising the above-mentioned PRRSV recombinant multi-epitope fusion protein and the intramolecular adjuvant.

[0009] Preferably, the intramolecular adjuvant includes any one of a p22T vector carrying Pelb and Tufisn, a p28T vector carrying PT8A and Tufisn, a p28ND vector carrying flagellin CD0 / 1 and flagellin ND0 / 1, a p28fcM vector carrying a mouse Fc fragment and Tufisn, a p28FcP vector carrying a porcine Fc fragment and Tufisn, a p28cTB vector carrying CTB and Tufisn, a p28T4 vector carrying T4-Fd and Tufisn, a p28GCN vector carrying GCN4 and Tufisn, and a p28Fe vector carrying ferritin and Tufisn.

[0010] Preferably, the amino acid sequence of Pelb is as shown in SEQ ID NO:7; the amino acid sequence of Tufisn is as shown in SEQ ID NO:8; the amino acid sequence of PT8A is as shown in SEQ ID NO:9; the amino acid sequence of flagellin CD0 / 1 is as shown in SEQ ID NO:10; the amino acid sequence of flagellin ND0 / 1 is as shown in SEQ ID NO:11; the amino acid sequence of the polymer peptide T4-Fd is as shown in SEQ ID NO:12; the amino acid sequence of the polymer peptide GCN4 is as shown in SEQ ID NO:13; the amino acid sequence of the CTB protein is as shown in SEQ ID NO:14; the amino acid sequence of ferritin is as shown in SEQ ID NO:15; the amino acid sequence of the mouse Fc fragment is as shown in SEQ ID NO:16; the amino acid sequence of the porcine Fc fragment is as shown in SEQ ID NO:17.

[0011] The present invention also provides the use of the multi-epitope fusion protein containing the intramolecular adjuvant in the preparation of porcine reproductive and respiratory syndrome virus vaccine.

[0012] The present invention also provides a porcine reproductive and respiratory syndrome virus subunit vaccine, comprising the multi-epitope fusion protein containing an intramolecular adjuvant and a pharmaceutically acceptable adjuvant.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects:

[0014] In the technical solution of the present invention, with GP5 protein as the core, bioinformatics analysis is used to assist protein design, screen epitopes, intramolecular adjuvants and tandem methods, and the obtained PRRSV recombinant multi-epitope fusion protein is then introduced with intramolecular adjuvants to improve the immune level, and the neutralizing epitope antibody level is enhanced through reasonable truncation, tandem, etc., reducing the ADE effect, and further strengthening humoral immunity and cellular immunity. The immunogenicity of the protein candidate is enhanced, and it has better immunogenicity than traditional subunit vaccines. DETAILED DESCRIPTION

[0015] The present invention provides a PRRSV recombinant multi-epitope fusion protein, including PRRSV recombinant multi-epitope fusion protein 1, PRRSV recombinant multi-epitope fusion protein 2, PRRSV recombinant multi-epitope fusion protein 3, PRRSV recombinant multi-epitope fusion protein 4, PRRSV recombinant multi-epitope fusion protein 5 or PRRSV recombinant multi-epitope fusion protein 6.

[0016] In the present invention, the PRRSV recombinant multi-epitope fusion protein 1 is obtained by combining GP5 fragment+M fragment+GP5 extracellular region+M extracellular region. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 1 is shown in SEQ ID NO: 1, and the specific sequence is NANNSSSHLQLIYNLTICELNGTDWLKKQNFDEAAAKMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQEAAAKDDFCNDSTAAEAAAKAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNEAAAKSNNSSSHIQLIYNLTLCELNGTDWLTQKFDWAEAAAKMGSSIDDFCNDSTAPQ.

[0017] In the present invention, the PRRSV recombinant multi-epitope fusion protein 2 is obtained by combining GP5 fragment+GP5 extracellular region+M fragment+M extracellular region. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 2 is shown in SEQ ID NO: 2, and the specific sequence is NANNSSSHLQLIYNLTICELNGTDWLKKQNFDEAAAKMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQEAAAKSNNSSSHIQLIYNLTLCELNGTDWLTQKFDWAEAAAKDDFCNDSTAAEAAAKAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNEAAAKMGSSIDDFCNDSTAPQ.

[0018] In the present invention, the PRRSV recombinant multi-epitope fusion protein 3 is obtained by combining GP2a fragment + GP3 fragment + GP4 fragment + GP2a extracellular region + GP3 extracellular region + GP4 extracellular region. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 3 is shown in SEQ ID NO: 3, and the specific sequence is.

[0019] In the present invention, the PRRSV recombinant multi-epitope fusion protein 4 is obtained by combining GP2a fragment + GP3 fragment + GP4 fragment + GP5 fragment + M fragment. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 4 is shown in SEQ ID NO: 4, and the specific sequence is AYLSQCRVDIPSWGTKHPLGMFWHHKVSTLIDEMVSRRMYHTMERAGQAAWKQVVSEATLSRISGLDVVAHFEAAAKAWLAFLSFTYTTQFHPEIFGIGNVSQVYVDIKNQLICAKHDGQNTEAAAKGMATRPLRRFAKVLSAAEAAAKISCLRHGDSSSPTIRKSSEAAAKNANNSSSHLQLIYNLTICELNGTDWLKKQNFDEAAAKMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQ.

