CN-122011200-A - Preparation and application of bispecific nano antibody targeting PEDV and PDCoV

Abstract

The invention relates to the technical field of biology, and discloses preparation and application of a bispecific nano antibody targeting PEDV and PDCoV. The invention constructs the bispecific nanometer antibody targeting PEDV and PDCoV by utilizing flexible connecting peptide (G4S) 3 aiming at the S protein RBD region of PEDV and PDCoV, can synchronously and specifically bind with RBD antigens of PEDV and PDCoV, does not generate cross reaction with other diarrhea related viruses such as TGEV, SADS-CoV and the like, has excellent specificity and applicability, can be used as an ideal detection tool for 'one-tube double detection', remarkably improves the diagnosis accuracy of PEDV/PDCoV mixed infection samples, effectively breaks the technical bottleneck of the current PEDV and PDCoV prevention and control, and has outstanding innovation, practical value and industrialization conversion prospect.

Inventors

• HU XIANGYUN
• CAO YANHONG
• CHEN SHAOMEI
• Xuan Zeyi
• WANG JIAN
• LI HUI

Assignees

• 广西农业职业技术大学
• 广西大学

Dates

Publication Date

20260512

Application Date

20260330

Claims (10)

1. 1. Bispecific nanobody targeting PEDV and PDCoV, characterized in that it comprises a first antigen binding domain PEDV-VHH specific for PEDV and a second antigen binding domain PDCoV-VHH specific for PDCoV, the amino acid sequence of PEDV-VHH being shown in SEQ ID No.1, and the amino acid sequence of PDCoV-VHH being shown in SEQ ID No. 2.
2. 2. The bispecific nanobody according to claim 1, wherein the first antigen binding domain PEDV-VHH and the second antigen binding domain PDCoV-VHH are connected by a linker.
3. 3. The bispecific nanobody according to claim 2, wherein the amino acid sequence of the linker is shown in SEQ ID No. 4.
4. 4. The bispecific nanobody of any of claims 1-3, wherein the amino acid sequence of said bispecific nanobody is shown in SEQ ID No. 3.
5. 5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the bispecific nanobody of any of claims 1-3 or the bispecific nanobody of claim 4.
6. 6. A vector comprising the nucleic acid molecule of claim 5.
7. 7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.
8. 8. A method of preparing a bispecific nanobody according to any one of claims 1 to 3 or a bispecific nanobody according to claim 4, wherein the method is to culture the host cell according to claim 7 under conditions allowing expression of the protein and to recover the bispecific antibody from the cultured host cell culture.
9. 9. A reagent or kit for detecting PEDV and/or PDCoV, comprising the bispecific nanobody of claim 1.
10. 10. Use of a bispecific nanobody according to claim 1 or a bispecific nanobody according to claim 2 for the manufacture of a medicament for the prophylaxis or treatment of PEDV and/or PDCoV infections.

Description

Preparation and application of bispecific nano antibody targeting PEDV and PDCoV Technical Field The invention relates to the field of biotechnology, in particular to preparation and application of a bispecific nanometer antibody targeting PEDV and PDCoV. Background Porcine Epidemic Diarrhea Virus (PEDV) and Porcine delta coronavirus (Porcine Deltacoronavirus, PDCoV) are Porcine enterocoronaviruses, are two important pathogens causing acute watery diarrhea, vomiting, dehydration and even death of piglets, and often present mixed infection situations in farms, which seriously threatens the healthy development of the global pig industry and causes huge economic loss. PEDV is a member of the coronaviridae genus a, whose Receptor-Binding Domain (RBD) of Spike (S) protein initiates infection primarily by interaction with sialic acid receptors on the host cell membrane. PDCoV belong to the genus Deltacoronavirus of the family Coronaviridae, and the RBD of the encoded S protein specifically recognizes and binds to the aminopeptidase N (Aminopeptidase N, APN) receptor on the surface of host cells and mediates viral invasion. The S protein RBD of two viruses is a core functional area of virus invasion, is a main antigen target for inducing an organism to generate a neutralizing antibody, and is a core target for disease diagnosis and antiviral strategy development. At present, PEDV is mainly controlled by traditional vaccines, but has the defects of limited cross protection and insufficient coverage of variant strains, and PDCoV has no commercial vaccine lot, and researches show that the PEDV has wide host tropism (including human cell lines), reveals potential cross-species transmission risks, and has been brought into the focus of attention in the field of public health. Aiming at prevention and control of PEDV/PDCoV mixed infection, a novel molecular tool with high speed, accuracy and broad spectrum is lacking at present. The existing diagnosis method depends on polyclonal antibody or monoclonal antibody, is difficult to distinguish PEDV, PDCoV and other co-epidemic porcine enterocoronavirus (such as porcine transmissible gastroenteritis virus TGEV and porcine acute diarrhea syndrome virus SADS-CoV), is easy to cause false positive or missed detection, and cannot meet the accurate diagnosis requirement of mixed infection. The Nanobody (Nanobody) is derived from a camelid natural heavy chain antibody variable region (VHH), has the advantages of small molecular weight (about 12-15 kDa), high stability (extreme pH and high temperature tolerance), strong tissue penetrability, easiness in genetic engineering transformation and the like, and has good application prospects in animal epidemic disease detection and treatment. The bispecific nanometer antibody can be connected with two specific VHH structural domains targeting different antigens in series through connecting peptide, so that the simultaneous identification of two pathogenic targets is realized, and a new research direction is provided for mixed infection prevention and control. However, no nanobody targeting both PEDV and PDCoV S protein RBD domains has been reported at present, and there is a significant technical gap in this field. Disclosure of Invention In view of the above, it is necessary to construct a nanobody for PDCoV and the S protein RBD domain of PEDV, which can simultaneously target and verify PEDV and PDCoV, can avoid cross reaction with other viruses causing diarrhea in pigs such as TGEV, SADS-CoV, and the like, has high stability and double-targeting recognition capability, can be used as an ideal detection tool for one-tube double-detection, and can significantly improve the diagnosis accuracy of PEDV/PDCoV mixed infection samples. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: Bispecific nanobodies targeting PEDV and PDCoV comprising a first antigen binding domain PEDV-VHH specific for PEDV and a second antigen binding domain PDCoV-VHH specific for PDCoV, wherein the amino acid sequence of PEDV-VHH is shown in SEQ ID No.1 and the amino acid sequence of PDCoV-VHH is shown in SEQ ID No. 2. Further, the first antigen binding domain PEDV-VHH and the second antigen binding domain PDCoV-VHH are linked by a linker. Furthermore, the amino acid sequence of the connector is shown as SEQ ID NO. 4. The amino acid sequence of the bispecific nanometer antibody is shown as SEQ ID NO. 3. The invention also includes an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the bispecific nanobody. The invention also includes vectors comprising said nucleic acid molecules. The invention also includes a host cell comprising said nucleic acid molecule or said vector. The invention also includes a method of making the bispecific nanobody by culturing the host cell under conditions that allow expression of the protein, and recovering the bispecific antibody from the cultured host cell cul