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CN-121992038-A - Recombinant plasmid and application thereof

CN121992038ACN 121992038 ACN121992038 ACN 121992038ACN-121992038-A

Abstract

The invention discloses a recombinant plasmid and application thereof, and belongs to the technical field of genetic engineering. The invention constructs a recombinant plasmid which comprises a signal peptide, a receptor binding domain RBD, heptad repeat sequences HR1 and HR2 and a flexible connecting peptide. Wherein the signal peptide has the function of guiding protein transportation and localization, and can accurately guide the relevant peptide segment to the proper cell position. And the HR1 and HR2 peptide fragments can automatically assemble into a 6-helix bundle structure. The S1 protein plays a central role in viral infection and immune response, and proper folding is the basis for ensuring the functional integrity of the S1 protein. The S1 protein is prepared by the recombinant plasmid constructed by the invention, and the S1 protein can be correctly folded in cells and is effectively anchored on cell membranes. This not only allows the S1 protein to better display its epitope, but also provides advantages for subsequent immune responses.

Inventors

  • ZHANG QUAN
  • LIU HANPING
  • QIU WENYING
  • XU JING
  • LI CHENGYI
  • DU DEYAN

Assignees

  • 华派生物技术(集团)股份有限公司

Dates

Publication Date
20260508
Application Date
20260206

Claims (10)

  1. 1. The ZY-2 PEDV S1-RBD-HR/Trimer recombinant plasmid is characterized by comprising a signal peptide, a receptor binding domain RBD of a ZY-2-PEDV S1 subunit, heptapeptide repeated sequences HR1 and HR2 of ZY-2 PEDV S2 and a flexible connecting peptide, wherein the signal peptide and the RBD are connected in series through the flexible connecting peptide and the HR1 and HR 2; wherein the HR1 has any one of the following nucleotide sequences: (A1) A nucleotide sequence as shown in SEQ ID NO. 4; (A2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases in the nucleotide sequence shown as SEQ ID NO.4 and codes for the same functional protein; (A3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 4; the HR2 has any one of the following nucleotide sequences: (B1) A nucleotide sequence as shown in SEQ ID NO. 5; (B2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases of the nucleotide sequence shown as SEQ ID NO.5 and codes for the same functional protein; (B3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 5; The receptor binding domain RBD has any of the following nucleotide sequences: (C1) A nucleotide sequence as shown in SEQ ID NO. 12; (C2) A nucleotide sequence encoding the same functional protein obtained by substitution, insertion or deletion of one or more bases of the nucleotide sequence shown as SEQ ID NO. 12; (C3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 12; the signal peptide is a signal peptide capable of guiding the secretory expression of the protein.
  2. 2. The ZY-2 PEDV S1-RBD-HR/primer recombinant plasmid of claim 1, wherein the signal peptide is GP67 signal peptide having any one of the following nucleotide sequences: (D1) A nucleotide sequence as shown in SEQ ID NO. 3; (D2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases in the nucleotide sequence shown as SEQ ID NO.3 and codes for the same functional protein; (D3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 3.
  3. 3. The ZY-2 PEDV S1-RBD-HR/primer recombinant plasmid of claim 1, wherein the flexible linker peptide has any of the following nucleotide sequences: (E1) A nucleotide sequence as shown in SEQ ID NO. 2; (E2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases in the nucleotide sequence shown as SEQ ID NO.2 and codes for the same functional protein; (E3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 2.
  4. 4. The ZY-2 PEDV S1-RBD-HR/primer recombinant plasmid of claim 1, wherein the C-terminus of the ZY-2 PEDV S1-RBD-HR/primer recombinant plasmid is further fused with a his histone tag.
  5. 5. The ZY-2 PEDV S1-RBD-HR/primer recombinant plasmid according to any one of claims 1 to 4, wherein the ZY-2 PEDV S1-RBD-HR/primer has any one of the following nucleotide sequences: (F1) A nucleotide sequence shown as SEQ ID NO. 1; (F2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases in the nucleotide sequence shown as SEQ ID NO.1 and codes for the same functional protein; (F3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 1.
  6. 6. Use of the ZY-2 PEDV S1-RBD-HR/primer recombinant plasmid according to any one of claims 1 to 5 for the preparation of porcine epidemic diarrhea virus S1 protein.
  7. 7. A method for preparing porcine epidemic diarrhea virus S1 protein, comprising the steps of: (1) Inserting the ZY-2 PEDV S1-RBD-HR/Trimer sequence according to any one of claims 1-5 into the XhoI/KpnI site of the Bac-to-Bac baculovirus vector pFastBac-Dual to construct a recombinant vector; (2) Transforming competent cells with the recombinant vector of the step (1) to obtain recombinant bacmid, screening the recombinant bacmid, and then transfecting Sf9 cells to obtain recombinant baculovirus; (3) And (3) infecting the High Five cells with the recombinant baculovirus in the step (2), collecting virus supernatant, and purifying to obtain the recombinant baculovirus.
  8. 8. The method according to claim 7, wherein the construction of the recombinant vector in step (1) specifically comprises the steps of: ① The method comprises the steps of using ZY-2 PEDV S1-RBD-HR/Trimer as a template, and using primers to amplify PEDV-S1-RBD-FL-F and PEDV-S1-RBD-FL-R, wherein the nucleotide sequences of PEDV-S1-RBD-FL-F and PEDV-S1-RBD-FL-R are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7; ② The pFastBac-Dual vector is digested by XhoI enzyme and KpnI enzyme, the digested product is purified and recovered, and then is transformed into competent cells after being connected with ZY-2 PEDV S1-RBD-HR/Trimer by using seamless cloning, and is selectively cultured on a culture medium of amp+ and then positive clones are identified by primer pairs P10-F and P10-R, thus obtaining the recombinant vector pFastBac-Dual-ZY-2 PEDV-S1/RBD-HR/Trimer, wherein the nucleotide sequences of the P10-F and the P10-R are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.
  9. 9. The method according to claim 8, wherein the promoter of the recombinant vector pFastBac-Dual-ZY-2 PEDV-S1/RBD-HR/Trimer is baculovirus late promoter p10.
  10. 10. The method according to claim 7, wherein the High Five cells are infected in step (3) when moi=0.1 to 10.

