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CN-116535517-B - Construction and application of PRRSV live vector vaccine strain for recombinant expression of PEDV S protein receptor RBD structural domain

CN116535517BCN 116535517 BCN116535517 BCN 116535517BCN-116535517-B

Abstract

The invention provides construction and application of a PRRSV live vector vaccine strain for recombinant expression of a PEDV S protein receptor RBD structural domain, and results show that the rescued recombinant virus rHuN-F112-SRBD 1 can express a dominant antigen region of the PEDV S protein RBD functional domain, and the biological characteristics of the recombinant virus are similar to those of a parent virus. The nucleotide sequence of SRBD gene introduced in each generation of recombinant virus and the amino acid sequence coded by the same are not deleted or mutated, and IFA and Western Blot detection results show that SRBD genes introduced in different generations of recombinant viruses rHuN-F112-SRBD 1 can be stably expressed, and the expression of self protein of the parent virus is not influenced, so that the recombinant virus rHuN-F112-SRBD 1 strain has genetic stability. The result of immunogenicity analysis on recombinant virus rHuN-F112-SRBD 1 shows that the recombinant virus rHuN-F112-SRBD strain can induce the generation of specific antibodies aiming at PEDV S protein and PRRSV2 in the bodies of piglets after immunization, and the induced antibodies have neutralization effect on PEDV epidemic strains, so that the recombinant virus can be used for preventing development and development of PRRS and PED novel genetic engineering vaccines.

Inventors

  • ZHOU YANJUN
  • LI GUOXIN
  • YU HAI
  • LI LIWEI
  • ZHENG HAO
  • TONG GUANGZHI
  • Lao Mengqin
  • CHEN PENGFEI
  • YU LINGXUE
  • GAO FEI
  • JIANG YIFENG
  • LIU CHANGLONG
  • TONG WU

Assignees

  • 中国农业科学院上海兽医研究所

Dates

Publication Date
20260512
Application Date
20220712
Priority Date
20220706

Claims (10)

  1. 1. A fusion epitope peptide aiming at an S protein receptor RBD domain of PEDV, wherein a dominant antigen region of the S protein RBD domain is a Gene sequence of a PEDV epidemic variation strain FJzz strain with accession number MK288006 published in Gene Bank, amino acid sequence analysis is carried out by DNASTAR software, a C-terminal domain of an S1 subunit in the S Gene is selected, nucleotide positions are 1495-2377nt, encoding amino acid positions are aa 496-792, the region is mainly distributed on the surface of an S protein extracellular domain trimer, the fusion epitope peptide comprises the PEDV potential RBD domain, an antigenicity index is up to 3.4, and a signal peptide sequence of the S Gene is introduced at the front end of the fusion epitope peptide, and the target Gene of the fusion epitope peptide aiming at the S protein receptor RBD domain of the PEDV is named RBD1 as shown in SEQ ID NO. 1.
  2. 2. The fusion epitope peptide of claim 1, wherein the N-terminus or the C-terminus of the epitope peptide is further coupled with a polypeptide label, and the polypeptide label is a biotin label or a fluorescent label.
  3. 3. Use of the fusion epitope peptide of the RBD domain of the S protein receptor for PEDV according to claim 1 for the preparation of a bivalent genetically engineered live vaccine against PRRS and PED.
  4. 4. A recombinant PRRSV pHuN-F112-SRBD 1 vaccine strain expressing the fusion epitope peptide of claim 1 directed against the RBD domain of the PEDV S protein receptor, wherein the dominant antigen region of PEDV S protein is the fusion epitope peptide of claim 1, and the full-length nucleotide sequence of the recombinant PRRSV pHuN-F112-SRBD vaccine strain is shown in SEQ ID No. 12.
  5. 5. Use of the fusion epitope peptide of the RBD domain of the S protein receptor of PEDV according to claim 1 for the preparation of a medicament for preventing PEDV.
  6. 6. Use of the recombinant PRRSV pHuN-F112-SRBD 1 vaccine strain of claim 4 in the manufacture of a medicament for the prophylaxis of PRRS and PED.
  7. 7. Use of the fusion epitope peptide of the RBD domain of the S protein receptor for PEDV according to claim 1 as a screening target for anti-PED infection drugs.
  8. 8. A pharmaceutical composition for preventing PED infection comprising the fusion epitope peptide of claim 1 directed against the RBD domain of the S protein receptor of PEDV and a pharmaceutically acceptable adjuvant.
  9. 9. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is a dosage form for nasal administration, and the dosage form is any one selected from the group consisting of a spray, a nose drop, a powder, a gel preparation and a microsphere preparation.
  10. 10. A diagnostic reagent for PED infection comprising the S protein fusion epitope peptide of claim 1, the fusion epitope peptide of the S protein receptor RBD domain for PEDV of claim 1 coated on a detection carrier selected from any one of a polystyrene microplate, a colloidal gold reagent strip, magnetic beads and a microfluidic chip.

