Recombinant novel coronavirus RBD tripolymer protein vaccine capable of generating broad-spectrum cross-neutralization activity as well as preparation method and application of recombinant novel coronavirus RBD tripolymer protein vaccine

A coronavirus and trimer technology, applied in the field of biomedicine, can solve the problems of weakening the affinity of neutralizing antibodies, enhancing the virulence and infectivity of the virus, and reducing the protective effect of vaccines.

Active Publication Date: 2021-08-24
NAT VACCINE & SERUM INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the mutation may increase the affinity of the virus to the ACE2 receptor and weaken the effect of neutralizing antibodies,...

Method used

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  • Recombinant novel coronavirus RBD tripolymer protein vaccine capable of generating broad-spectrum cross-neutralization activity as well as preparation method and application of recombinant novel coronavirus RBD tripolymer protein vaccine
  • Recombinant novel coronavirus RBD tripolymer protein vaccine capable of generating broad-spectrum cross-neutralization activity as well as preparation method and application of recombinant novel coronavirus RBD tripolymer protein vaccine
  • Recombinant novel coronavirus RBD tripolymer protein vaccine capable of generating broad-spectrum cross-neutralization activity as well as preparation method and application of recombinant novel coronavirus RBD tripolymer protein vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1: Novel coronavirus RBD trimeric protein designed based on protein structure and computational biology

[0109]By comparing and analyzing the representative mutant strains and prototype strains of the new coronavirus (as shown in Table 1), the Beta (B.1.351) mutant strain had 3 amino acid mutations, namely K417N, E484K and N501Y; The Kappa (B.1.617.1) mutant had 2 amino acid mutations, namely L452R and E484Q. Among them, E484K is considered to be the most important site mutation leading to immune escape. Through molecular dynamics simulation and free energy calculation, it is found that the E484K mutation will lead to a significant decrease in the affinity of RBD and multiple neutralizing monoclonal antibodies, listed by WHO Among the 10 mutant strains classified as VOC and VOI, 6 mutant strains all contained the E484K mutation. In addition, the L452R mutation has also been proven to be able to escape neutralizing antibodies and the serum of recovered patients...

Embodiment 2

[0116] Example 2: Trimeric protein expression, purification and identification

[0117] According to the codon bias of the CHO cell expression system, the nucleotide sequence encoding protein A to protein E (amino acid sequence shown in SEQ ID NO.4 to SEQ ID NO.8) is codon optimized, protein A (amino acid sequence The optimized nucleotide sequence for SEQ ID NO.4) is shown in SEQ ID NO.9, and the optimized nucleotide sequence for protein B (amino acid sequence is SEQ ID NO.5) is shown in SEQ ID NO.10, The optimized nucleotide sequence of protein C (amino acid sequence is SEQ ID NO.6) is shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14, protein D (amino acid The optimized nucleotide sequence of sequence is SEQ ID NO.7) as shown in SEQ ID NO.15, and the optimized nucleotide sequence of protein E (amino acid sequence is SEQ ID NO.8) is as SEQ ID NO.16 or SEQ ID NO.15 Shown in ID NO.17.

[0118] After constructing the CHO cell expression vector, it was transfect...

Embodiment 3

[0120] Example 3: Binding Biological Analysis with Neutralizing Monoclonal Antibodies

[0121] Purified trimeric protein A, protein B, protein C, and trimeric protein (by connecting 3 amino acid fragments shown in SEQ ID No.1 to form amino acid sequence shown in SEQ ID No.18) Protein, obtained by recombinant expression and chromatographic purification of 293FT cells or CHO cells) and dimeric protein (composed of 2 amino acid fragments shown in SEQ ID No.1 connected sequentially to form the amino acid sequence shown in SEQ ID No.19 Protein, recombinantly expressed and chromatographically purified in 293FT cells or CHO cells), RBD protein (manufacturer: Beijing Yiqiao Shenzhou Technology Co., Ltd.; article number: 40592-V08B), consistent with the mutation site of Beta (B.1.351) strain virus RBD proteins (K417N, E484K, N501Y; manufacturer: Beijing Yiqiao Shenzhou Technology Co., Ltd.; article number: 40592-V08H85), RBD proteins (L452R, E484Q; Manufacturer: Beijing Yiqiao Shenzho...

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Abstract

The invention discloses a recombinant RBD trimer protein capable of simultaneously generating cross neutralization activity aiming at multiple novel coronavirus (SARS-CoV-2) epidemic strains, the trimer protein is composed of subunits of three novel coronavirus S protein RBD regions, and the amino acid sequences of the three novel coronavirus RBD regions are the same or at least one is different; and when the amino acid sequences of the three novel coronavirus RBD regions are the same, the amino acid sequences are the amino acid sequences shown as SEQ ID No.2 or SEQ ID No.3, or sequences with more than 95% of homology with the amino acid sequences shown as SEQ ID No.2 or SEQ ID No.3. The RBD trimer protein is taken as an antigen and supplemented with an adjuvant to immunize an organism, so that a high-titer neutralizing antibody aiming at various novel coronavirus epidemic strains can be generated at the same time, and the antibody has certain broad spectrum and can be used for treating and/or preventing novel coronavirus infection and/or novel coronavirus diseases.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant novel coronavirus RBD trimer protein vaccine producing broad-spectrum cross-neutralizing activity, its preparation method and application. Background technique [0002] Novel coronavirus (SARS-CoV-2), belonging to Nidovirales, Coronaviridae, Orthocoronavirus subfamily, Betacoronavirus genus, Sarbecovirus subgenus, SARS-like virus species, single-stranded positive-sense RNA virus , with an envelope, the full length of the genome is about 29.9kb, most of which encode non-structural proteins, involved in virus replication and translation, and a small part of the sequence encodes structural proteins, such as: S protein (spike protein,), M protein (membrane protein), E protein (envelope protein) and N protein (nucleo protein). In addition, there are several accessory proteins: 3a, 3b, p6, 7a, 7b, 8b, 9b and orf14, which are all involved in virus assembly. The S, M and E prot...

Claims

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Application Information

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IPC IPC(8): C07K14/165C07K19/00C12N15/62C12N15/85C12N5/10A61K39/215A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61P31/14C12N2770/20022C12N2770/20034C07K2319/00A61K2039/53Y02A50/30
Inventor 李启明梁宇张靖苏计国韩子泊邵帅侯亚楠张浩陈实靳玉琴张学峰杜丽芳侯俊伟马智静雷泽华郑凡唐芳刘兆明刘宁
Owner NAT VACCINE & SERUM INST
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