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CN-121971594-A - Hepatitis E virus recombinant annular RNA nanoparticle vaccine taking MS2 phage pseudovirus particles as packaging vectors and construction method thereof

CN121971594ACN 121971594 ACN121971594 ACN 121971594ACN-121971594-A

Abstract

The invention belongs to the technical field of vaccines, in particular to a hepatitis E virus recombinant circular RNA nanoparticle vaccine taking MS2 phage pseudovirus particles as packaging vectors and a construction method thereof, and the construction method of the hepatitis E virus recombinant circular RNA nanoparticle vaccine successfully constructs a novel hepatitis E circular RNA vaccine taking MS2 phage pseudovirus particles as vectors, thereby providing a new thought for research and development and application of the hepatitis E vaccine. The method is based on the combined application of an armored RNA technology and a Clean-PIE technology to realize the delivery of HEVp annular mRNA vaccine particles, and nanoparticle molecular vaccines are specifically combined with PAC aptamer sequences embedded in annular RNA (circRNA) molecules through PAC binding sites on the inner surfaces of MS2 phage pseudovirus particles, so that the annular RNA is efficiently packaged and encapsulated in the particles to form MS2DIMERTATHIS-HEV CIRCRNA nanoparticle vaccines, and a novel technical idea and method are provided for preventing, controlling and controlling hepatitis E.

Inventors

  • XU YIFAN
  • CHEN ZELIANG
  • MA MINGXIAO
  • GAO SHENYANG
  • XUAN HAO
  • LI HUI
  • LI MENGYING
  • ZHANG YIXIN
  • Sha Xingliang
  • LI SHUANG
  • CHI YANG
  • LI DANDAN

Assignees

  • 锦州医科大学

Dates

Publication Date
20260505
Application Date
20260129

Claims (10)