[0020] In the present invention, the PRRSV recombinant multi-epitope fusion protein 5 is obtained by combining the GP2a extracellular region + the GP3 extracellular region + the GP4 extracellular region + the GP5 extracellular region + the M extracellular region. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 5 is shown in SEQ ID NO: 5, and the specific sequence is SQSPDGWWYFASDWFAPRYSVRALPFTLSNYRRSYEAYLSQCRVDIPSWGTKHPLGMFWHHKVSTLIDEMVSRRMYHTMERAGQAAWKQVVSEATLSRISGLDVVAHFQHLAAIEAETCKYLASRLPMLHNLRMAGSNVTIEYNSTLGRVFAIFPTPGSRPKLHDFQQGGGSGGGSGSNNTFCFWFPLVRGNFSFELTVNYTVCPPCLTRQAAEEIYEPGRSLWCRIGNDRCEETDHDELESKVPPG LSSEGHLTSVYAWLAFLSFTYTTQFHPEIFGIGNVSQVYVDIKNQLICAKHDGQNTTLSRHGNITAVYQTYYQHQVDGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPVSRVSVRVFQTLRPTPPAHQVPL SSKTSAALGMATRPLRRFARSLNVARREAAAKAAAGFAVLQDISCLRHRDSASEAIRKIPQCRTAIGTPVYVEAAAKSNNSSSHIQLIYNLTLCELNGTDWLTQKFDWAEAAAKMGSSIDDFCNDSTAPQ.

[0021] In the present invention, the PRRSV recombinant multi-epitope fusion protein 6 is obtained by combining GP5 fragment + TT + M fragment + Q11 + GP5 extracellular region + MDP + M extracellular region + PT8A. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 6 is shown in SEQ ID NO: 6, and the specific sequence is ANNSSSHLQLIYNLTICELNGTDWLKKQNFDSGSGMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQQYIKANSKFIGITESGSGDDFCNDSTAASGSGAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNQQKFQFQFEQQSGSGSNNSSSHIQLIYNLTLCELNGTDWLTQKFDWADEHGTAVMLKSGSGMGSSIDDFCNDSTAPQHIDSQKKA.

[0022] In the present invention, the GP5 fragment is screened from the sequence of GENBANK ID: AXF36025.1. The amino acid sequence of the GP5 fragment is shown in SEQ ID NO: 7, and the specific sequence is NANNSSSHLQLIYNLTICELNGTDWLKKQNFDEAAAKMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQ.

[0023] In the present invention, the M fragment is screened from the sequence of GENBANK ID: AXF36026.1. The amino acid sequence of the M fragment is shown in SEQ ID NO: 8, and the specific sequence is DDFCNDSTAAEAAAKAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRA VKRGVVN.

[0024] In the present invention, the GP5 extracellular region is screened from the sequence of GENBANK ID: AOW71948.1. The amino acid sequence of the GP5 extracellular region is shown in SEQ ID NO: 9, and the specific sequence is SNNSSSHIQLIYNLTLCELNGTDWLTQKFDWA.

[0025] In the present invention, the M extracellular region is screened from the sequence of GENBANK ID: AOW71949.1. The amino acid sequence of the M extracellular region is shown in SEQ ID NO: 10, and the specific sequence is MGSSIDDFCNDSTAPQ.

[0026] In the present invention, the GP2a fragment is screened from the sequence of GENBANK ID: AXF36020.1. The amino acid sequence of the GP2a fragment is shown in SEQ ID NO: 11, and the specific sequence is AYLSQCRVDIPSWGTKHPLGMFWHHKVSTLIDEMVSRRMYHTMERAGQAAWKQVVSEATLSRISG LDVVAHF.

[0027] In the present invention, the GP3 fragment is screened from the sequence of GENBANK ID: AXF36022.1. The amino acid sequence of the GP3 fragment is shown in SEQ ID NO: 12, and the specific sequence is AWLAFLSFTYTTQFHPEIFGIGNVSQVYVDIKNQLICAKHDGQNTEAAAKGMATRPLRRFAKVLSA A.

[0028] In the present invention, the GP4 fragment is screened from the sequence of GENBANK ID: AXF36023.1. The amino acid sequence of the GP4 fragment is shown in SEQ ID NO: 13, and the specific sequence is ISCLRHGDSSSPTIRKSS.

[0029] In the present invention, the GP2a extracellular region is screened from the sequence of GENBANK ID: AAG49621.1. The amino acid sequence of the GP2a extracellular region is shown in SEQ ID NO: 14, and the specific sequence is SQSPDGWWYFASDWFAPRYSVRALPFTLSNYRRSYEAYLSQCRVDIPSWGTKHPLGMFWHHKVST LIDEMVSRRMYHTMERAGQAAWKQVVSEATLSRISGLDVVAHFQHLAAIEAETCKYLASRLPMLH NLRMAGSNVTIEYNSTLGRVFAIFPTPGSRPKLHDFQQ.