Description

Construction and application of PRRSV live vector vaccine strain for recombinant expression of PEDV S protein receptor RBD structural domain Technical Field The invention relates to the field of biology, and relates to construction and application of a PRRSV live vector vaccine strain for recombinant expression of dominant antigen epitope peptide of PEDV S protein receptor RBD structural domain. Background Porcine epidemic diarrhea (Porcine EPIDEMIC DIARRHEA, PED) is a highly contagious intestinal infectious disease that causes huge economic losses in the world pig industry, and is widely and severely prevalent in newborn piglet groups, causing significant economic losses to the world pig industry. Porcine Epidemic Diarrhea Virus (PEDV) is the main pathogen causing the disease, PEDV can infect pigs of all ages and cause clinical symptoms, but the severity and mortality of infected pigs are inversely proportional to the age of pigs, wherein the influence on newborn suckling piglets within 10 days of age is the greatest, and the disease incidence and mortality of newborn suckling piglets can be even as high as 80-100% mainly due to watery diarrhea, vomiting, dehydration, emaciation, death and the like. PEDV is a enveloped single-stranded positive-stranded RNA virus belonging to the order of the family of the mantle viridae, the genus coronaviridae, a member of the subgroup of the alpha-coronaviruses. A variant PEDV strain newly appears at the end of 2010, has strong pathogenicity to newborn suckling piglets and high transmission speed, and causes large-area outbreak of acute diarrhea epidemic situation of the suckling piglet group, and the morbidity and mortality rate are high. The mutated PEDV has high transmission speed and strong infectivity, so that the mutated PEDV becomes the first viral infectious disease which threatens newborn piglets most at present. PEDV isolates before 2010 are currently classified as a G1 subgroup, whereas PEDV variant strains after 2010 are classified as a G2 subgroup. PEDV genome is about 28kb in length and comprises 5 'and 3' untranslated regions (UTR) and at least 7 Open Reading Frames (ORFs), in order ORF1a, ORF1b, spike protein (S), helper protein ORF3, envelope protein (E), membrane protein (M) and nucleocapsid protein (N). Wherein ORF1 occupies about two thirds of the full length of the genome at the 5' end, encodes two multimeric proteins pp1a and pp1ab, pp1a and pp1ab being cleaved by papain and 3C-like serine protease into 16 nonstructural proteins NSP1-16, which are primarily involved in viral replication, transcription, translation and viral protein processing. PEDV S glycoprotein is a homotrimeric membrane glycoprotein located on the surface of viral particles, consisting of two subunits S1 and S2, S1 mediating PEDV binding to host receptors, S2 inducing membrane fusion and PEDV invasion. The N-terminus (NTD) of the S1 protein has neuraminidase binding activity, the C-terminus (CTD) comprises a cell Receptor Binding Domain (RBD) which is mainly responsible for recognizing host cell surface receptors and mediating virus adsorption, and antibodies to PEDV RBD can inhibit PEDV S protein from recognizing host cell receptors, so RBD region is also a main target of host immune defense during virus infection and is generally used as a target for vaccine development. Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is an infectious disease of great economic significance, and the main clinical symptoms are respiratory disease and sow reproductive dysfunction of pigs of all ages. The causative agent of PRRS is Porcine Reproductive and Respiratory Syndrome (PRRSV), which has been one of the major challenges facing the global pig industry since the first report in 1987, and is still constantly mutating to result in new epidemics, PRRSV infection has been reported to cause losses of about $6.64 billion per year to the pig industry in the united states alone. PRRSV is also a single-stranded positive strand RNA virus, and PRRSV-infected target cells are predominantly monocytes-macrophages, the most typical of which are alveolar macrophages (PAMs). PRRSV is mainly of two genotypes, namely PRRSV1 and PRRSV2, and the nucleotide and amino acid homology between the two genotype strains is only 60% and 56% respectively, but the clinical symptoms, pathological injury characteristics, tissue tropism and the like caused by the two types of strains are similar. PRRSV1 is prevalent in europe and PRRSV2 is prevalent in america and asia, but there is also a report of isolation of both types of strains in europe, north america and asia. At present, PRRSV2 strains are mainly popular in the pig group in China, and the PRRSV strains which are popular in clinic are complex and various because the PRRSV is easy to mutate and recombine, and the phenomenon that different subtype strains including classical CH-1a PRRSV, highly pathogenic PRRSV, NADC30-