  1. 1. A method for constructing a hepatitis e virus recombinant circular RNA nanoparticle vaccine, comprising: (1) Synthesizing in vitro hepatitis E circular mRNA comprising an antigen gene of hepatitis E; (2) And constructing MS2 phage pseudovirus reconstruction particles, and packaging the annular mRNA by using the MS2 phage pseudovirus reconstruction particles to obtain the hepatitis E virus recombinant annular RNA nanoparticle vaccine by taking the MS2 phage pseudovirus particles as packaging vectors.
  2. 2. The method of constructing a hepatitis e virus recombinant circular RNA nanoparticle vaccine of claim 1, wherein the in vitro synthesis of hepatitis e circular mRNA comprises: Screening HEV-g4P239 gene sequences and constructing plasmids, wherein the structural form of the screened HEV-g4P239 gene sequences is that a T4td intron fragment II-eGFP truncated fragment II-EV29-P239-T2A-eGFP truncated fragment I-T4td intron fragment I, the sequence of the T4td intron fragment I is shown as SEQ ID NO.1, the sequence of the T4td intron fragment II is shown as SEQ ID NO.2, the sequence of the eGFP truncated fragment I is shown as SEQ ID NO. 3, the sequence of the eGFP truncated fragment II is shown as SEQ ID NO. 4, the sequence of the Ev29 is shown as SEQ ID NO. 5, the sequence of the P239 is shown as SEQ ID NO. 6, the sequence of the T2A is shown as SEQ ID NO. 7, and connecting the HEV-g4P239 gene sequences to pUC57 vectors to obtain pUC 57-4 HEVp plasmids shown as follows; constructing a primer for amplifying the plasmid pUC57-gt4HEVp239, carrying out PCR amplification on the plasmid pUC57-gt4HEVp239, recovering a PCR product, preparing linear mRNA by in vitro transcription of a purified linear plasmid template, and cyclizing the linear mRNA to obtain circular mRNA.
  3. 3. The method for constructing the recombinant circular RNA nanoparticle vaccine for hepatitis E virus according to claim 2, wherein the primers for constructing the amplification plasmid pUC57-gt4HEVp239 comprise the steps of designing upstream and downstream primers for T4td intron fragment II and T4td intron fragment I at two ends of circular mRNA to obtain amplification primers, namely, a circRNA amplification F and a circRNA amplification R, wherein the sequence of the circRNA amplification F is shown as SEQ ID NO. 8, and the sequence of the circRNA amplification R is shown as SEQ ID NO. 9.
  4. 4. The method for constructing a recombinant circular RNA nanoparticle vaccine for hepatitis E virus according to claim 3, further comprising performing circular identification on the circular RNA, and verifying whether the circular RNA is successfully cyclized by three circular identification methods, namely RNase R cleavage identification, RT-PCR identification and sequencing identification; in the RT-PCR identification, the upstream and downstream primers of the eGFP cut-off fragment I and the eGFP cut-off fragment II are designed, wherein the primers comprise a circRNA identification F and a circRNA identification R, the sequence of the circRNA identification F is shown as SEQ ID NO. 10, and the sequence of the circRNA identification R is shown as SEQ ID NO. 11.
  5. 5. The method for constructing the recombinant circular RNA nanoparticle vaccine for hepatitis E virus according to claim 1, wherein the construction of the MS2 phage pseudovirus recombinant particle comprises the steps of referring to MS2 phage pseudovirus particle and HEV genome sequences, synthesizing a target gene MS2 dimer-HEV by connecting an MS2 dimer gene and an HEV ORF2/3 conserved region in series, subcloning the target gene MS2 dimer-HEV into an expression vector for plasmid synthesis and cloning, and obtaining the MS2 phage pseudovirus recombinant particle through plasmid expression and purification.
  6. 6. The method for constructing a recombinant circular RNA nanoparticle vaccine against hepatitis E virus according to claim 5, wherein the subcloning of the target gene MS2 dimer-HEV into the expression vector for plasmid synthesis and cloning comprises subcloning the MS2 dimer-HEV between NcoI and HindIII of pET28b, inserting TAT peptide between a.a.14-15 of the first MS2 monomer, inserting two Kpnl cleavage sites and 6 XHis tag between a.a.13-14 of the second MS2 monomer, and then synthesizing and cloning the plasmid to obtain the plasmid pET28bMS DIMERTATHIS.
  7. 7. The method according to claim 6, further comprising verifying the MS2 phage pseudovirus recombinant particle, treating the MS2 phage pseudovirus recombinant particle with a universal nuclease, extracting RNA with a viral genome DNA/RNA rapid extraction kit, performing reverse transcription, and respectively performing qPCR and RT-qPCR detection with the RNA and cDNA extraction products as templates, wherein the primer JVHEVF, JVHEVR and probe sequences for qPCR and RT-qPCR detection are shown as SEQ ID NO. 12, the JVHEVR sequence is shown as SEQ ID NO. 13, and the probe sequence is shown as SEQ ID NO. 14.
  8. 8. The method for constructing a recombinant circular RNA nanoparticle vaccine for hepatitis E virus according to claim 1, wherein the step of packaging the circular mRNA by using the MS2 bacteriophage pseudovirus reconstitution particles comprises the steps of adjusting pH to depolymerize the MS2 bacteriophage pseudovirus reconstitution particles, centrifuging to remove impurities, collecting supernatant, adding the supernatant into a ultrafilter tube to obtain ultrafiltrate, and packaging the circular mRNA.
  9. 9. The method for constructing a recombinant circular RNA nanoparticle vaccine for hepatitis E virus according to claim 1, further comprising verifying the expression of the circular mRNA in vitro, transfecting the circular mRNA into 293T cells, and detecting the expression of the circular mRNA in the 293T cells; And (3) verifying the expression of the hepatitis E virus recombinant circular RNA nanoparticle vaccine taking MS2 phage pseudovirus particles as packaging vectors in vitro, namely, transfecting the constructed nanoparticle vaccine into 293T cells, and detecting the expression of the nanoparticle vaccine in the 293T cells.
  10. 10. A hepatitis e virus recombinant circular RNA nanoparticle vaccine using MS2 phage pseudovirus particles as packaging vectors, which is prepared according to the construction method of the hepatitis e virus recombinant circular RNA nanoparticle vaccine of any one of claims 1 to 9.

