CN113514440A - Baculovirus fluorescent nanoprobe - Google Patents
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Abstract
The invention discloses a baculovirus fluorescent nano probe, which is characterized in that baculovirus envelope glycoprotein GP64 is labeled by self biotinylation, and then a ruthenium complex [ Ru (phen) ] is labeled on a genome of the baculovirus envelope glycoprotein GP64 by utilizing the replication and self-assembly processes of the baculovirus2dppz]2+And preparing the fluorescent signal amplification nano probe. The invention can be used for detecting and analyzing nucleic acid and biological imaging according to the fluorescence signal value of the ruthenium complex combined in the viral genome.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a baculovirus fluorescent nanoprobe for cell imaging, pathogen detection, nucleic acid detection and the like.
Background
Baculovirus is a double-stranded circular DNA virus with envelope package, its genome size is 80-180kb, and it can be used as specific host by arthropod to make infection and transmission in nature. Baculoviruses have two distinct virion morphologies: one type of virion, a Budded Virus (BV), primarily mediates cell-to-cell systemic infection, invading cells by receptor-mediated endocytosis; the other is embedded virus particles (ODV), and in the process of oral infection of the virus, the shell of the embedded virus particles is separated by the alkaline environment of the intestinal tract of the arthropod, so that the embedded virus particles germinate into the virus with infection capability and infect intestinal cells to achieve virus transmission.
At present, the known nuclear polyhedrosis viruses with complete genome sequences include Autographa californica nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Flammulina yezoensis hubner nuclear polyhedrosis virus, gypsy moth nuclear polyhedrosis virus, beet armyworm nuclear polyhedrosis virus, cotton bollworm nuclear polyhedrosis virus and Spodoptera litura nuclear polyhedrosis virus, wherein AcMNPV is the most deep research, and the nuclear polyhedrosis virus has important application value in the fields of biological pesticides, eukaryotic expression systems and the like.
The gene expression of AcMNPV is divided into 4 stages, namely immediate early gene expression, late gene expression and extremely late gene expression.
The first two stages of gene expression precede DNA replication, while the latter two stages of gene expression are accompanied by a series of viral DNA syntheses. In the process of extremely late gene expression, there are two kinds of high-efficiency expressed proteins, namely polyhedrin and P10 protein. Polyhedrin is the main component for forming inclusion body virus, can be accumulated in cells in the late stage of infection by 30-50 percent, is a non-essential component for virus replication, and has a certain protective effect on virus particles.
Another highly expressed very late protein is the P10 protein, which is also a non-essential component of viral replication and can form a fibrous mass in cells, which may be associated with cell lysis. The promoters of the polyhedron gene and the P10 gene have strong starting capability and are non-essential components for virus replication, so the two gene sites become ideal foreign gene insertion sites of a baculovirus expression vector system.
However, the application of the method is limited to the fields of biological pesticides, eukaryotic expression and the like.
Disclosure of Invention
Based on the above, the primary object of the present invention is to provide a baculovirus fluorescent nanoprobe, which can be used for nucleic acid detection and analysis and biological imaging according to the fluorescence signal value of ruthenium complex bound in the viral genome, has an ultra-sensitivity detection capability, and can improve the sensitivity and accuracy of DNA detection.
The invention also aims to provide a baculovirus fluorescent nano probe which emits light under the condition of existence of a hydrated polymer, thereby generating good marking effect and being widely applied to cell imaging, pathogen detection and nucleic acid detection.
