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CN-122012622-A - Baculovirus expression vector and construction method and application thereof

CN122012622ACN 122012622 ACN122012622 ACN 122012622ACN-122012622-A

Abstract

The invention discloses a baculovirus expression vector, a construction method and application thereof. The vector takes pFastBacDual as a framework, modifies a promoter by connecting 2-9 repeated BS sequences in series at the downstream of a polh promoter, and introduces a very late transcription factor VLF-1 gene, and the two synergistically enhance transcription activity. The invention constructs pBSX-eGFP, pBSX-eV and pBSX-GV recombinant vectors, determines that the optimal combination is 2 repeated BS sequences and VLF-1 over-expression, and can improve the expression quantity of target proteins. The construction flow of the vector is standardized, enzyme cutting sites such as BamHI and EcoRI are reserved, and the high-expression protein can be obtained after Sf9 cells are transfected and cultured for 4-5 days. The invention improves the expression efficiency of the recombinant protein of the baculovirus expression system, retains the advantages of correct folding and post-translational modification of the recombinant protein, is suitable for large-scale industrialized recombinant protein production scenes such as biopharmaceuticals, vaccine research and development and the like, and has important application value.

Inventors

  • LIANG CHANGYONG
  • ZHANG HAO
  • ZHAO SHULING
  • LIU MINGMING

Assignees

  • 扬州大学

Dates

Publication Date
20260512
Application Date
20260212

Claims (9)

  1. 1. A recombinant baculovirus expression vector pBSX-eGFP is characterized in that X repeated BS sequences are inserted into the downstream of a polh promoter by taking a pFastBacDual plasmid as a basic skeleton, the nucleotide sequence of the burst sequence is shown as SEQ ID No.1, the eGFP target gene is inserted into the vector through XhoI and BamHI enzyme cleavage sites, and X=2, 3, 5,7 and 9.
  2. 2. A method of constructing the recombinant baculovirus expression vector pBSX-eGFP of claim 1, comprising the steps of: (1) The double enzyme cutting fragment containing multiple BS sequences is connected with pFastBacDual plasmid, and the double enzyme cutting fragment is converted to obtain recombinant plasmid pBS2 containing multiple BS sequences; (2) Then, respectively carrying out double enzyme digestion on the pBS2 plasmid, recovering a target fragment, carrying out enzyme linked conversion, and extracting a recombinant plasmid to obtain pBS3; (3) Repeating the steps to construct pBS5, pBS7 and pBS9; (4) Then XhoI and BamHI double enzyme cutting pBSX and pBD-eGFP plasmid containing eGFP gene are used to recover fragment and enzyme-link so as to obtain recombinant plasmid pBSX-eGFP.
  3. 3. A recombinant baculovirus expression vector pBSX-eV is characterized in that a coding gene of a very late transcription factor VLF-1 is introduced on the basis of pBSX-eGFP of claim 1, VLF-1 is specifically combined with a BS sequence, the nucleotide sequence of the VLF-1 is shown as SEQ ID No.3, and X=2, 3 and 5.
  4. 4. A method of constructing the recombinant baculovirus expression vector pBSX-eV as defined in claim 3, comprising the steps of: (1) Amplifying the VLF-1 gene fragment by PCR with AcMNPV as a template and VLF-1F and VLF-1R as primers to construct an intermediate vector pBD-IE2-VLF-1 containing VLF-1; (2) And finally, the XhoI/BamHI double enzyme digestion is used for cutting pBD-eV and pBSX, the fragments with different BS numbers are connected with the vector fragments, and the recombinant baculovirus expression vector pBSX-eV is obtained after transformation and screening.
  5. 5. A recombinant baculovirus expression vector pBSX-GV is characterized in that 2 repeated BS sequences and VLF-1 coding genes are integrated, an SV40-IE1-GFP fusion fragment is introduced as a marking element, bamHI and EcoRI cleavage sites are reserved, the nucleotide sequence of the VLF-1 coding genes is shown as SEQ ID No.3, the nucleotide sequence of the 2 repeated BSs is shown as SEQ ID No.2, and X=2, 3 and 5.
  6. 6. A method of constructing the recombinant baculovirus expression vector pBSX-GV as defined in claim 5, comprising the steps of: (1) PCR amplifying SV40 fragment with pFastBac Dual as template, PCR amplifying IE1-GFP fragment with pFastBac-PH-GFP as template, mixing, amplifying SV40-IE1-GFP fusion fragment by overlapping PCR, connecting with pMD19-T carrier, and recovering by SalI and NotI double enzyme digestion; (2) Enzymatically ligating the fusion fragment with the pBD-eV vector of claim 4 after cleavage to obtain pBD-GV; (3) pBD-GV was digested with XhoI/BamHI and subjected to the enzymatic ligation with pBSX of claim 1, and screened to obtain pBSX-GV.
  7. 7. The use of the recombinant baculovirus expression vector pBSX-GV of claim 5 for the efficient expression of heterologous proteins.
  8. 8. The use according to claim 7, wherein the exogenous gene of interest is inserted into pBSX-GV through BamHI and EcoRI cleavage sites to construct a recombinant expression vector and transfected into Sf9 insect cells to achieve efficient expression of the exogenous gene of interest.
  9. 9. A recombinant baculovirus comprising the recombinant plasmid pBSX-eGFP, pBSX-eV or pBSX-GV of claim 1.

