Engineering and application of proteins based on heterotrimeric motifs

Abstract

The application belongs to the technical field of biological medicine, and discloses modification and application of a protein based on a heterologous trimer motif, wherein the trimer motif protein comprises a set of positive and negative salt bridge sequences, and the amino acid sequences of the positive and negative salt bridges are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively. When the positive and negative salt bridge sequences exist in RSV fusion protein F, the thermal stability and storage stability of the RSV fusion protein F can be significantly improved. The scheme disclosed in the application can maintain the stability and broad spectrum of the important immune antigen pre F protein of respiratory syncytial virus, and simultaneously express two subtypes of antigens, thereby reducing the production process of the bivalent RSV vaccine and reducing the production cost.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the modification and application of proteins based on heterotrimeric motifs. Background Technology

[0002] Respiratory syncytial virus (RSV) is a major pathogen causing serious lung and respiratory diseases. In children, the vast majority experience multiple RSV infections before the age of two. Meanwhile, immunocompromised adults and the elderly are also susceptible to RSV.

[0003] RSV fusion protein F belongs to class I fusion proteins and plays a crucial role in viral infection. It mediates viral entry into cells through a metastable pre-fusion conformation (pre-F) transition to a stable post-fusion conformation (post-F). Notably, the pre-F conformation possesses a neutralizing sensitive site absent in the post-F conformation, and most potent neutralizing antibodies are effective only against the pre-F conformation. However, the pre-F conformation itself is unstable, making the preservation of this conformation and its important antigenic sites a key challenge and core issue in RSV vaccine development.

[0004] Currently, there are many problems with related technologies. On the one hand, although traditional trimeric motifs can maintain the pre-fusion conformation of the F protein to a certain extent, they have significant defects in thermal stability and storage stability, making it difficult to meet the needs of practical applications. On the other hand, the bivalent vaccine strategy adopted by companies like Pfizer involves expressing two subtypes of antigen separately and then mixing them evenly in a specific ratio. This approach may face many challenges in terms of production processes, cost control, and vaccine efficacy. Summary of the Invention

[0005] To overcome the problems of existing technologies, this invention proposes the modification and application of proteins based on heterotrimeric motifs. While maintaining the stability and broad spectrum of the important respiratory syncytial virus (RSV) immunogenic preF protein, it achieves the simultaneous expression of two subtype antigens, thereby reducing the number of production steps and lowering production costs for bivalent RSV vaccines.

[0006] The objective of this invention is achieved as follows:

[0007] The first aspect of this invention provides a heterotrimeric motif protein, wherein the trimeric motif protein includes a set of positive and negative salt bridge sequences, and the amino acid sequence of the positive salt bridge (+α1) is shown in SEQ ID NO.1:

[0008] MFSFVDLRLLLLLAATALLTHGPKGPPGPKGPPGPKGPPGPKGPPGPKGPPGP

[0009] KGPPGPKGPPGPKGPPGPKGPPGPKGPPGGSSGRANVVRDRDLEVDTTLKS

[0010] LSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKV

[0011] FCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGG

[0012] QGSDPADVAIQLTFLRRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQ

[0013] GSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL.

[0014] The amino acid sequence of the negative salt bridge (-α2) is as SEQ ID Shown in NO.2: MLSFVDTRTLLLLAVTLCLATCEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGPRSAPSLRPKDYEVDATLKSLNNQIETLLTPEGSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAIKVYCDFST GETCIRAQPENIPAKNWYRSSKDKKHVWLGETINAGSQFEYNVEGVTSKEMATQLAFMRLLANYASQNITYHCKNSIAYMDEETGNLKKAVILQGSNDVELVAEGNSRFTYTVLVDGCSKKTNEWGKTIIEYKTNKPSRLPFLDIAPLDIGGADQEFFVDIGPVCFK.

[0015] The second aspect of the present invention provides the application of the heterotrimeric motif protein described in the first aspect in improving the stability of respiratory syncytial virus fusion protein F. Specifically, when the positive and negative salt bridge sequences are present in RSV fusion protein F, the thermal stability and storage stability of RSV fusion protein F can be significantly improved.