[0030] In the present invention, the GP3 extracellular region is screened from the sequence of GENBANK ID: AAG49622.1. The amino acid sequence of the GP3 extracellular region is shown in SEQ ID NO: 15, and the specific sequence is GSNNTFCFWFPLVRGNFSFELTVNYTVCPPCLTRQAAEEIYEPGRSLWCRIGNDRCEETDHDELESKVPPGLSSEGHLTSVYAWLAFLSFTYTTQFHPEIFGIGNVSQVYVDIKNQLICAKHDGQNTTLSRHGNITAVYQTYYQHQVDGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPVSRVSVRVFQTLRPTPPAHQVPLSSKTSAALGMATRPLRRFARSLNVARR.

[0031] In the present invention, the GP4 extracellular region is screened from the sequence of GENBANK ID: AAG49623.1. The amino acid sequence of the GP4 extracellular region is shown in SEQ ID NO: 16, and the specific sequence is AAAGFAVLQDISCLRHRDSASEAIRKIPQCRTAIGTPVYV.

[0032] In the present invention, the amino acid sequence of TT is shown in SEQ ID NO: 17, and the specific sequence is QYIKANSKFIGITE.

[0033] In the present invention, the amino acid sequence of Q11 is shown in SEQ ID NO: 18, and the specific sequence is QQKFQFQFEQQ.

[0034] In the present invention, the amino acid sequence of the MDP is shown in SEQ ID NO: 19, and the specific sequence is DEHGTAVMLK.

[0035] In the present invention, the amino acid sequence of PT8A is shown in SEQ ID NO: 20, and the specific sequence is HIDSQKKA.

[0036] The present invention also provides a multi-epitope fusion protein containing an intramolecular adjuvant, comprising the above-mentioned PRRSV recombinant multi-epitope fusion protein and the intramolecular adjuvant.

[0037] In the present invention, the internal adjuvant includes any one of the vectors p22T, p28T, p28ND, p28FcM, p28FcP, p28cTB, p28T4, p28GCN and p28Fe.

[0038] In the present invention, the vector p22T is obtained by synthesizing Pelb and Tufisn sequences onto the PET28a vector, the amino acid sequence of Pelb is shown in SEQ ID NO: 21, and the specific sequence is MKYLLPTAAAGLLLLAAQPA; the amino acid sequence of Tufisn is shown in SEQ ID NO: 22, and the specific sequence is TKPR.

[0039] In the present invention, the vector p28T is obtained by synthesizing PT8A and Tufisn sequences onto the PET28a vector, the amino acid sequence of PT8A is shown in SEQ ID NO: 20, and the specific sequence is HIDSQKKA.

[0040] In the present invention, the vector p28ND is obtained by synthesizing flagellin CD0 / 1 and flagellin ND0 / 1 sequences onto a PET28a vector, the amino acid sequence of the flagellin CD0 / 1 is shown in SEQ ID NO: 23, and the specific sequence is MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNA NDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSN; the amino acid sequence of the flagellin ND0 / 1 is shown in SEQ ID NO: 24, and the specific sequence is KSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQ ILQQAGTSVLAQANQVPQNVLSLL.

[0041] In the present invention, the vector p28T4 is obtained by synthesizing T4-Fd and Tufisn sequences onto a PET28a vector, and the amino acid sequence of the polymer peptide T4-Fd is shown in SEQ ID NO: 25, and the specific sequence is MQALQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLSPA.

[0042] In the present invention, the vector p28GCN is obtained by synthesizing GCN4 and Tufisn sequences onto a PET28a vector, and the amino acid sequence of the polymer peptide GCN4 is shown in SEQ ID NO: 26, and the specific sequence is MKQIEDKIEEILSKIYHIENEIARIKKLIGEV.

[0043] In the present invention, the vector p28cTB is obtained by synthesizing CTB and Tufisn sequences onto the PET28a vector, and the amino acid sequence of the CTB protein is shown in SEQ ID NO: 27, and the specific sequence is MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATF QVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN.

[0044] In the present invention, the vector p28Fe vector is obtained by synthesizing ferritin and Tufisn sequences onto a PET28a vector, and the amino acid sequence of the ferritin is shown in SEQ ID NO: 28, and the specific sequence is MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENN VPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLF KDILDKIELIGNENHGLYLADQYVKGIAKSRKS.