Description

Recombinant plasmid and application thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to a recombinant plasmid and application thereof. Background Porcine epidemic diarrhea (PED, porcine EPIDEMIC DIARRHEA) is an important and challenging disease in the pig industry. The disease has the characteristics of acute attack and high infectivity, and brings great economic loss to pig industry. Infection with Porcine Epidemic Diarrhea Virus (PEDV) destroys the digestive system of the pig, resulting in symptoms such as loss of appetite, vomiting, and severe diarrhea in the pig. Even if the medicine is used for treatment, piglets can be seriously affected by factors such as poor growth and the like, and serious economic loss is brought to the pig industry. Thus, prevention and control of porcine epidemic diarrhea is a vital task in the pig industry. PEDV S proteins belong to the class I transmembrane glycoproteins, which are about 1383 amino acids in length, and comprise an S1 subunit responsible for receptor binding (e.g., sialic acid), an N-terminal domain (NTD), a C-terminal domain (CTD), and a plurality of neutralizing epitopes. S2 subunit mediates membrane Fusion and comprises Fusion Peptide (Fusion Peptide), heptad repeat region (HR 1/HR 2) and transmembrane domain. Trimerization domain located in the HR1/HR2 region of the S2 subunit, drives trimer assembly by the formation of a coiled coil structure (Coiled-coil). Different expression systems significantly affect the correct assembly and stability of PEDV S proteins due to differences in post-translational modification ability and folding mechanism. The existing part of research team uses prokaryotic expression system to express and purify PEDV-S1 protein, but the recombinant protein obtained by the method does not have natural conformation and can not simulate PEDV-S1 protein molecule on virus surface. Therefore, it is very necessary to develop a recombinant plasmid for efficiently preparing porcine epidemic diarrhea virus S1 protein. Disclosure of Invention In order to solve the defects in the prior art, the invention aims to provide a recombinant plasmid and application thereof, so as to efficiently prepare porcine epidemic diarrhea virus S1 protein. The technical scheme of the invention for solving the technical problems is that a ZY-2 PEDV S1-RBD-HR/Trimer recombinant plasmid is provided, which comprises a signal peptide, a receptor binding domain RBD of ZY-2-PEDV S1 subunit, heptapeptide repetitive sequences HR1 and HR2 of ZY-2 PEDV S2 and a flexible connecting peptide, wherein the signal peptide and the RBD are connected in series through the flexible connecting peptide and HR1 and HR 2; Wherein HR1 has any one of the following nucleotide sequences: (A1) A nucleotide sequence as shown in SEQ ID NO. 4; (A2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases in the nucleotide sequence shown as SEQ ID NO.4 and codes for the same functional protein; (A3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 4; HR2 has any one of the following nucleotide sequences: (B1) A nucleotide sequence as shown in SEQ ID NO. 5; (B2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases of the nucleotide sequence shown as SEQ ID NO.5 and codes for the same functional protein; (B3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 5; the receptor binding domain RBD has any of the following nucleotide sequences: (C1) A nucleotide sequence as shown in SEQ ID NO. 12; (C2) A nucleotide sequence encoding the same functional protein obtained by substitution, insertion or deletion of one or more bases of the nucleotide sequence shown as SEQ ID NO. 12; (C3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 12; The signal peptide is a signal peptide capable of directing secretory expression of the protein. Further, the signal peptide is GP67 signal peptide having any one of the following nucleotide sequences: (D1) A nucleotide sequence as shown in SEQ ID NO. 3; (D2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases in the nucleotide sequence shown as SEQ ID NO.3 and codes for the same functional protein; (D3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO. 3. Further, the flexible linker peptide has any one of the following nucleotide sequences: (E1) A nucleotide sequence as shown in SEQ ID NO. 2; (E2) A nucleotide sequence which is obtained by substituting, inserting or deleting one or more bases in the nucleotide sequence shown as SEQ ID NO.2 and codes for the same functional protein; (E3) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