[0016] A third aspect of the present invention provides a recombinant respiratory syncytial virus (RSV) trimer protein constructed using the heterotrimeric motif protein described in the first aspect, wherein the RSV includes, but is not limited to, RSV-A2 strain and RSV-CH18537 strain.

[0017] The amino acid sequence of the RSV-A2 strain is shown in SEQ ID NO.3.

[0018] The amino acid sequence of the RSV-CH18537 strain is shown in SEQ ID NO.4.

[0019] A fourth aspect of the present invention provides a nucleic acid molecule that encodes the heterotrimeric motif protein of the first aspect or the recombinant respiratory syncytial virus trimer protein of the second aspect.

[0020]

[0021]

[0022] The fifth aspect of this invention provides a method for preparing recombinant respiratory syncytial virus trimer protein as described in the third aspect above, comprising the following steps:

[0023] Step A) Prepare the nucleic acid molecule as described in the fourth aspect, construct an expression vector containing the nucleic acid molecule, and transform or transfect the expression vector into expression cells;

[0024] Step B), use the product of step A) to express protein;

[0025] Step C) involves purifying the expression product obtained in step B) to obtain the recombinant respiratory syncytial virus trimer protein. The purification in step C) can be performed using any suitable method, such as anion exchange chromatography.

[0026] The sixth aspect of the present invention provides the use of the heterotrimeric motif protein as described in the first aspect, the recombinant respiratory syncytial virus trimer protein as described in the third aspect, and the nucleic acid molecule as described in the fourth aspect in the preparation of a medicament for treating and / or treating respiratory syncytial virus infection and / or diseases caused by respiratory syncytial virus.

[0027] The seventh aspect of the present invention provides a recombinant protein vaccine comprising the heterotrimeric motif protein of the first aspect or the recombinant respiratory syncytial virus trimer protein of the third aspect.

[0028] Furthermore, the method of administration of the vaccine includes, but is not limited to, intramuscular injection. The vaccine may be a recombinant protein vaccine or a nucleic acid vaccine.

[0029] The advantages and beneficial effects of this invention are:

[0030] 1. Through continuous screening of the lengths of positive and negative salt bridges, this invention provides a set of positive and negative salt bridge sequences for improving the stability of respiratory syncytial virus fusion protein F. This salt bridge design can form unique salt bridges in three collagen chains. When the positive and negative salt bridge sequences are present in RSV fusion protein F, they can significantly improve the thermal stability and storage stability of RSV fusion protein F.

[0031] 2. The recombinant respiratory syncytial virus (RSV) trimer protein constructed using the positive and negative salt bridge sequences described in this application exhibits significantly better storage stability at 4°C for 10 days than the currently commonly used trimer motif—T4 fibrin (T4Foldon). Furthermore, this recombinant RSV trimer protein retains various important antigenic epitopes, such as I, II, III, IV, and V. After being expressed and purified using the eukaryotic expression system 293F, this trimeric protein demonstrated the generation of antibodies against the antigen in animal experiments.

[0032] 3. Animal experiments have confirmed that the recombinant respiratory syncytial virus trimer protein constructed in this invention can effectively protect the body from RSV infection, avoid viral attack on lung tissue, and significantly reduce lung tissue lesions. It can be used to treat and / or prevent diseases caused by respiratory syncytial virus. Attached Figure Description

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0034] Figure 1 This is a schematic diagram showing the change in the form of trimer after the trimer motif modification described in Example 1; wherein, A represents the formation of two trimer proteins, (α1)3 and (α1)2(α2)1, after not adding a specific length of positive and negative design; B represents the formation of only one trimer protein, (α1)2(α2)1, after adding a specific length of positive and negative design.

[0035] Figure 2 This is a schematic diagram of the Western blot results described in Example 1;

[0036] Figure 3 The diagram illustrates the trimer formation patterns of the different respiratory syncytial virus mutants Q74 described in Example 2 under the heterotrimer motif;

[0037] Figure 4 Example 2 shows the evaluation results of T4 Foldon and heterotrimer under thermal stability and storage stability.