[0045] In the present invention, the vector p28fcM is obtained by synthesizing a mouse Fc fragment and a Tufisn sequence onto a PET28a vector, the amino acid sequence of the mouse Fc fragment is shown in SEQ ID NO: 29, and the specific sequence is VRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0046] In the present invention, the vector p28fcP is obtained by synthesizing a porcine Fc fragment and a Tufisn sequence onto a PET28a vector. The amino acid sequence of the porcine Fc fragment is shown in SEQ ID NO: 30, and the specific sequence is MWQLLSPTALLLLVSVPGTHAEDPPKSVVILDPPWDRLLEKDSVTLKCQGAYPPRDDSTEWRWNGTLISNKASSYSITDATVGNSGEYTCKTGLSAQSDPLRLEVYKGWLLLQAPRWVVQEGESIRLRCHTWKNITIQKVQYFQNGMGKKFSHQNFEYHIPNATLKDGGSYFCRGIIKNYDLSSEPVKVTVQGSKSPSPILSFFLPWHQIIFCLVMGFLFAVDTGLYFSVRKVLRSSKEDWRNGKVTWSRDPADKGG.

[0047] The present invention also provides the use of the multi-epitope fusion protein containing the intramolecular adjuvant in the preparation of porcine reproductive and respiratory syndrome virus vaccine.

[0048] The present invention also provides a porcine reproductive and respiratory syndrome virus subunit vaccine, comprising the multi-epitope fusion protein containing an intramolecular adjuvant and a pharmaceutically acceptable adjuvant.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] (1) Identification of candidate vaccine strains

[0052] The amino acid sequences of the required GP2, GP3, GP4, GP5, and M proteins were collected from the NCBI database in the fasta file format and used as templates. The sequence conservation was analyzed using MEGA7.0 software, and the resulting comparative data set was then visualized and analyzed for conservation using the WebLogo server.

[0053] (2) Immunoinformatics design

[0054] Epitope prediction and screening: ABCpred server was used to predict candidate linear B lymphocyte epitopes of proteins. MHC-II epitopes were predicted using NetMHCIIpan-4.0 server. The potential for epitope combination with MHC-II was evaluated by EpiTOP server. Prediction was performed by IFN epitope server and IL4pred server. MHC-I was combined with NetMHCpan4.1 server and IEDB server for CTL epitope prediction. All predicted epitopes were determined by IEDB server. Non-toxic immunogenicity test was performed by ToxinPred server.

[0055] Identification of protective epitopes and assessment of antigenic epitope conservation: Dominant epitopes were assessed by the conservation analysis tool on the IEDB server.

[0056] Concatenation of candidate epitopes: Sequences collected from literature and predicted sequences are concatenated with candidate vaccines using flexible linkers to extend the duration of protein action in vivo, effectively avoiding the introduction of new binding epitopes. GGPPG is used for CTL epitopes, AAY is used for HTL epitopes, and KK is used for B lymphocyte epitopes. In order to improve the immunogenicity of candidate vaccines, intramolecular adjuvants are added and connected to the N-terminus of the construct through an EAAAK linker, and a Flynn-sensitive linker (RVRR) is used for cross-protein connection.

[0057] By adjusting the number of repeated copies and the construction position, the neutralizing epitopes selected from known literature were designed to enhance antigenicity.

[0058] Construction of candidate vaccines and prediction of antigenicity, sensitization, solubility and physicochemical properties: The final antigenicity analysis of the candidate vaccine was performed using the ANTIGENpro server and the VaxiJen v2.0 server; the AllerTOPv2.0 server was used for the final allergenicity analysis of the candidate vaccine; and the SOLpro server was used for the final solubility analysis of the candidate vaccine. The physicochemical properties of the candidate vaccine were analyzed using the ExPASy server, and several candidate combinations were selected.

[0059] Secondary and tertiary structure modeling, refinement and disulfide engineering of candidate vaccines: The secondary structure prediction of the final candidate vaccine was completed using PSIPRED4.0 server and SOPMA server. The tertiary structure prediction of the final candidate vaccine was completed using trRosetta server. The structure was optimized using Galaxy-WEB server. Ramachandran plots were generated by EBI-PDBSum server to evaluate the quality of the model before and after optimization. Disulfide engineering of the candidate vaccine was performed using DisulfidebyDesign32.2 server. Global detection of the model was performed using ProSA-Web server.

[0060] Tertiary epitope prediction and protein docking analysis: The tertiary structure of the vaccine protein was predicted by ElliPro server. The interaction between TLR3 as receptor and protein vaccine as ligand was predicted using PyDockWEB server. LigPLot+ was used to determine the amino acid residues with hydrogen bonds and hydrophobic interactions after docking.

[0061] Example 2

[0062] (I) Synthesis, expression and purification of recombinant multi-epitope fusion proteins

[0063] Based on the above screening, the dominant sequence was obtained, and then PRRSV recombinant multi-epitope fusion protein 1, PRRSV recombinant multi-epitope fusion protein 2, PRRSV recombinant multi-epitope fusion protein 3, PRRSV recombinant multi-epitope fusion protein 4, PRRSV recombinant multi-epitope fusion protein 5 and PRRSV recombinant multi-epitope fusion protein 6 were designed.

