Recombinant bluetongue virus for fusion expression of TC tag on NS2 protein and construction method

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a recombinant bluetongue virus for fusion expression of a tetracysteine (TC) tag on an NS2 protein and a construction method of the recombinant bluetongue virus. The recombinant bluetongue virus BTV1-NS2-354TC is constructed by inserting a TC tag into the C terminal (behind the 354 amino acid) of a wild type bluetongue virus non-structural protein NS2; the biological characteristics of the recombinant bluetongue virus and the biological characteristics of a wild type bluetongue virus are not obviously different, the TC tag-carrying NS2 protein expressed by the recombinant bluetongue virus can be dyed by a double-arsenic dye (FlAsH-EDTA), green fluorescence is displayed under a fluorescence microscope, and the recombinant bluetongue virus can be used for visual quantitative detection of BTV infection and positioning and tracing research of the NS2 protein.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a recombinant bluetongue virus with a TC tag fused and expressed on an NS2 protein and a construction method thereof. Background Art

[0002] Bluetongue disease, caused by the bluetongue virus (BTV), is a highly contagious disease that severely infects ruminants such as sheep, cattle, and deer. The World Organization for Animal Health (WOAH) lists it as a notifiable zoonotic disease, and my country classifies it as a Category II zoonotic disease. BTV, a representative member of the Orbivirus genus in the Reoviridae family, is an arbovirus transmitted by the blood-sucking midge (Culicoides bitingmidges). Currently, 29 different serotypes have been identified, and there is no cross-immunity between serotypes.

[0003] The BTV particle has icosahedral symmetry and lacks an envelope. Its genome consists of 10 linear, double-stranded RNA (dsRNA) segments (S1-S10). The BTV genome encodes seven structural proteins (VP1-VP7) and four nonstructural proteins (NS1, NS2, NS3 / NS3A, and NS4). The BTV particle has a double capsid, with VP2 and VP5 forming the outer capsid and VP3 and VP7 forming the inner capsid. After BTV sheds the outer capsid, the viral core particle is formed. Three enzymatic proteins, VP1, VP4, and VP6, are located within the core particle. The four nonstructural proteins, NS1, NS2, NS3 / NS3A, and NS4, are primarily involved in viral replication, assembly, maturation, and release. The nonstructural protein NS2 forms viral inclusion bodies in infected cells, the site of viral replication and assembly, recruiting multiple viral proteins and BTV transcripts. NS2 is also the only BTV protein that is phosphorylated, and NS2 phosphorylation is crucial for protein function during viral assembly and replication. NS2 also functions as a nucleotidase, breaking down NTPs to provide the energy required for BTV genome packaging and transport.

[0004] In recent years, the visualization and quantification of viruses has primarily relied on the quantification of recombinant viruses encoding fluorescent proteins (FPs). This involves genetically engineering the GFP gene to fuse with viral protein genes, enabling fluorescent labeling of specific viral proteins. However, this labeling approach has significant limitations for BTV. The BTV genome consists of 10 double-stranded RNA fragments of varying sizes (0.8-3.9 kb), which have a very limited capacity to accommodate foreign genes. Since fluorescent proteins typically have a molecular weight of 27 to 37 ku, viral expression of fluorescent proteins can affect the infectivity and replication of the labeled virus.

[0005] Previous researchers inserted TC tags after amino acids 156 and 493 of the NS1 protein, respectively, to construct recombinant bluetongue viruses (BTV-1S6-156TC and BTV-1S6-493TC) with biological properties indistinguishable from wild-type bluetongue virus. However, insertion of a TC tag at the C-terminus of the NS1 protein (after amino acid 552) failed to rescue recombinant bluetongue virus. Regarding the NS2 protein, the present inventors unexpectedly discovered that insertion of a TC tag at the C-terminus of the NS2 protein (after amino acid 354) successfully rescued bluetongue virus. Summary of the Invention

[0006] In response to the above technical problems, the present invention designed to insert a TC tag and an mCherry tag at the C-terminus of NS2 (after the 354th amino acid), and at the same time insert a TC tag after the 323rd amino acid of NS2 to perform virus rescue. The recombinant virus BTV1-S8-354TC with the TC tag inserted at the C-terminus of NS2 was successfully rescued. However, the recombinant bluetongue virus was not rescued after the TC tag was inserted after the 323rd amino acid of NS2 and the mCherry tag was inserted at the C-terminus. Therefore, the present invention unexpectedly discovered that the recombinant bluetongue virus BTV1-NS2-354TC was successfully constructed by inserting a TC tag sequence at the C-terminus (after the 354th amino acid) of the non-structural protein NS2 of the wild-type bluetongue virus. Specifically, it includes the following contents:

[0007] In the first aspect, the present invention provides a recombinant bluetongue virus expressing a TC tag fused to the NS2 protein, wherein the recombinant bluetongue virus is obtained by inserting a TC tag after the 354th amino acid of the wild-type bluetongue virus non-structural protein NS2; the TC tag is a hairpin structure composed of four cysteines, and its amino acid sequence is CCPGCC.