Description

Hepatitis E virus recombinant annular RNA nanoparticle vaccine taking MS2 phage pseudovirus particles as packaging vectors and construction method thereof Technical Field The invention belongs to the technical field of vaccines, and in particular relates to a hepatitis E virus recombinant annular RNA nanoparticle vaccine taking MS2 phage pseudovirus particles as packaging vectors and a construction method thereof. Background Hepatitis E Virus (HEV) is abbreviated as hepatitis E Virus, and is one of the main causes of acute viral hepatitis in the world. Although usually a self-limiting disease, severe acute renal failure can result in mortality rates as high as 20% -30% in pregnant women, the elderly and chronic liver disease patients. There are four major genotypes of hepatitis E Virus that cause human disease, hepatitis E Virus genotypes 1 and 2 primarily infect humans, while genotypes 3, 4 and 7 primarily infect non-human mammals, occasionally causing zoonotic disease in humans. Currently, only recombinant protein vaccines (such as Hecolin) based on HEV virus-like particles (VLPs) are marketed in batches in China globally, and the vaccines are effective, but have some limitations, such as limited applicable population, lack of sufficient data support for high-risk population, potential blind areas in genotype coverage and incomplete protection. Disclosure of Invention The invention aims to provide a hepatitis E virus recombinant circular RNA nanoparticle vaccine taking MS2 phage pseudovirions as packaging vectors and a construction method thereof, which are used for providing a new technical idea and method for preventing, controlling and controlling hepatitis E. The cyclic RNA (circRNA) is an RNA molecule with a covalent closed cyclic structure, which is discovered in recent years, has no 5' -end cap and 3' -end poly (A) tail, has longer half life in cells than the existing mRNA due to the fact that the cyclic RNA is closed and has resistance to degradation of exonuclease, so that more durable and efficient antigen protein expression can be realized, the circRNA can directly start protein translation through an Internal Ribosome Entry Site (IRES) without depending on a 5' -end cap structure, efficient and continuous expression of a target antigen can be realized, and meanwhile, the circRNA can furthest avoid activating an innate immunosensor of cells, realize invisible translation, reduce side effects and improve protein yield. Therefore, the circRNA technology platform is considered as an ideal candidate of the next generation nucleic acid vaccine, and is expected to overcome the problems of stability, immunogenicity and persistence of the traditional mRNA vaccine. MS2 is a male-specific coliphage whose capsid proteins self-assemble into icosahedral virus-like particles of about 27 nanometers. The MS2 capsid protein naturally has a mechanism for packaging its own RNA, and the packaging site can be fused with the circRNA sequence through genetic engineering, so that the MS2 capsid can efficiently and specifically package the circRNA in vitro to form a 'pseudo virus particle'. Based on the thought, the invention aims to apply the leading-edge circRNA technology to the development of hepatitis E vaccine, construct the annular RNA vaccine targeting HEV core capsid protein p239, and provide a new strategy and experimental basis for developing a new generation of efficient and broad-spectrum hepatitis E vaccine. The recombinant gene type 4 hepatitis E circular RNA vaccine using MS2 phage pseudovirus particles as a vector provided by the invention comprises (i) circRNA-p239 (circular mRNA) and (ii) MS2DIMERTATHIS pseudovirus particles by using circular RNA technology and armored RNA technology. The circRNA-p239 is prepared by introducing p239 and PAC aptamer based on the Clean PIE method disclosed in the prior art such as Zuo et al, wherein the circRNA-p239 is circular RNA comprising the T4td intron fragment II-eGFP fragment II-EV29-p239-T2A-eGFP fragment I-T4td intron fragment I along the 5'-3' direction, and inserting the elements formed by these gene fragments into pUC57 vector. Here, the p239 gene is selected from the HEV genome open reading frame 2 (ORF 2) full-length sequence (a.a.Met1-Ser 660) gene (gt) 4 (Genbank number: OP 185389). The invention selects p239-gt4 and the unique hepatitis E vaccine HEV239 Hecolin currently on the market worldwide, a.a.368-606 to carry out amino acid sequence and nucleotide sequence comparison, and obtains the p239 nucleotide sequence of the coding gene type 4 hepatitis E. PAC aptamer is introduced in the construction process of circRNA-p239, and RNA is mediated to be packaged into virus-like particles. The pET28b-MS2DIMERTATHIS (plasmid of MS2DIMERTATHIS pseudo-virus particle) constructed by the invention comprises the following three parts of connecting two MS2 capsid protein monomers in series to form an MS2dimer, inserting an HEV ORF2/3 conservation region behind an