[0064] The PRRSV recombinant multi-epitope fusion protein 1 is composed of GP5 fragment+M fragment+GP5 extracellular region+M extracellular region. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 1 is shown in SEQ ID NO: 1, and the specific sequence is NANNSSSHLQLIYNLTICELNGTDWLKKQNFDEAAAKMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQEAAAKDDFCNDSTAAEAAAKAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNEAAAKSNNSSSHIQLIYNLTLCELNGTDWLTQKFDWAEAAAKMGSSIDDFCNDSTAPQ.

[0065] The PRRSV recombinant multi-epitope fusion protein 2 is composed of GP5 fragment+GP5 extracellular region+M fragment+M extracellular region. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 2 is shown in SEQ ID NO: 2, and the specific sequence is NANNSSSHLQLIYNLTICELNGTDWLKKQNFDEAAAKMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQEAAAKSNNSSSHIQLIYNLTLCELNGTDWLTQKFDWAEAAAKDDFCNDSTAAEAAAKAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNEAAAKMGSSIDDFCNDSTAPQ.

[0066] The composition of the PRRSV recombinant multi-epitope fusion protein 3 is GP2a fragment+GP3 fragment+GP4 fragment+GP2a extracellular region+GP3 extracellular region+GP4 extracellular region. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 3 is shown in SEQ ID NO: 3, and the specific sequence is.

[0067] The PRRSV recombinant multi-epitope fusion protein 4 is composed of GP2a fragment+GP3 fragment+GP4 fragment+GP5 fragment+M fragment. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 4 is shown in SEQ ID NO:4, and the specific sequence is AYLSQCRVDIPSWGTKHPLGMFWHHKVSTLIDEMVSRRMYHTMERAGQAAWKQVVSEATLSRISGLDVVAHFEAAAKAWLAFLSFTYTTQFHPEIFGIGNVSQVYVDIKNQLICAKHDGQNTEAAAKGMATRPLRRFAKVLSAAEAAAKISCLRHGDSSSPTIRKSSEAAAKNANNSSSHLQLIYNLTICELNGTDWLKKQNFDEAAAKMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQ.

[0068] The composition of the PRRSV recombinant multi-epitope fusion protein 5 is GP2a extracellular region + GP3 extracellular region + GP4 extracellular region + GP5 extracellular region + M extracellular region. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 5 is shown in SEQ ID NO: 5, and the specific sequence is SQSPDGWWYFASDWFAPRYSVRALPFTLSNYRRSYEAYLSQCRVDIPSWGTKHPLGMFWHHKVSTLIDEMVSRRMYHTMERAGQAAWKQVVSEATLSRISGLDVVAHFQHLAAIEAETCKYLASRLPMLHNLRMAGSNVTIEYNSTLGRVFAIFPTPGSRPKLHDFQQGGGSGGGSGSNNTFCFWFPLVRGNFSFELTVNYTVCPPCLTRQAAEEIYEPGRSLWCRIGNDRCEETDHDELESKVPPGL SSEGHLTSVYAWLAFLSFTYTTQFHPEIFGIGNVSQVYVDIKNQLICAKHDGQNTTLSRHGNITAVYQTYYQHQVDGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPVSRVSVRVFQTLRPTPPAHQVPLS SKTSAALGMATRPLRRFARSLNVARREAAAKAAAGFAVLQDISCLRHRDSASEAIRKIPQCRTAIGTPVYVEAAAKSNNSSSHIQLIYNLTLCELNGTDWLTQKFDWAEAAAKMGSSIDDFCNDSTAPQ.

[0069] The composition of the PRRSV recombinant multi-epitope fusion protein 6 is GP5 fragment+TT+M fragment+Q11+GP5 extracellular region+MDP+M extracellular region+PT8A. The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 6 is shown in SEQ ID NO:6, and the specific sequence is NANNSSSHLQLIYNLTICELNGTDWLKKQNFDSGSGMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQQYIKANSKFIGITESGSGDDFCNDSTAASGSGAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNQQKFQFQFEQQSGSGSNNSSSHIQLIYNLTLCELNGTDWLTQKFDWADEHGTAVMLKSGSGMGSSIDDFCNDSTAPQHIDSQKKA.

[0070] The above designed sequence forms were sent to BGI for synthesis, and BGI connected the synthesized sequence to pET28a to prepare the corresponding recombinant plasmids (dry powder). The recombinant plasmid was centrifuged at 5000rpm / min for 5min, then taken into the clean bench and dissolved by adding 40μL of double distilled water. Take the competent cells of Escherichia coli BL21 (DE3) in the -80℃ refrigerator and place them in ice for 10min, add 3μL of recombinant plasmid to the competent cells, place them in ice for 30min, heat shock them in a 42℃ water bath for 90s, and immediately place them in ice for 2min. Then add 750μL of LB liquid medium pre-warmed at 37℃ to the EP tube and shake and culture at 37℃ for 1h. 5000rpm / min, centrifuge for 5min, discard 800μL of supernatant, resuspend the remaining bacteria and apply them to the plate containing kanamycin, and invert the plate in a 37℃ incubator for overnight culture.