The applicant researches and discovers that: the biotin contains tryptophan residues, the binding force Kd value is as high as 10-15mol/L, the binding force is ten thousand times higher than that between antigen and antibody, the affinity is high, the sensitivity is high, the specificity is strong, the stability is good, and the multi-stage amplification effect is realized. The biotin receptor polypeptide (AP-Tag or Avi-Tag for short) comprises a short peptide label consisting of 15 amino acid residues, and the lysine residues have the advantages of mild reaction conditions, extremely high specificity, high efficiency, extremely low influence on protein conformation and activity and convenient operation; activation of biotin to form Bioacyl-5' -adenylate transfers biotin to the biotin receptor protein to form a ruthenium complex [ Ru (phen)2dppz]2+The ruthenium complex does not emit light in water, but emits light in a non-aqueous solvent (ethanol and acetonitrile) and in the presence of a hydrated polymer (such as DNA), and has good marking effect. It has good detection ability if it can be combined with baculovirus.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a baculovirus fluorescent nano probe is characterized in that baculovirus envelope glycoprotein GP64 is labeled through self biotinylation, and then a ruthenium complex [ Ru (phen) ] is labeled on a genome of the baculovirus envelope glycoprotein by utilizing the replication and self-assembly processes of the baculovirus2dppz]2+And preparing the fluorescent signal amplification nano probe.
Specifically, genes encoding biotin ligase (BirA) and biotin receptor polypeptide (AP-tag) and the GP64 gene, which is an envelope protein of baculovirus (AcMNPV), were fused together to give recombinant bacmid; the recombinant bacmid is transfected into a baculovirus host Sf9 insect cell to be cultured, and a ruthenium complex ([ Ru (phen) is added in the culture process2dppz]2+) (ii) a GP64 fused with biotin receptor polypeptide can be specifically generated in the presence of biotin ligase, biotin and ATPBiotinylation, thus obtaining recombinant virus with biotinylated envelope protein, and ruthenium complex ([ Ru (phen))2dppz]2+) The baculovirus genome DNA replicated in Sf9 cells can be combined with the ruthenium complex, so that the recombinant virus with the genome DNA combined with the ruthenium complex can be obtained, namely the finally obtained recombinant virus comprises the biotinylated envelope protein and the genome DNA combined with the ruthenium complex. The biotin on the recombinant virus envelope protein can be combined with streptavidin, and further combined with a biotinylated DNA probe to prepare a signal probe, and the signal probe is used for detecting and analyzing nucleic acid and biological imaging according to a fluorescence signal value of a ruthenium compound combined in a virus genome.
The addition of ruthenium complex ([ Ru (phen))2dppz]2+) During the culture, 1mM of [ Ru (phen) ]is added to the suspension cell culture species2dppz]2+In solution, baculovirus genomic DNA replicated in Sf9 cells can be bound to the ruthenium complex.
The invention has the beneficial effects that:
the invention firstly carries out self biotinylation to mark baculovirus envelope glycoprotein GP64, and then marks the genome thereof with [ Ru (phen) ]by utilizing the replication and self-assembly processes of baculovirus2dppz]2+The oligonucleotide sequence obtained by in vitro screening of the small segment can be combined with the corresponding ligand with high affinity and strong specificity, because of [ Ru (phen) ]2dppz]2+Has low background excitation, special photophysical properties (long-lasting metal-ligand charge transfer excited state and red shift excitation), so when [ Ru (phen)2dppz]2+The expected enhancement of luminescence is achieved after binding to DNA>104And thus has ultra-sensitivity detection capability.
The application direction of the baculovirus fluorescent nano probe is as follows: cell imaging, pathogen detection, nucleic acid detection, and the like.
Drawings
FIG. 1 is a schematic illustration of the generation of probes achieved by the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in FIG. 1, the present invention is a schematic diagram of a baculovirus fluorescent nanoprobe realized by labeling baculovirus envelope glycoprotein GP64 by self-biotinylation and then labeling its genome with ruthenium complex using replication and self-assembly process of baculovirus [ Ru (phen ]2dppz]2+And (3) preparing.