Description

Baculovirus expression vector and construction method and application thereof Technical Field The invention relates to a baculovirus expression vector, a construction method and application thereof, belonging to the field of genetic engineering. Background Baculovirus Expression Vector Systems (BEVS) are eukaryotic expression tools commonly used in biopharmaceuticals, vaccine development, and recombinant protein production. The method has the core advantages that the recombinant protein can be correctly folded to finish post-translational modification processing, and meanwhile, large-scale industrialized culture can be realized, so that the method has wide application prospects in the related fields. The polh promoter is used as a baculovirus extremely late-stage strong promoter, can efficiently drive the expression of exogenous proteins, and is a core element for constructing BEVS. However, the baculovirus expression system constructed on the basis of the polh promoter still has the problem of low protein expression efficiency, which limits the further application of the baculovirus expression system in industrial production to a certain extent. It was found that the transcriptional activity of the polh promoter is dependent on the Burst Sequence (BS) upstream of its open reading frame, the integrity of which directly influences the transcriptional efficiency. If the BS sequence is mutated or deleted, the expression level of the protein driven by the polh promoter is remarkably reduced. However, up to the present, the regulation and control rule of the BS sequence copy number on the polh promoter activity is not clear, and whether increasing the number of BS sequences can continuously improve transcription efficiency is also lack of experimental verification of the system. In addition, very late transcription factor VLF-1 is a key protein regulating the activity of the polh promoter, and can be specifically combined with BS sequence to jointly enhance the transcription capacity of the polh promoter. However, in the prior art, the combination of the 'over-expression of VLF-1' and the 'optimized BS sequence copy number' is not applied, and a mature method for maximizing the expression efficiency of the polh promoter through the synergistic effect of the two is not available. Meanwhile, the insertion of too many BS sequences may cause abnormality in the spacer sequence between the promoter and the target gene, rather inhibiting the transcriptional activity. Therefore, aiming at the problems of undefined regulation mechanism and limited expression efficiency of the existing baculovirus polh promoter, a novel synergistic regulation method is urgently needed to be developed, namely, a high-activity polh promoter is constructed by optimizing the copy number of a BS sequence and combining with VLF-1 over-expression, so that the expression quantity of the foreign protein in BEVS is remarkably improved, and the requirements of the fields of biopharmaceutical, vaccine production and the like on a high-efficiency protein expression system are met. Disclosure of Invention The invention aims to provide a baculovirus expression vector, a construction method and application thereof. The invention provides a recombinant baculovirus expression vector pBSX-eGFP (comprising multiple repeated BS sequences and eGFP target genes), a pFastBacDual plasmid is taken as a basic skeleton, a plurality of repeated burst sequences are inserted into the downstream of a polh promoter, the nucleotide sequence of the burst sequences is shown as SEQ ID No.1, the nucleotide sequence of 2 repeated BSs is shown as SEQ ID No.2, the eGFP target genes are inserted into the vector through enzyme cleavage sites, and X=2, 3, 5, 7 and 9. The invention also provides a construction method of the recombinant baculovirus expression vector pBSX-eGFP, which comprises the following steps: (1) Firstly, a fragment containing multiple BS sequences is cut by XhoI and HindIII double enzyme and is connected with pFastBacDual plasmid by T4 DNA ligase, and then the recombinant plasmid pBS2 containing multiple BS sequences is obtained through the conversion; (2) Then XhoI/XbaI and XhoI/SpeI are used for respectively carrying out double enzyme digestion on the pBS2 plasmid and recovering target fragments, and after enzyme connection, the target fragments are converted, and recombinant plasmids are extracted to obtain pBS3; (3) Repeating the steps to construct pBS5, pBS7 and pBS9; (4) Then XhoI and BamHI double enzyme cutting pBSX and pBD-eGFP plasmid containing eGFP gene are used to recover fragment and enzyme-link so as to obtain recombinant plasmid pBSX-eGFP. The invention also provides a recombinant baculovirus expression vector pBSX-eV (comprising a multiple repetition BS sequence, an eGFP target gene and a VLF-1 gene), wherein a coding gene of a very late transcription factor VLF-1 is introduced on the basis of pBSX-eGFP, the VLF-1 is specifically combined with th