[0071] The next day, monoclonal colonies were picked and added to 5 mL of LB liquid medium containing kanamycin to verify their expression. Different gradients of induction temperature (16°C, 28°C, 37°C), different gradients of induction time (4h, 6h, 8h, 10h), and different gradients of inducer addition (0.2mmol / L, 0.4mmol / L, 0.6mmol / L, 0.8mmol / L, 1mmol / L) were set to explore the conditions for inducing the expression of recombinant proteins.

[0072] The results showed that the optimal induction temperature was 37℃, the optimal induction time was 6h, and the optimal inducer concentration was 0.4mmol / L.

[0073] The verified strain was fermented in a shake flask with 2.4 L of bacterial liquid, and the bacteria were harvested: centrifuged at 10,000 r / min for 10 min, the precipitate after centrifugation was broken with high-salt lysis buffer 1 (composed of NaCl, Tris-HCL, EDTA, glycerol and NP40) (3s, 3s, 30min), and then centrifuged at 10,000 r / min for 10 min, the supernatant was discarded, and the precipitate was broken with ordinary lysis buffer 2 (composed of Tris-HCL and Triton-X 100) (3s, 3s, 30min), and then centrifuged at 10,000 r / min for 10 min. The precipitate was suspended with 2 mL of 8 M urea and then dropped into 40 mL of 8 M urea for sedimentation for 30 min to allow the protein to better integrate into 8 M urea and then broken (3s, 3s, 30min), and then centrifuged at 10,000 r / min for 10 min. The precipitate was collected and washed with inclusion body washing solution to obtain inclusion body protein solution, and then the inclusion body protein solution was centrifuged at 10000r / min for 10min, and the supernatant protein was filtered with a 0.22μm filter membrane to remove significant precipitate and prevent clogging of the column filter membrane; the filtered supernatant was purified with Ni-NTA resin at 4°C, and after combination, gradient elution was performed with eluent containing different concentrations of imidazole (100, 300, 500mmol / L), and the optimal elution concentration was 300mmol / L. The purified protein after elution was collected and stored at -20°C.

[0074] (II) Identification and stability study of recombinant multi-epitope fusion proteins

[0075] The purified protein was extracted with TriitonX-114 to remove endotoxin. The initial protein endotoxin content was higher than 100EU / mL, and was less than 10EU / mL after two extractions.

[0076] Western-blot method was used to verify whether it was a recombinant protein, and Dot-ELISA method was used to identify the immunogenicity of the protein by reacting the antigen with PRRSV positive serum. The results showed that both had positive reactions.

[0077] Long-term stability experiments were conducted to investigate the storage stability of recombinant antigens at different temperatures (-80°C, -20°C, 4°C or 37°C for accelerated destruction). The results confirmed that the protein was most stable at -80°C, followed by -20°C, and could only be stably stored for two weeks at 4°C.

[0078] (III) Dialysis refolding of recombinant multi-epitope fusion protein

[0079] Cut the dialysis bag into a suitable size, put it into a beaker, add appropriate amount of pure water, boil it in a boiling water bath for 10 minutes, and take it out to cool.

[0080] Take 50mL of the above-filtered inclusion body protein solution and add refolding solution (20mmol / L Tris-HCl, 500mmol / L NaCl, 45mL / L glycerol, 1g / L arginine) and dithiothioethanol respectively, and put it into the pretreated dialysis bag for refolding. The volume of the refolding solution added is 10% of the volume of the inclusion body protein solution, and the volume of dithiothiothioethanol added is 0.5% of the volume of the inclusion body protein solution. After confirming that there is no leakage in the seal, place it at 4°C for refolding procedure, and stir the dialyzate with a magnetic stirrer. The refolding procedure is:

[0081] 1) Dialyze with 10 times the volume of external solution A for 1 hour (the external solution A is 8M urea diluted 100 times with 2 times the volume of dialysate), and then add 3 times the volume of external solution water for 1 hour;

[0082] 2) Change the volume of external solution B by 100 times and dialyze for 3 hours (the external solution B is the refolding solution diluted 1000 times);

[0083] 3) Change the volume of external solution C by 100 times and dialyze for 4 hours (the external solution C is 10 mM pH 9.5 CB, and the CB solution is obtained by diluting 0.318 g of sodium carbonate and 0.586 g of sodium bicarbonate to 100 ml).

[0084] The renatured protein was centrifuged at 5000 r / min for 10 min, and the supernatant was retained. Then, the protein before and after renaturation was subjected to 12% SDS-PAGE electrophoresis to determine its renaturation status.

[0085] The results showed that the target band existed after renaturation, and its size was consistent with that before renaturation, and no other impurity bands were generated.