[0008] Preferably, the wild-type bluetongue virus is bluetongue virus serotype 1.

[0009] Preferably, the wild-type bluetongue virus is BTV-1 isolate GS / 11.

[0010] In a second aspect, the present invention provides a method for constructing a recombinant bluetongue virus, which comprises inserting a TC tag after the 354th amino acid of the nonstructural protein NS2 of the wild-type bluetongue virus by genetic engineering technology.

[0011] Preferably, the method comprises the following steps:

[0012] (1) Inserting the TC tag gene sequence after base 1062 of the CDS sequence of the wild-type bluetongue virus S8 gene to construct a bluetongue virus S8 gene transcription plasmid containing the TC tag; in vitro transcription was performed to generate S8 mRNA transcripts containing the TC tag;

[0013] (2) constructing transcription plasmids for wild-type bluetongue virus genes S1-S7 and S9-S10, respectively, and transcribing them into mRNA transcripts in vitro;

[0014] (3) Co-transfecting cells with the S8 mRNA containing the TC tag described in step (1) and the S1-S7 and S9-S10 mRNA transcripts described in step (2) to screen and obtain recombinant bluetongue virus.

[0015] Preferably, the method for constructing the bluetongue virus S8 gene transcription plasmid containing the TC tag in step (1) is:

[0016] Using the wild-type bluetongue virus S8 gene as a template, the TC tag gene sequence was inserted after the 1062th base of the wild-type bluetongue virus S8 gene CDS sequence by PCR gene site-directed mutagenesis technology to construct a bluetongue virus S8 gene transcription plasmid containing the TC tag; the gene sequence of the TC tag is shown in SEQ ID NO.2.

[0017] Preferably, the wild-type bluetongue virus is bluetongue virus serotype 1.

[0018] Preferably, the wild-type bluetongue virus is BTV-1 isolate GS / 11.

[0019] In a third aspect, the present invention provides a recombinant bluetongue virus constructed and obtained by the method described in the second aspect.

[0020] In a fourth aspect, the present invention provides the recombinant bluetongue virus according to the first aspect or the third aspect having any of the following uses:

[0021] (1) Application in visual quantitative detection of bluetongue virus;

[0022] (2) Application in the study of dynamic expression, localization and tracing of bluetongue virus NS2 protein.

[0023] The present invention has the following beneficial effects: In the early stages, the inventors inserted TC tags after amino acids 156 and 493 of the NS1 protein, respectively, to construct recombinant bluetongue viruses BTV-1S6-156TC and BTV-1S6-493TC, whose biological properties were not significantly different from those of wild-type bluetongue virus. However, insertion of a TC tag at the C-terminus of the NS1 protein (after amino acid 552) failed to rescue the recombinant bluetongue virus. Regarding the NS2 protein, the inventors successfully rescued the bluetongue virus by inserting a TC tag sequence at the C-terminus of the NS2 protein (after amino acid 354). However, insertion of an mCherry tag at the C-terminus of the NS2 protein (after amino acid 354) and after amino acid 323 of the NS2 protein failed to rescue the bluetongue virus. Therefore, the present invention unexpectedly discovered that only by inserting the TC tag sequence at the C-terminus (after the 354th amino acid) of the non-structural protein NS2 of the wild-type bluetongue virus, the recombinant bluetongue virus BTV1-NS2-354TC was rescued; the biological characteristics of the recombinant bluetongue virus were no significantly different from those of the wild-type bluetongue virus, and the recombinant bluetongue virus expressing the TC tag could be stained with a double arsenic dye (FlAsH-EDT2), and the TC-labeled NS2 protein could exhibit fluorescence, which can be used for visual quantitative detection of BTV infection and localization and tracing studies of the NS2 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Passaging of the recombinant virus on BSR cells, where A represents normal BSR cells; B represents BSR cells inoculated with experimental group 1; C represents BSR cells inoculated with experimental group 2 (i.e., BSR cells infected with BTV1-NS2-354TC); D represents BSR cells inoculated with experimental group 3; and E represents BSR cells infected with wild-type BTV1.

