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CN-121991185-A - Nucleocapsid protein mutant, gene, recombinant expression vector, mutant virus strain and application

CN121991185ACN 121991185 ACN121991185 ACN 121991185ACN-121991185-A

Abstract

The invention provides a nucleocapsid protein mutant, a gene, a recombinant expression vector, a mutant virus strain and application thereof, belonging to the technical field of biological medicine. The invention is based on SARS-CoV-2 virus-like particle (VLP) pseudotyping technology, and discovers that the mutation of N protein alanine, L353A, H356A, I357A, D358A and Y360A break the hydrophobic pocket of N protein CTD, reduce the binding affinity of nucleic acid, and further discovers that F403A, F407A and M411A cause the increase of N protein monomer, the decrease of tetramer and the increase of abnormal high polymer N protein, and inhibit the replication of SARS-CoV-2 wild live virus and variant strain. Meanwhile, the invention designs chiral D-type polypeptide aiming at the CTD region, and discovers that the chiral D-type polypeptide can effectively inhibit the assembly of VLP pseudoviruses.

Inventors

  • WANG YOUCHUN
  • HUANG WEIJIN
  • NIE JIANHUI
  • LIU SHUO
  • ZHANG LI
  • Liang ziteng

Assignees

  • 中国食品药品检定研究院
  • 中国医学科学院医学生物学研究所

Dates

Publication Date
20260508
Application Date
20260210

Claims (10)

  1. 1. A nucleocapsid protein mutant, wherein said nucleocapsid protein mutant is obtained by amino acid mutation of nucleocapsid protein, said amino acid mutation comprises site mutation of CTD region or N3 region; The mutation site of the CTD region comprises one or more of L353A, H356A, I357A, D358A and Y360A; the mutation site of the N3 region comprises one or more of F403A, L A and M411A.
  2. 2. A gene encoding the nucleocapsid protein mutant of claim 1.
  3. 3. A recombinant expression vector comprising the gene of claim 2.
  4. 4. Use of a nucleocapsid protein mutant according to claim 1 for the construction of a mutant viral strain of severe acute respiratory syndrome coronavirus 2.
  5. 5. A mutant strain of severe acute respiratory syndrome coronavirus 2 comprising expression of the nucleocapsid protein mutant of claim 1 or the gene of claim 2.
  6. 6. Use of the nucleocapsid protein mutant of claim 1 or the mutant viral strain of claim 5 for the preparation of a severe acute respiratory syndrome coronavirus 2 vaccine.
  7. 7. Use of the nucleocapsid protein mutant of claim 1 or the mutant viral strain of claim 5 for screening for a medicament against severe acute respiratory syndrome coronavirus 2.
  8. 8. The application of the nucleocapsid protein amino acid site in screening medicines for resisting severe acute respiratory syndrome coronavirus 2 as target spots is characterized in that the nucleocapsid protein amino acid site comprises one or more of 353 th leucine, 356 th histidine, 357 rd isoleucine, 358 th aspartic acid and 360 th tyrosine of CTD region, or One or more of phenylalanine 403, leucine 407, and methionine 411, including the N3 region.
  9. 9. A method for reducing pathogenicity of severe acute respiratory syndrome coronavirus 2, wherein the amino acid sequence of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein is subject to a site mutation, said site mutation comprising a site mutation of CTD region or N3 region; The mutation site of the CTD region comprises one or more of L353A, H356A, I357A, D358A and Y360A; the mutation site of the N3 region comprises one or more of F403A, L A and M411A.
  10. 10. A chiral polypeptide is characterized in that the amino acid sequence of the chiral polypeptide is shown as SEQ ID No.1, and the chiral polypeptide has the effect of inhibiting the assembly of severe acute respiratory syndrome coronavirus 2.

