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CN-122011216-A - Respiratory syncytial virus F protein and preparation method and application thereof

CN122011216ACN 122011216 ACN122011216 ACN 122011216ACN-122011216-A

Abstract

The application relates to the technical field of respiratory tract virus prevention, in particular to a respiratory tract syncytial virus F protein, a preparation method and application thereof. The F protein is a single-chain form fusion pre-conformation F protein trimer, the truncated F2 protein and the truncated F1 protein are connected through a connector, the F protein is in preF conformation through a plurality of amino acid substitutions, and the trimer structure is further maintained through an extended C end and a plurality of amino acid substitutions. The recombinant respiratory syncytial virus F protein provided by the application has obvious advantages in three dimensions of structural characteristics, stability and immunogenicity, and realizes key breakthrough compared with the existing vaccine technology containing the T4 folden structural domain.

Inventors

  • LIU XIAOMAN
  • WANG QI
  • YANG LIXIAN
  • ZHAO CE
  • FU LILI
  • SONG LIQIANG

Assignees

  • 北京华诺泰生物医药科技股份有限公司

Dates

Publication Date
20260512
Application Date
20260402

Claims (10)

  1. 1. A respiratory syncytial virus F protein is characterized in that truncated F2 protein with an amino acid sequence shown as SEQ ID NO. 7 and truncated F1 protein with an amino acid sequence shown as SEQ ID NO. 9 are fused through a connector 1, the following 22 point mutations ：A149C、Y458C、S155C、S290C、S46G、E92D、P102A、S190F、V207L、S215P、L373R、I379V、I384V、M447V、K465Q、D486N、E487L、D489Y、F505W、S509F、G519V、T523I, are carried out on the basis of the fused sequence to obtain preF protein, a signal peptide is added at the N end of the preF protein, and a SpyTag tag sequence is fused at the C end through a connector 2.
  2. 2. The F protein of claim 1, wherein the preF protein has the amino acid sequence shown in SEQ ID No. 12.
  3. 3. The F protein of claim 1, wherein the amino acid sequence of the signal peptide is as shown in SEQ ID No. 13; alternatively, the amino acid sequence of the SpyTag tag sequence is shown as SEQ ID No. 15.
  4. 4. The F protein according to claim 1, wherein the amino acid sequence of linker 1 is shown in SEQ ID No. 10; alternatively, the amino acid sequence of the linker 2 is shown in SEQ ID No. 14.
  5. 5. The F protein of claim 1, wherein the amino acid sequence of the F protein is set forth in SEQ ID No. 16.
  6. 6. A gene encoding the F protein of any one of claims 1-5.
  7. 7. An expression vector, characterized in that, the expression vector comprises the gene of claim 6.
  8. 8. A host cell expressing the gene of claim 6 or the expression vector of claim 7.
  9. 9. Use of the F protein as defined in any one of claims 1 to 5 for the preparation of a composition for the prevention and/or treatment of respiratory syncytial virus-related disorders.
  10. 10. A vaccine for the prevention of respiratory syncytial virus, characterized in that the vaccine comprises the F protein of any one of claims 1-5, and a pharmaceutically acceptable carrier or excipient.

Description

Respiratory syncytial virus F protein and preparation method and application thereof Technical Field The application relates to the technical field of respiratory tract virus vaccines, in particular to a respiratory tract syncytial virus F protein, a preparation method and application thereof. Background Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is the leading pathogen worldwide responsible for severe lower respiratory tract infections (Lower Respiratory TRACT DISEASE, LRTI) in infants and elderly. The epidemiological characteristics of the infant are characterized by high incidence and wide prevalence, especially for premature infants, infants and the elderly, which pose a serious health threat. Despite significant progress in RSV vaccine development, both current marketed recombinant protein vaccines use the RSV fusion protein (F protein) as a core antigen, but the prior art approaches still have significant limitations. The RSV F protein is a key protein of virus invasion to host cell, and has two conformations of pre-fusion (pre-F) and post-fusion (post-F), wherein neutralizing epitopes and V are exposed in the pre-fusion conformations, the titer of the induced neutralizing antibodies is 10-100 times that of the post-fusion conformations, and the neutralizing antibodies are core targets for vaccine development. The (post-F) conformation is more stable in nature after fusion, but retains only epitope I, II, III, IV and is significantly less immunogenic. However, pre-F conformation is extremely prone to spontaneously convert to post-F conformation with significantly reduced immunogenicity when expressed in vitro, resulting in loss of immunogenicity, which conformational instability is the primary bottleneck in vaccine development. In order to maintain the key preF conformational trimer structure of the existing recombinant F protein vaccine, a strategy of fusion expression of a T4 folden trimerization domain at the C end of the F protein is generally adopted. Although the technical route can realize trimer assembly, the potential risk brought by exogenous structural domains is introduced. Studies show that the T4 folden structural domain as a non-RSV-derived heterologous protein possibly triggers specific immune response of an organism aiming at the structural domain, and the non-target immune response not only consumes immune resources, but also can reduce the immune protection effect aiming at the key neutralizing epitope of the F protein through antigen competition or epitope interference mechanism, thereby becoming an important bottleneck for restricting the immunogenicity of the vaccine. In addition, the problem of insufficient conformational stability of the existing recombinant F protein is not ignored. The preF conformation, which is the key functional form for inducing neutralizing antibodies, has structural stability directly affecting the immunogenicity and shelf life of the vaccine. During production and storage, the F protein is susceptible to conformational changes or trimeric dissociation, resulting in reduced vaccine efficacy. These technical drawbacks together highlight the necessity of developing novel recombinant F protein construction strategies to address the inherent limitations of existing vaccines in terms of safety and stability. Disclosure of Invention The application provides a respiratory syncytial virus F protein, a preparation method and application thereof. The recombinant respiratory syncytial virus F protein provided by the application has obvious advantages in three dimensions of structural characteristics, stability and immunogenicity, and realizes key breakthrough compared with the existing vaccine technology containing the T4 folden structural domain. In a first aspect, the present application provides a respiratory syncytial virus F protein, which adopts the following technical scheme: A respiratory syncytial virus F protein is prepared through fusing truncated F2 protein with amino acid sequence as shown in SEQ ID NO. 7 with truncated F1 protein with amino acid sequence as shown in SEQ ID NO. 9 by linker 1, performing the following 22 point mutations ：A149C、Y458C、S155C、S290C、S46G、E92D、P102A、S190F、V207L、S215P、L373R、I379V、I384V、M447V、K465Q、D486N、E487L、D489Y、F505W、S509F、G519V、T523I, to obtain preF protein, adding signal peptide to N end of preF protein, and fusing SpyTag tag sequence to C end by linker 2. The application aims to solve the core challenges faced by the preparation of recombinant respiratory syncytial virus F protein in the prior art, and specifically comprises three key targets, namely, firstly, overcoming the problems of potential safety hazard of vaccine and reduced immunospecificity caused by introducing exogenous trimerization structural domain, secondly, improving the structural stability of the conformation F protein (preF) before fusion, reducing the conformation heterogeneity in the in-vitro expression process, and finally, im