[0038] Figure 5 Example 3 shows the evaluation results of the formation of a trimer by the heterotrimer trimer motif in mice by the two subtypes A2 and CH18537.

[0039] Figure 6 Example 4 shows the results of an experiment verifying the protective effect of the A2 strain against mouse immunization against the A2 strain when the two subtypes, A2 and CH18537, form a trimer with the heterotrimer motif. Detailed Implementation

[0040] The implementation of this invention is not limited to the embodiments described below. Any modifications and / or alterations made to this invention will fall within the scope of protection of this invention. In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. Unless otherwise specified, the methods used in the embodiments are techniques generally applicable in the field.

[0041] For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0042] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The following is an explanation of some of the terms appearing in this invention.

[0043] The term "trimer" refers to a type of protein quaternary structure. Protein quaternary structure refers to the number and arrangement of protein subunits relative to each other. A protein containing three subunits is considered a trimer.

[0044] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cosmids; and artificial chromosomes.

[0045] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0046] Example 1: Modification and Verification of Heterotrimer Motifs Based on Positive and Negative Charges

[0047] like Figure 1 As shown in Figure B, this embodiment provides a heterotrimer motif protein, which is constructed based on a set of specific positive and negative salt bridges. The amino acid sequences of the positive salt bridge (+α1) and the negative salt bridge (-α2) are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0048] The preparation method of the heterotrimer includes the following steps:

[0049] Using pcDNA3.1 as a plasmid vector, the gene sequences of the positive salt bridge (+α1) and negative salt bridge (-α2) were directly synthesized to construct expression vectors. The expression vectors containing the two plasmids were co-transfected into 293T adherent cells. The 293T cells used for transfection were plated one day in advance in T25 flasks, 6-well cell culture plates and 48-well cell culture plates. Transfection was performed when the cell confluence was 75%-85%.

[0050] According to the instructions for the plasmid transfection reagent, take an appropriate amount of DNA, add a certain amount of Opti-MEM cell culture medium, mix thoroughly, and incubate at room temperature for 5 minutes. According to the instructions for the liposome transfection reagent, take a certain amount of transfection reagent, add a certain amount of Opti-MEM cell culture medium, mix thoroughly, and incubate at room temperature for 5 minutes. Mix the obtained DNA mixture with the transfection reagent mixture, gently pipette to mix thoroughly, and incubate at room temperature for 15 minutes to allow the transfection complex to form. Change the medium for the transfected cells, adding a small amount of cell culture medium. Add the transfection complex to the cells to be transfected, and gently shake to ensure that the transfection complex and the culture medium are thoroughly mixed. After 6 hours of transfection, discard the transfection solution and replace it with cell culture medium. Continue culturing for 24-48 hours, then observe under a fluorescence microscope or collect the cell culture supernatant to prepare a Western blot sample.

[0051] In addition, trimeric proteins were prepared using the same method described above. Figure 1 The difference is that the positive and negative salt bridges are replaced by (α1) and (α2).

[0052] The amino acid sequence of (α1) is:

[0053] MFSFVDLRLLLLLAATALLTHGANVVRDRDLEVDTTLKSLSQQIENIRSPEGS

[0054] RKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYP

[0055] TQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLT

[0056] FLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEIEIRAEGNSR

[0057] FTYSVTVDGCTSTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL.

[0058] The amino acid sequence of (α2) is:

[0059] MLSFVDTRTLLLLAVTLCLATCPRSAPSLRPKDYEVDATLKSLNNQIETLLTPE

[0060] GSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAIKVYCDFSTGETCIR

[0061] AQPENIPAKNWYRSSKDKKHVWLGETINAGSQFEYNVEGVTSKEMATQLAF

[0062] MRLLANYASQNITYHCKNSIAYMDEETGNLKKAVILQGSNDVELVAEGNSRF

[0063] TYTVLVDGCSKKTNEWGKTIIEYKTNKPSRLPFLDIAPLDIGGADQEFFVDIGPVCFK.