Description

Nucleocapsid protein mutant, gene, recombinant expression vector, mutant virus strain and application Technical Field The invention relates to the technical field of biological medicine, in particular to a nucleocapsid protein mutant, a gene, a recombinant expression vector, a mutant virus strain and application. Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a novel coronavirus infection (COVID-19), spread worldwide since the end of 2019. Despite the great efforts made by researchers in developing vaccines and antiviral drugs, effective control of viruses has not been achieved. Currently, blocking studies on COVID-19 transmission have focused mainly on viral spike protein (S protein) because its receptor domain (RBD region) is a critical region for binding to the human angiotensin converting enzyme 2 (ACE 2) receptor, directly mediating viral invasion into host cells, and it is the primary antigen that induces the body to produce neutralizing antibodies. However, due to the extremely high mutation rate of the S protein, effective neutralizing antibodies have been developed which are extremely susceptible to rapid loss of neutralizing activity. Therefore, searching for new therapeutic targets for SARS-CoV-2, developing broad-spectrum antiviral vaccines and drugs is an urgent need for current research. Today, the development of broad-spectrum antiviral vaccines and drugs is mainly focused on two methods, the first being targeting viral proteins, mainly viral enzymes, interrupting the viral life cycle by interfering with the function of the viral proteins and the second being targeting host proteins involved in the viral life cycle, such as ACE2 receptors, which have potential broad-spectrum prevention and control potential for coronaviruses interacting with ACE2 receptors. However, ACE2 receptors are responsible for important physiological functions in humans that regulate respiration and heartbeat, and thus, safety issues remain to be studied and addressed when drugs are developed for ACE2 receptors. Viral targeting inhibitors are aimed at disrupting various stages of the SARS-CoV-2 lifecycle, and therefore virally encoded proteins that serve the core functions of each link are naturally considered as the primary drug targets. In combination with the division of the virus life cycle, the research status of each target point and the corresponding inhibitor is that spike protein (S protein) is the key to mediating virus adsorption and membrane fusion in the virus entry stage, and the inhibitors aimed at the spike protein (S protein) are mainly divided into two types, wherein one type is an inhibitor for blocking the interaction of the S protein and ACE2 receptor, such as ensovibep, FSR m and FSR22, and the other type is a peptide inhibitor for targeting the S protein heptapeptide repeat alpha helical region, such as EK1; papain-like protease cleaves the polyproteins pp1a and pp1ab into various non-structural proteins such as nsp1 to nsp3 during the protein processing phase, so far more than 30 papain-like protease inhibitors have been developed, 3C-like protease can cleave pp1a and pp1ab polyproteins, releasing viral proteins nsp4 to nsp16, thus protease inhibitors targeting 3C-like protease such as nimat Lei Wei, entetavir and SIM0417 have been used for the treatment of new coronavirus infections, core targets RNA-dependent RNA polymerase (RdRp), also known as nsp12, which is a highly conserved enzyme playing a vital role in replication and transcription of viral RNA, can catalyze template DNA helicity, RNA synthesis, RNA proofreading and RNA end-capping, rdRp inhibitors developed based on this target can block RNA synthesis by targeting key components of replication transcript complexes, representative drugs including REMDESIVIR, MOLNUPIVIR, FAVIPIRAVIR and Bemnifosbuvir, core targets are RNA-dependent RNA polymerase (RNA-dependent RNA polymerase) which is a highly conserved enzyme, the core target protein is a major domain for viral protein (CTD) is a major domain for viral protein-binding domain in the three-terminal domain of the viral capsid domain, therefore, the current research focus has turned to the direction of "mediating abnormal high aggregation of N protein" to achieve antiviral effects by disrupting its normal assembly process. Replication-defective SARS-CoV-2 Virus-like particles (pseudoviruses) comprise 4 structural proteins (Spike protein, membrane glycoprotein Membrane glycoprotein, envelope protein, nucleocapsid phosphoprotein Nucleocapsid phosphoprotein) of a novel coronavirus, which can self-assemble to form infectious Virus-like particles (viruses LIKE PARTICLES, VLPs) resembling a true Virus, the interior of which is bound to N proteins by a segment of RNA of ORF1ab, thereby achieving the encapsulation and delivery of foreign genes. By utilizing the tool, the influence of N protein mutation on virus assembly can be deeply studied, so that mutatio