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CN-122012621-A - Universal baculovirus transfer vector and application thereof

CN122012621ACN 122012621 ACN122012621 ACN 122012621ACN-122012621-A

Abstract

The invention provides a general type baculovirus transfer vector and application thereof, wherein the general type baculovirus transfer vector comprises elements (a) a 3CD protease coding gene containing Q184L mutation, (b) a baculovirus homologous repeat region enhancer hr1, (c) a baculovirus late expression factor lef5 gene and an expression regulation element thereof, (d) a polh-pSel promoter for driving P1 protein expression, and (e) a target serotype enterovirus P1 protein, wherein the elements (a) - (d) form a general core framework, and the element (e) allows insertion or replacement of a specific P1 protein gene. The invention remarkably enhances the stability of 3CD, greatly improves the yield and the assembly efficiency of VLP, has outstanding universality and provides a high-efficiency technical platform for developing broad-spectrum enterovirus vaccines.

Inventors

  • SHEN SHUO
  • PEI JIE
  • LI BINGTUAN
  • Liu Ruilun
  • XIAO AO
  • WANG MENGJUN
  • GUO JING

Assignees

  • 武汉生物制品研究所有限责任公司

Dates

Publication Date
20260512
Application Date
20260128

Claims (10)

  1. 1. A universal baculovirus transfer vector comprising the elements of: (a) A 3CD protease encoding gene comprising a Q184L mutation; (b) Baculovirus homologous repeat enhancer hr1; (c) Baculovirus late expression factor lef5 gene and expression regulatory element thereof; (d) The polh-pSel promoter for driving the expression of P1 protein; (e) Target serotype enterovirus P1 protein; wherein elements (a) - (d) constitute a general core backbone and element (e) allows for insertion or replacement of a specific P1 protein gene.
  2. 2. The universal baculovirus transfer vector of claim 1, wherein said 3CD protease encoding gene containing Q184L mutation is derived from EV71 virus.
  3. 3. The universal baculovirus transfer vector as claimed in claim 1, wherein the target serotype in element (e) comprises EV71, CVA6, CVA10 or CVA16.
  4. 4. Recombinant baculovirus obtained by transposition of recombinant bacmid comprising the universal baculovirus transfer vector as defined in any one of claims 1-3 through the Bac-to-Bac system.
  5. 5. The method for constructing a recombinant baculovirus as defined in claim 4, comprising the steps of: S1, cloning a P1 protein coding gene of a target serotype enterovirus to a P1 expression site (e) of the general type baculovirus transfer vector; S2, transforming the complete transfer vector obtained in the step (1) into DH10Bac competent cells containing baculovirus shuttle vectors; S3, obtaining recombinant pole grains through resistance and blue and white spot screening; S4, transfecting the recombinant bacmid into an insect host cell, and saving to obtain the recombinant baculovirus.
  6. 6. The method of producing enterovirus-like particles in insect cells from recombinant baculovirus of claim 4, comprising the steps of: SA, infecting insect host cells with recombinant baculovirus; SB, culturing the infected insect host cell; SC, stable 3CD protease containing Q184L mutation in infected insect host cells, effectively cleaves P1 protein, producing VP0, VP3 and VP1 structural proteins; SD, structural proteins self-assemble to form enterovirus VLPs of the target serotype; SE, collecting and purifying VLPs.
  7. 7. The method of claim 6, wherein the insect host cell is an Sf9 or High Five cell.
  8. 8. An enterovirus-like particle prepared by the method of claim 6 or 7.
  9. 9. Use of an enterovirus like particle according to claim 8 in the manufacture of a vaccine for preventing a disease caused by EV71, CVA6, CVA10 or CVA16 enterovirus infection.
  10. 10. Use of an enterovirus like particle according to claim 8 in the preparation of a diagnostic reagent for detecting EV71, CVA6, CVA10 or CVA16 enterovirus infection.