[0064] For detection purposes, in this embodiment, tag sequences are added to the (+α1), (-α2), (α1), and (α2) sequences, respectively.

[0065] The amino acid sequence of (HA-+α1) is:

[0066] MFSFVDLRLLLLLAATALLTHGYPYDVPDYAPKGPPGPKGPPGPKGPPGPKG

[0067] PPGPKGPPGPKGPPGPKGPPGPKGPPGPKGPPGPKGPPGGSGRANVVRDRD

[0068] LEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQ

[0069] GCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMT

[0070] DGFQFEYGGQGSDPADVAIQLTFLLRLMSTEASQNITYHCKNSVAYMDQQTG

[0071] NLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL.

[0072] The amino acid sequence of (FLAG-α2) is as follows:

[0073] MLSFVDTTRTLLLLAVTLCLATCDYKDDDDKEPGEPGEPGEPGEPGEPGEPGEP

[0074] GEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGEPGPRSAPSLRPKDYEVDAT

[0075] LKSLNNQIETLLTPEGSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAI

[0076] KVYCDFSTGETCIRAQPENIPAKNWYRSSKDKKHVWLGETINAGSQFEYNVE

[0077] GVTSKEMATQLAFMRLLANYASQNITYHCKNSIAYMDEETGNLKKAVILQG

[0078] SNDVELVAEGNSRFTYTVLVDGCSKKTNEWGKTIIEYKTNKPSRLPFLDIAPLDIGGADQEFFVDIGPVCFK.

[0079] The amino acid sequence of (HA-α1) is:

[0080] MFSFVDLRLLLLLAATALLTHGYPYDVPDYAGSGSGRANVVRDRDLEVDTT

[0081] LKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAI

[0082] KVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEY

[0083] GGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALL

[0084] LQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL.

[0085] The amino acid sequence of (FLAG-α2) is:

[0086] MLSFVDTRTLLLLAVTLCLATCDYKDDDDKPRSAPSLRPKDYEVDATLKSLN

[0087] NQIETLLTPEGSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAIKVYC

[0088] DFSTGETCIRAQPENIPAKNWYRSSKDKKHVWLGETINAGSQFEYNVEGVTS

[0089] KEMATQLAFMRLLANYASQNITYHCKNSIAYMDEETGLKKAVILQGSNDV

[0090] ELVAEGNSRFTYTVLVDGCSKKTNEWGKTIIEYKTNKPSRLPFLDIAPLDIGGADQEFFVDIGPVCFK.

[0091] The comparison test results are as follows:

[0092] (1) Trimer motif characterization: After co-transfection of 293T adherent cells with two plasmids, Western blot (WB) was performed for verification.

[0093] Specifically Figure 2 In lane A, the bands are obtained after adding loading buffer containing SDS and β-mercaptoethanol to the sample, heating at 100°C for 10 min, and electrophoresis using an SDS-free PAGE gel in an SDS-containing electrophoresis buffer. Lane 1 represents the negative control GFP, lane 2 represents HA-α1 transfection alone, lane 3 represents Flag-α2 transfection alone, lane 4 represents co-transfection of HA-α1 and Flag-α2, lane 5 represents +HA-α1 transfection alone, lane 6 represents -Flag-α2 transfection alone, and lane 7 represents the positions of the bands under co-transfection of +HA-α1 and -Flag-α2, respectively, using HA antibody and Flag antibody.

[0094] Figure 2 In lane B, the bands are obtained after loading the sample directly without adding a loading buffer containing SDS and β-mercaptoethanol, and electrophoresis using an SDS-free PAGE gel in an SDS-containing electrophoresis buffer. Lane 1 represents the negative control GFP, lane 2 represents HA-α1 transfection alone, lane 3 represents Flag-α2 transfection alone, lane 4 represents co-transfection of HA-α1 and Flag-α2, lane 5 represents +HA-α1 transfection alone, lane 6 represents -Flag-α2 transfection alone, and lane 7 represents the positions of the bands under co-transfection of +HA-α1 and -Flag-α2, respectively, using HA antibody and Flag antibody.