Specifically, genes encoding biotin ligase (BirA) and biotin receptor polypeptide (AP-tag) and envelope protein GP64 gene of baculovirus (AcMNPV) were fused together to obtain recombinant bacmid; the recombinant bacmid is transfected into a baculovirus host Sf9 insect cell for culture, and GP64 fused with biotin receptor polypeptide can be specifically biotinylated in the presence of biotin ligase, biotin and ATP (note: since the N end of GP64 extends on the outer surface of the envelope protein, the biotin receptor polypeptide at the N end is also on the outer surface of the recombinant virus), so that the recombinant virus with the envelope protein biotinylated can be obtained, and meanwhile, a ruthenium complex (Ru (phen) is added in the culture process) (the N end of GP64 extends on the outer surface of the envelope protein)2dppz]2+) Baculovirus genomic DNA replicated in Sf9 cells was then bound to the ruthenium complex (note: the ruthenium complex can be combined with double-stranded DNA), so that the recombinant virus with the ruthenium complex combined with the genome DNA can be obtained, namely the finally obtained recombinant virus comprises the biotinylated envelope protein and the genome DNA combined with the ruthenium complex. The biotin on the recombinant virus envelope protein can be combined with streptavidin, and further combined with a biotinylated DNA probe to prepare a signal probe, and the signal probe is used for detecting and analyzing nucleic acid and biological imaging according to a fluorescence signal value of a ruthenium compound combined in a virus genome.
Wherein, the ruthenium complex ([ Ru (phen))2dppz]2+) Has the molecular formula of RuC42H26N8The structural formula is as follows:
the construction of the reference recombinant bacmid (BAC-BirAAP-GFP) is shown in FIG. 1.
Wherein baculovirus genomic DNA replicated in Sf9 cells was combined with the ruthenium complex by adding 1mM [ Ru (phen) ] to the suspension cell culture species during the culture2dppz]2+Solution so that the baculovirus genomic DNA replicated in Sf9 cells can bind to the ruthenium complex.
In summary, the present invention self-biotinylates and labels baculovirus envelope glycoprotein GP64, and then utilizes the replication and self-assembly process of baculovirus to label its genome [ Ru (phen) ]2dppz]2+The oligonucleotide sequence obtained by in vitro screening of the small segment can be combined with the corresponding ligand with high affinity and strong specificity, because of [ Ru (phen) ]2dppz]2+Has low background excitation, special photophysical properties (long-lasting metal-ligand charge transfer excited state and red shift excitation), so when [ Ru (phen)2dppz]2+The expected enhancement of luminescence is achieved after binding to DNA>104And thus has ultra-sensitivity detection capability.
The application direction of the baculovirus fluorescent nano probe is as follows: cell imaging, pathogen detection, nucleic acid detection, and the like.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (3)
1. A baculovirus fluorescent nano probe is characterized in that baculovirus envelope glycoprotein GP64 is labeled through self biotinylation, and then a ruthenium complex [ Ru (phen) ] is labeled on a genome of the baculovirus envelope glycoprotein by utilizing the replication and self-assembly processes of the baculovirus2dppz]2+And preparing the fluorescent signal amplification nano probe.
2. The baculovirus fluorescent nanoprobe of claim 1, wherein genes encoding biotin ligase and biotin receptor polypeptide are fused with baculovirus envelope protein GP64 gene to obtain a recombinant bacmid; transfecting the recombinant bacmid into a baculovirus host Sf9 insect cell for culture, and adding a ruthenium compound in the culture process; in the presence of biotin ligase, biotin and ATP, GP64 fused with a biotin receptor polypeptide can be specifically biotinylated, and recombinant viruses with ruthenium complexes bound to genomic DNA can be obtained.
3. Baculovirus fluorescent nanoprobe according to claim 2, characterized in that said ruthenium complex ([ ru (phen) is added2dppz]2+) During the culture of Sf9 insect cells, [ Ru (phen) ] is added to the suspension cell culture species2dppz]2+Solution, baculovirus genomic DNA replicated in Sf9 cells was combined with ruthenium complexes.
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| CN101952436A (en) * | 2008-01-09 | 2011-01-19 | 建国大学校产学协力团 | Baculovirus-based vaccines |
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| CN108728397A (en) * | 2017-04-17 | 2018-11-02 | 中国科学院微生物研究所 | The method that the foreign protein that insect protein or insect expression system are expressed is marked |
| FR3081474A1 (en) * | 2018-05-25 | 2019-11-29 | Association Pour La Recherche Et Le Developpement Des Methodes Et Processus Industriels - Armines | RECOMBINANT BACULOVIRUS MARKED WITH A FLUORESCENT DNA MARKING SYSTEM AND ITS APPLICATIONS |
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