[0086] Example 3

[0087] 1. Vaccine preparation and mouse immunization

[0088] The renatured protein was quantitatively set to 200ug / mL and mixed with 201 adjuvant at a ratio of 1:1. After mixing, a homogenizer was used to emulsify for 5 minutes. After centrifugation, no stratification occurred, and the vaccine preparation was obtained. Balb / c mice (purchased from Henan Sikebeisi Biotechnology Co., Ltd.) were selected for immunization. The mice were selected to be over two months old. The mice were immunized subcutaneously. Three points were selected for immunization, including subcutaneous injection in the neck and subcutaneous injection in the hind legs. Each mouse received a total of 200μL, including 100μL in the neck and 50μL in the inner side of the hind legs. The immunization was performed three times, with an interval of 14 days each time. Blood was collected from the tail vein one week after each immunization, and blood was collected from the orbits two weeks after the three immunizations. The corresponding immunization times when the titer was 1:100,000 were the common immunization endpoints. If the titer did not meet the requirements, enhanced immunization was performed. Blood was collected from the tail vein one week after immunization. The operation was the same as the previous three times. Enhanced immunization was performed until the titer was reached, and a maximum of 5 immunizations were performed.

[0089] (II) Humoral Immunity Testing

[0090] The blood of the immunized mice was collected, and the serum obtained was separated and tested for specific antibody IgM and IgG indicators according to the ELISA method. The results are shown in Table 1.

[0091] Table 1 The titers of different PRRSV recombinant multi-epitope fusion proteins in immunoassay

[0092]

[0093]

[0094] The results showed that the highest relative titer of the PRRSV recombinant multi-epitope fusion protein immunoassay consisting of GP5 fragment + M fragment + GP5 extracellular region + M extracellular region and GP5 fragment + GP5 extracellular region + M fragment + M extracellular region was 256,000.

[0095] (III) Evaluation of cellular immune effects

[0096] The mice were killed by cervical dislocation after blood was drawn from the eyeballs, and spleen tissues were obtained by dissection. Splenic single cell suspensions were prepared and cell stimulation and culture (1×10 6 cells / mL), cell surface staining, fixation, membrane permeabilization, cell activation stimulation, extracellular factor staining after blocking the antibody Fc-terminal receptor, fixation, membrane permeabilization and washing, resuspended and then flow cytometry detection, detection of CD3, CD4, CD8 indicators.

[0097] The results showed that CD3, CD4 and CD8 all increased.

[0098] (IV) Safety evaluation

[0099] The mice were observed after immunization, and their appearance, behavior, spirit, and food intake were observed every day during the immunization period. No abnormalities were found.

[0100] Three days after the last immunization, a histopathological examination was performed: the mice were killed by cervical dislocation, and the lungs, hearts, spleens, kidneys, etc. were taken out on a sterile operating table. No pathological changes such as edema and congestion were observed with the naked eye.

[0101] Then, the above tissues were rinsed twice with PBS and fixed in 10% formalin solution. The fixed tissues were paraffin-embedded for 24 hours, cut into 4 μm slices under a sliding microtome, and dehydrated step by step with different concentrations of ethanol. The obtained slices were stained with hematoxylin-eosin (HE), and the inflammatory cell infiltration of the tissues was observed under an optical microscope. The results showed that no inflammatory cell infiltration was observed in any tissue.

[0102] (V) In vitro neutralization test evaluation

[0103] The serum was placed in a 56°C water bath and inactivated for 30 min; the in vitro cell virus neutralization test was evaluated according to the following operating steps.

[0104] 1. Centrifuge the mouse serum 42 days after the initial immunization at 12000rpm / min for 2min, take 200uL of each sample, incubate in 56℃ water or metal bath for 30min, and dilute the serum with DMEM with double antibody at 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256. Prepare 250uL for each dilution.

[0105] 2. Dilute the virus to 200 TCID50 using DMEM with double antibodies. For example, the virus titer is 10 -5 TCID50 / mL, after diluting 500 times, take 250 μL and add it to the diluted serum.

[0106] 3. Vortex the diluted virus solution and treated serum evenly (250 μL diluted serum + 250 μL diluted virus) and place in a 37°C incubator for 1.5 hours.

[0107] 4. Remove the culture medium from the 96-well plate with Marc145 cells, wash once with 1×PBS, add 100 μL of serum-virus mixture to each well, and culture in a 37°C, 5% CO2 incubator for 1.5 hours. Then discard the culture medium, wash once with 1×PBS, add 100uL of DMEM containing 2% FBS+double antibody to each well, continue to culture for 4-6 days, observe the cytopathic effect, record the results, and calculate the neutralization index according to the Reed-Muench method.

[0108] Note: After inoculating cells, 0.1 TCID50 should not cause any cell pathology, while 100 TCID50 must cause cell pathology; blank control cells should always maintain good morphology and characteristics.

[0109] Table 2 Neutralization titers of different recombinant multi-epitope fusion proteins in cell experiments

[0110]

[0111] The results are shown in Table 3. It can be seen that the best is the recombinant multi-epitope fusion protein 2 composed of GP5 fragment + GP5 extracellular region + M fragment + M extracellular region, followed by the recombinant multi-epitope fusion protein 1 composed of GP5 fragment + M fragment + GP5 extracellular region + M extracellular region.