[0095] like Figure 2As shown, the magnitude of α1 is approximately 35 kDa, and the magnitude of α2 is approximately 37 kDa; the magnitude of +α1 is approximately 37 kDa, and the magnitude of -α2 is approximately 42 kDa. Figure 2 (A). Denaturing nonreducing gel electrophoresis demonstrated that both HA-α1 and +HA-α1 can trimerize individually, forming trimers of approximately 100 kDa and 140 kDa, respectively, consistent with previous reports that α1 can form trimers alone. α2 alone cannot trimerize. When α1 and α2 are co-transfected, two types of trimers are formed: (α1)3 and (α1)2(α2)1. However, when +α1 and -α2 are co-transfected, denaturing nonreducing gel electrophoresis showed that +α1 and -α2 only form one type of trimer, (+α1)2(-α2)1. Figure 2 (B)

[0096] Example 2: Modification and Validation of the F Protein of Respiratory Syncytial Virus Based on Heterotrimer

[0097] In this embodiment, the recombinant respiratory syncytial virus (RSV) trimeric protein (Q74-heterotrimer) was constructed using the heterotrimer motif protein heterotrimer-(+α1)2(-α2)1 described in Example 1, wherein the RSV used was the Q74 sequence of RSV.

[0098] The preparation method of the Q74-heterotrimer includes the following steps:

[0099] Gene sequences such as positive salt bridge (Q74-+α1) and negative salt bridge (Q74--α2) were directly synthesized using pcDNA3.1 as a plasmid vector, and expression vectors were constructed. The expression vectors containing the two plasmids were co-transfected into 293T adherent cells.

[0100] The 293T cells to be transfected were plated one day in advance in T25 flasks, 6-well cell culture plates and 48-well cell culture plates. Transfection was performed when the cell confluence was 75%-85%.

[0101] According to the instructions for the plasmid transfection reagent, take an appropriate amount of DNA, add a certain amount of Opti-MEM cell culture medium, mix thoroughly, and incubate at room temperature for 5 minutes. According to the instructions for the liposome transfection reagent, take a certain amount of transfection reagent, add a certain amount of Opti-MEM cell culture medium, mix thoroughly, and incubate at room temperature for 5 minutes. Mix the obtained DNA mixture with the transfection reagent mixture, gently pipette and mix thoroughly, and incubate at room temperature for 15 minutes to allow the transfection complex to form. Change the medium for the transfected cells, adding a small amount of cell culture medium. Add the transfection complex to the cells to be transfected, and gently shake to ensure that the transfection complex and the culture medium are thoroughly mixed. After 6 hours of transfection, discard the transfection solution and replace it with cell culture medium. Continue culturing for 24-48 hours, then observe under a fluorescence microscope and collect the expression supernatant of the cells for ELISA and Western blot (WB) verification.

[0102] (1) Trimer motif characterization: After co-transfection of 293T adherent cells with two plasmids, Western blot verification was performed.

[0103] Specifically, Figure 3 In lane A, the bands are obtained after adding loading buffer containing SDS and β-mercaptoethanol to the sample, heating at 100°C for 10 min, and electrophoresis using an SDS-free PAGE gel in an SDS-containing electrophoresis buffer. Lane 1 represents the negative control GFP, lane 2 represents Q74-+α1 transfection alone, lane 3 represents Q74--α2 transfection alone, and lane 4 represents the position of the bands under anti-RSV F protein in the case of co-transfection of Q74-+α1 and Q74--α2. Figure 3 In lane B, the bands are obtained after loading the sample directly without adding loading buffer containing SDS and β-mercaptoethanol, and electrophoresis using an SDS-free PAGE gel in an SDS-containing electrophoresis buffer. Lane 1 represents the position of the band after transfection of Q74-+α1 alone, lane 2 represents the position of the band after transfection of Q74--α2 alone, and lane 3 represents the position of the band under anti-RSV F protein in the case of co-transfection of Q74-+α1 and Q74--α2.