Description

Universal baculovirus transfer vector and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a universal baculovirus transfer vector and application thereof. Background Enteroviruses (especially EV71, CVA6, CVA16, CVA10, etc.) are the major causative agents of hand-foot-and-mouth disease, constituting a serious threat to children's health. The virus-like particle (VLP) has no virus genetic material, retains natural conformational epitope, can excite high-efficiency neutralizing antibody, has excellent safety, and becomes an ideal vaccine candidate form. However, large-scale production of VLPs faces common bottlenecks such as low expression levels, poor assembly efficiency, cumbersome preparation of multiple serotypes, and the like. Baculovirus-insect cell expression systems (such as Bac-to-Bac) are common platforms for producing complex VLPs, but have the problems of poor stability of 3CD protease, insufficient expression level of exogenous proteins, low serotype switching efficiency and the like in enterovirus VLP preparation. There are studies attempting to improve stability through 3CD mutation, but lacking systematic verification of Q184L key site and not solving the problem of multi-serotype applicability, the prior art multi-focusing single element optimization (such as hr enhancer addition or promoter replacement), neglecting the synergistic mechanism of each element, and no integrated vector design integrating "stability mutation+expression enhancement+serotype rapid switching" at present, resulting in high multivalent vaccine development cost. The invention aims to break through the bottleneck through systematic transformation of transfer vectors and realize the production of high-yield, stable and broad-spectrum enterovirus VLPs. Disclosure of Invention In view of the above, the invention provides a general type baculovirus transfer vector and application thereof, which improves the stability of 3CD protease and improves the expression stability and yield of VLP. In order to achieve the above purpose, the invention adopts the following technical scheme: First, the present invention provides a universal baculovirus transfer vector comprising the elements of: (a) A 3CD protease encoding gene comprising a Q184L mutation; (b) Baculovirus homologous repeat enhancer hr1; (c) Baculovirus late expression factor lef5 gene and expression regulatory element thereof; (d) The polh-pSel promoter for driving the expression of P1 protein; (e) Target serotype enterovirus P1 protein; wherein elements (a) - (d) constitute a general core backbone and element (e) allows for insertion or replacement of a specific P1 protein gene. In the technical scheme, the expression of the lef5 gene optimizes the expression environment of late proteins, improves the expression stability of P1 and 3CD proteins, and the synergistic effect of the hr1 enhancer and the polh-pSel promoter remarkably improves the final yield of VLP, and simultaneously inserts the enhancer hr1 of the homologous repeat region of baculovirus and improves the transcription efficiency of exogenous genes. The mutation Q184L was introduced to significantly inhibit its self-degradation. The replacement of the promoter driving the expression of P1 protein with polh-pSel ensures high level expression of P1. The invention integrates the transformation elements into a fixed core skeleton and is provided with a replaceable P1 expression unit, and the recombinant baculovirus corresponding to VLP can be quickly constructed by only inserting the P1 coding genes of target serotypes (such as CVA6, CVA10 and CVA 16) into the skeleton. Preferably, the 3CD protease encoding gene containing the Q184L mutation is derived from EV71 virus. Preferably, the target serotype in element (e) comprises EV71, CVA6, CVA10 or CVA16. In a second aspect, the present invention provides a recombinant baculovirus obtained by transposition of a Bac-to-Bac system from recombinant bacmid comprising said universal baculovirus transfer vector. In a third aspect, the present invention provides a method for constructing the recombinant baculovirus, comprising the steps of: S1, cloning a P1 protein coding gene of a target serotype enterovirus to a P1 expression site (e) of the general type baculovirus transfer vector; S2, transforming the complete transfer vector obtained in the step (1) into DH10Bac competent cells containing baculovirus shuttle vectors; S3, obtaining recombinant pole grains through resistance and blue and white spot screening; S4, transfecting the recombinant bacmid into an insect host cell, and saving to obtain the recombinant baculovirus. In a fourth aspect, the present invention provides a method for producing enterovirus-like particles in insect cells from said recombinant baculovirus, comprising the steps of: SA, infecting insect host cells with recombinant baculovirus; SB, culturing the infected insect host ce