[0112] Example 4

[0113] The sequences of the dominant components screened out in Example 1 were sent to BGI, which synthesized the sequences of the dominant components into PET28a vectors, and the corresponding new vectors obtained were used as intramolecular adjuvants.

[0114] Table 3 Intramolecular adjuvant

[0115]

[0116] Table 4 Sequences of the main dominant components in intramolecular adjuvants

[0117]

[0118]

[0119]

[0120] The optimal recombinant multi-epitope fusion protein sequence screened above was synthesized by subcloning using two restriction sites, BamHI and HindIII, on each of the above-mentioned intramolecular adjuvants, to obtain a multi-epitope fusion protein containing an intramolecular adjuvant. The same vaccine preparation and mouse immunization were then performed, and the in vitro cell virus neutralization experiment was evaluated.

[0121] Table 5 Neutralization titer of multi-epitope fusion proteins containing different intramolecular adjuvants in cell experiments

[0122] Multi-epitope fusion protein containing the following built-in adjuvants Potency Cell assay neutralization titer p2T 128000 1：8 p28 128000 1：8 p28ND 128000 1：8 p28FcM 128000 1：10 p28FcP 128000 1：8 p28cB 128000 1：8 p28T4 256000 1：12 p28GCN 256000 1：12 p28Fe 256000 1：16

[0123] The results showed that when the internal adjuvants were p28T4, p28GCN and p28Fe, the neutralization titer in the cell experiment was relatively the highest, which means that the multi-epitope fusion proteins containing the three internal adjuvants p28T4, p28GCN and p28Fe respectively had the best immunogenicity, and the multi-epitope fusion protein containing the internal adjuvant p28Fe had the best immunogenicity.

[0124] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A PRRSV recombinant multi-epitope fusion protein, characterized in that: Including PRRSV recombinant multi-epitope fusion protein 1, PRRSV recombinant multi-epitope fusion protein 2, PRRSV recombinant multi-epitope fusion protein 3, PRRSV recombinant multi-epitope fusion protein 4, PRRSV recombinant multi-epitope fusion protein 5 or PRRSV recombinant multi-epitope fusion protein 6; The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 1 is shown in SEQ ID NO: 1; The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 2 is shown in SEQ ID NO: 2; The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 3 is shown in SEQ ID NO: 3; The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 4 is shown in SEQ ID NO: 4; The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 5 is shown in SEQ ID NO: 5; The amino acid sequence of the PRRSV recombinant multi-epitope fusion protein 6 is shown in SEQ ID NO:

6.

2. A multi-epitope fusion protein containing an intramolecular adjuvant, characterized in that: It comprises the PRRSV recombinant multi-epitope fusion protein as claimed in claim 1 and an intramolecular adjuvant.

3. The multi-epitope fusion protein containing an internal adjuvant according to claim 2, characterized in that: The intramolecular adjuvant includes any one of a p22T vector carrying Pelb and Tufisn, a p28T vector carrying PT8A and Tufisn, a p28ND vector carrying flagellin CD0 / 1 and flagellin ND0 / 1, a p28fcM vector carrying a mouse Fc fragment and Tufisn, a p28FcP vector carrying a porcine Fc fragment and Tufisn, a p28cTB vector carrying CTB and Tufisn, a p28T4 vector carrying T4-Fd and Tufisn, a p28GCN vector carrying GCN4 and Tufisn, and a p28Fe vector carrying ferritin and Tufisn.

4. The multi-epitope fusion protein containing an internal adjuvant according to claim 3, characterized in that: The amino acid sequence of the Pelb is shown in SEQ ID NO:7; the amino acid sequence of the Tufisn is shown in SEQ ID NO:8; the amino acid sequence of the PT8A is shown in SEQ ID NO:9; the amino acid sequence of the flagellin CD0 / 1 is shown in SEQ ID NO:10; the amino acid sequence of the flagellin ND0 / 1 is shown in SEQ ID NO:11; the amino acid sequence of the polymer peptide T4-Fd is shown in SEQ ID NO:12; the amino acid sequence of the polymer peptide GCN4 is shown in SEQ ID NO:13; the amino acid sequence of the CTB protein is shown in SEQ ID NO:14; the amino acid sequence of the ferritin is shown in SEQ ID NO:15; the amino acid sequence of the mouse Fc fragment is shown in SEQ ID NO:16; the amino acid sequence of the porcine Fc fragment is shown in SEQ ID NO:

17.

5. Use of the multi-epitope fusion protein containing an intramolecular adjuvant according to claim 2 in the preparation of a porcine reproductive and respiratory syndrome virus vaccine.

6. A porcine reproductive and respiratory syndrome virus subunit vaccine, characterized in that: It comprises the multi-epitope fusion protein containing an intramolecular adjuvant as claimed in claim 2 and a pharmaceutically acceptable adjuvant.

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