[0104] Western blot results showed that co-transfection with Q74-+α1 and Q74--α2 resulted in the formation of a single trimeric protein with a size of approximately 240 kDa. Figure 3 (B)

[0105] (2) Transfection ratio optimization: This embodiment optimized the co-transfection ratio of the two plasmids. When the ratio of Q74-+α1 to Q74--α2 was 2:1, D25 in the cell culture supernatant was detected by ELISA. The absorbance value of Palivizumab (II) is higher. Figure 4(B)

[0106] (3) Thermal stability verification: The cell culture supernatant was treated at 50℃ and 60℃ for 1 h and at 4℃ for 10 days, respectively, and then the two cell culture supernatants were detected by ELISA.

[0107] Specifically, Figure 4 In the middle section, A refers to the situation where Q74-+α1 and Q74--α2, at a total plasmid dose of 4 μg, are co-transfected in different proportions to form heterotrimer, and the formation of RSV F protein is detected by D25 and Palivizumab antibody. Figure 4 B is the post-F conformational reaction of the expression supernatant after co-transfection of 293T with Q74-+α1 and Q74--α2 in a 2:1 ratio and storage at 4°C for 10 days. Figure 4 The expression supernatant of 293T cells co-transfected with Q74-+α1 and Q74--α2 in a 2:1 ratio underwent Palivizumab(Ⅱ) epitope conformation reactions after untreated, heated at 50℃ and 60℃ for 1 h. Figure 4 D25 was produced after co-transfection of 293T cells with Q74-+α1 and Q74--α2 in a 2:1 ratio, and the supernatant was subjected to untreated, 50℃, and 60℃ heating for 1 h. Epitope conformation reaction. Figure 4 E represents the 4D7 (post F) epitope conformation reaction of expression supernatant after co-transfection of 293T with Q74-+α1 and Q74--α2 in a 2:1 ratio, followed by heating at 50℃ and 60℃ for 1 h in the untreated, 50℃, and 60℃ environments. Figure 4 The expression supernatant of 293T after co-transfection with Q74-+α1 and Q74--α2 in a 2:1 ratio underwent hRSV90(V) epitope conformation reactions after untreated, heated at 50℃ and 60℃ for 1 h.

[0108] The results demonstrate that heterotrimer has a greater advantage in thermal stability than T4 Foldon, with various pre-F epitopes D25. Neither Palivizumab (II) nor hRSV90 (V) showed a significant decrease, and the post F epitope 4D7 did not show a significant increase. Regarding storage stability, Q74-heterotrimer did not enhance the conversion from pre-F to post-F after treatment at 4°C for 10 days, demonstrating that Q74-heterotrimer has a certain advantage over T4 Foldon in maintaining the pre-F conformation of the respiratory syncytial virus (RSV) F protein.

[0109] The recombinant respiratory syncytial virus trimer protein constructed in this embodiment showed almost no significant difference in ELISA reactivity after treatment at 50℃ and 60℃ for 1 h, and the post F conformation was not significantly higher than that of T4-Foldon after storage at 4℃ for 10 days.

[0110] Example 3: Evaluation of initiating antibodies in a protein vaccine based on Bi-F-Trimer antigen

[0111] This embodiment utilizes the heterotrimer-(+α1)2(-α2)1 trimeric protein described in Example 1 to construct a recombinant respiratory syncytial virus (RSV) trimeric protein (Bi-F-Trimer). The RSV strains used are RSV-A2 and RSV-CH18537. The amino acid sequences of RSV-A2 and RSV-CH18537 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, and the nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The preparation method includes the following steps:

[0112] Gene sequences of two trimeric motifs, A2-+α1 and CH18537--α2, were synthesized using pcDNA3.1 as the plasmid vector. Expression vectors were constructed, and the expression vectors containing the two plasmids were co-transfected into 293F suspension cells. The 293F cells used for transfection were cultured to 1×10⁻⁶ cells one day in advance. 6 Prepare 300ml of cell suspension with cells / ml.

[0113] According to the instructions for the plasmid transfection reagent, take an appropriate amount of A2-+α1 and CH18537--α2 plasmids, add a certain amount of Opti-MEM cell culture medium, mix thoroughly, and incubate at room temperature for 5 min. According to the instructions for the PEI transfection reagent, take a certain amount of transfection reagent, add a certain amount of Opti-MEM cell culture medium, mix thoroughly, and incubate at room temperature for 5 min. Mix the obtained DNA mixture with the transfection reagent mixture, gently pipette and mix thoroughly, and incubate at room temperature for 15 min to allow the transfection complex to form. Add the transfection complex to the cells to be transfected, gently shake to ensure that the transfection complex and culture medium are thoroughly mixed. Continue culturing for 72 h, then observe under a fluorescence microscope and collect the expression supernatant of the cells for evolution.

[0114] Centrifuge the cell expression suspension to obtain the expression supernatant. Concentrate the supernatant using a 30kDa protein concentration ultrafiltration tube, and replace the buffer with anion exchange purification solution A. Load the protein concentrate onto a sample and purify and separate the protein.

[0115] The purified protein sample was quantified. 5 μg of antigen was combined with adjuvant and used to immunize mice. A booster immunization was performed on day 28. Blood samples were collected 14 days after the second immunization to detect the antibodies produced by the mice.

[0116] Figure 5 The samples in section A are those collected after anion exchange purification of the A2 and CH18537 subtypes expressed as heterotrimer in 293F suspension cells. The samples were loaded directly without the addition of loading buffer containing SDS and β-mercaptoethanol and electrophoresed on an SDS-free PAGE gel in an SDS-containing electrophoresis buffer. Lane 1 represents GFP and lane 2 represents the position of the Bi-F-Trimer band formed after co-transfection of Q74-+α1 and Q74--α2. Figure 5 Figure B is an immunization diagram showing that each mouse was immunized with 5μg of antigen, and a second immunization was performed in the fourth week. Figure 5 C represents the titer of specific binding antibodies against RSV F induced in mice, measured in blood samples taken two weeks after the second immunization. Figure 5 D is the type of specific binding antibody against RSVF induced by blood samples taken from mice two weeks after the second immunization.

[0117] The results are analyzed as follows:

[0118] (1) Anion exchange column purification to collect the formation of trimeric proteins: such as Figure 5 As shown in Figure A, in this embodiment, the size of the purified trimer protein was identified using non-reducing polyacrylamide gel electrophoresis. The size was approximately 250 kDa as expected, and only one band was formed.

[0119] (2) Examination of the formation of specific binding antibodies after immunization of mice with antigen and adjuvant: such as Figure 5 As shown in Figure B, in this embodiment, each injection administered 5 μg of antigen on day 0 and day 28. Blood was collected from mice 14 days after the second immunization to detect the antibody titer in the serum. The results showed that Bi-F-Trimer stimulated a high production of binding antibodies against the RSVF antigen. Figure 5 (C). Among them, the type of IgG-specific binding antibody produced by mice against RSVpre-F also tends to be IgG2a ( Figure 5 (D).

[0120] Example 4: Observation and verification of the protective effect of Bi-F-Trimer antigen against respiratory syncytial virus (RSV) challenge.

[0121] In this embodiment, to verify whether Bi-F-Trimer-induced humoral immunity can protect mice from respiratory syncytial virus (RSV) attack, a challenge experiment was conducted on mice 28 days after their second immunization and at rest. The specific method is as follows:

[0122] Mice that had undergone two immunizations and were at rest 28 days after immunization were intranasally injected with 5 × 10⁵ mice. 5 The A2 strain of PFU virus was used to monitor changes in mouse weight and body temperature daily after challenge. Four days after challenge, mice were sacrificed to assess lung pathological changes.

[0123] A catheter containing 1 ml of PBS solution was used for bronchoalveolar lavage. After repeated blowing and aspiration three times, bronchoalveolar lavage fluid was collected, and the volume of the lavage fluid was recorded. The fluid was centrifuged at 500×g for 5 min to obtain the supernatant containing the lavage fluid and cells. The cells were resuspended in an equal volume of PBS. One portion was counted using a red blood cell counting chamber, and the other portion (10 μl) was smeared and stained with Giemsa stain.

[0124] After separation of the left lung, it was fixed with 4% paraformaldehyde and subjected to HE sectioning.

[0125] Experimental results are as follows Figure 6 As shown, A is the flowchart of the experimental design. B shows the change in body temperature of mice after challenge on day 56. C shows the change in the rate of weight change of mice after challenge on day 56. D shows the cell infiltration in the lung interstitial spaces of mice after challenge on day 60. E shows the infiltration of cytokine IFN-γ in the lung interstitial spaces of mice after challenge on day 60. F shows the infiltration of cytokine IL-2 in the lung interstitial spaces of mice after challenge on day 60. G shows the relative quantitative comparison of viral load in the lungs of mice after challenge on day 60. H shows the pathological examination of the left lung of mice after HE staining.

[0126] 50 μl nasal drops per mouse (1 × 10) 7 Mice were challenged with the A2 strain at PFU / ml, and their body weight, body temperature, and other changes were monitored daily after the challenge. Figure 6 (A). In the PBS group, the body temperature of mice decreased more sharply compared to those in the Bi-F-Trimer group. Figure 6 In the Bi-F-Trimer group, the rate of change in body weight was significantly lower compared to that in the PBS group (B). Figure 6 (C). No significant difference in cell infiltration was found in the bronchoalveolar lavage fluid of mice sacrificing PBS and Bi-F-Trimer mice four days after challenge with the virus. Figure 6 (D), but the viral load in the lungs was relatively higher in the PBS group ( Figure 6(G). IFN-γ and IL-2 are immune-biased cytokines that prevent ERD effects in mice. The Bi-F-Trimer group showed higher IFN-γ secretion in bronchoalveolar lavage fluid. Figure 6 (E&F). HE results showed that the Bi-F-Trimer group mice had weaker pathological conditions such as lung inflammation, congestion, and alveolar septal thickening, indicating that Bi-F-Trimer played a role in protecting mice from respiratory syncytial virus (RSV) attack. Figure 6 (H).

[0127] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. The application of a heterotrimeric motif protein in improving the stability of respiratory syncytial virus fusion protein F, characterized in that, The trimer motif protein is formed by the self-assembly of two positive salt bridges and one negative salt bridge, and the amino acid sequences of the positive and negative salt bridges are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

2. A recombinant respiratory syncytial virus trimer protein constructed using a heterotrimeric motif protein, characterized in that, The recombinant respiratory syncytial virus trimer protein consists of two polypeptides shown in SEQ ID NO. 3 and one polypeptide shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant respiratory syncytial virus trimer protein as described in claim 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence encoding the polypeptide shown in SEQ ID NO. 3 is shown in SEQ ID NO. 5, and the nucleotide sequence encoding the polypeptide shown in SEQ ID NO. 4 is shown in SEQ ID NO.

6.

5. A method for preparing the recombinant respiratory syncytial virus trimer protein as described in claim 2, characterized in that, The preparation method includes the following steps: Step A), prepare the nucleic acid molecule as described in claim 3 or 4, construct an expression vector containing the nucleic acid molecule, and transform or transfect the expression vector into expression cells; Step B), using the product of step A) for protein expression; Step C) purifies the expression product obtained in step B) to obtain the recombinant respiratory syncytial virus trimer protein.

6. Use of the recombinant respiratory syncytial virus trimer protein of claim 2, or the nucleic acid molecule of claim 3 or 4, in the preparation of a medicament for treating respiratory syncytial virus infection and / or diseases caused by respiratory syncytial virus.

7. A recombinant protein vaccine, characterized in that, The vaccine contains the recombinant respiratory syncytial virus trimer protein as described in claim 2.

8. The recombinant protein vaccine according to claim 7, characterized in that, The vaccine is administered via intramuscular injection.

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