[0025] Figure 2 BSR cells infected with BTV1-NS2-354TC recombinant virus were stained with double arsenic dye FlAsH-EDT2 and Hoechst33342 for NS2 protein and cell nucleus, respectively;

[0026] Figure 3 Results of native polyacrylamide gel electrophoresis of dsRNA of wild-type BTV1 and BTV1-NS2-354TC recombinant virus;

[0027] Figure 4 Western blot was used to detect the expression of NS2 protein in BSR cells infected with wild-type virus and recombinant virus for 24 hours; lane 1 shows the expression of NS2 protein in BSR cells infected with wild-type BTV1; lane 2 shows the expression of NS2 protein in BSR cells infected with BTV1-NS2-354TC;

[0028] Figure 5 Laser confocal microscopy was used to observe the co-localization of the NS2 protein of the recombinant virus and the TC tag; the green fluorescence was the recombinant NS2 protein labeled with the double arsenic dye FlAsH-EDT2, the red fluorescence was the recombinant NS2 protein labeled with the NS2 antibody, and the blue fluorescence was the cell nucleus labeled with the Hoechst33342 dye.

[0029] Figure 6 Twelve hours after BSR cells were infected with the BTV1-NS2-354TC recombinant virus, images were taken continuously for 4 hours using a spinning disk confocal live cell workstation. The green fluorescence indicates recombinant NS2 protein labeled with the double arsenic dye FlAsH-EDT2, and the blue fluorescence indicates cell nuclei labeled with the Hoechst33342 dye. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0031] The following examples use bluetongue virus serotype 1 (BTV-1 (GS / 11)) as an example. Using bioinformatics software and published literature on BTV1-NS2 (encoded by the S8 gene), the present invention designed two insertion sites for a tetracysteine ​​(TC) tag sequence (amino acid sequence CCPGCC) and one mCherry tag insertion site on the S8 gene. It was found that only by inserting the TC tag after amino acid position 354 (C-terminus) of the NS2 protein could bluetongue virus be successfully rescued. The amino acid sequence of the NS2 protein is shown in SEQ ID NO. 1, the CDS sequence of the S8 gene is shown in SEQ ID NO. 2; the gene sequence of the TC tag is shown in SEQ ID NO. 3; and the gene sequence of the mCherry tag protein is shown in SEQ ID NO. 4.

[0032] Example 1 Construction of recombinant bluetongue virus

[0033] A TC tag (TGTTGTCCCGGGTGTTGT) was inserted at the C-terminus (after amino acid position 354) of the BTV-1 NS2 protein encoding the 1062nd base, and at the 323rd amino acid, and at the 969th base of the S8 gene CDS sequence. An mCherry tag was inserted at the C-terminus (after amino acid position 354) of the BTV-1 NS2 protein encoding the 1062nd base of the S8 gene CDS sequence.

[0034] Constructing NS2 gene transcription plasmid containing TC tag by PCR gene point mutation technology: design site-directed mutation primer with TC tag sequence, use wild type S8 gene transcription plasmid as template, use high-fidelity DNA polymerase for PCR, gel recovery and purification of PCR product and use DpnI enzyme to eliminate template plasmid. The treated PCR product is transformed into competent E. coli Trans5a, cultured on LB agar plate (containing ampicillin) overnight, single colonies are picked and expanded, and the plasmid is extracted for sequencing verification, obtaining S8 gene in vitro transcription plasmid with TC tag inserted gene sequence (S8-TC323, S8-TC354).

[0035] Constructing NS2 gene transcription plasmid containing mCherry tag by homologous recombination technology: design primers for insertion fragment and linearized vector on Novozyme primer design website (https: / / crm.vazyme.com / cetool / singlefragment.html), use wild type S8 gene transcription plasmid and plasmid containing mCherry tag as templates, use high-fidelity DNA polymerase for PCR, gel recovery and purification of PCR product, and homologous recombination of purified PCR product. The homologous recombination product is transformed into competent E. coli Trans5a, cultured on LB agar plate (containing ampicillin) overnight, single colonies are picked and expanded, and sequencing verification is performed, obtaining S8 gene transcription plasmid with mCherry tag inserted gene sequence (S8-mCherry354).

[0036] S8-TC323, S8-TC354, S8-mCherry354 and wild type BTV in vitro transcription plasmid (S1-S10) are linearized by enzyme digestion, and mRNA transcripts are obtained by in vitro transcription.

[0037] BSR cells were cultured in 12-well plates. When the cell confluence reached 70% to 90%, S8-TC323, S8-TC354 or S8-mCherry354 mRNA and the mRNAs of nine other wild-type genes (S1, S2, S3, S4, S5, S6, S7, S9, and S10) were co-transfected into BSR cells. The specific groups are as follows: experimental group 1: transfection of S8-TC323 and wild-type BTV1 gene (S1, S2, S3, S4, S5, S6, S7, S9, S10) mRNA; experimental group 2: transfection of S8-TC354 and wild-type BTV1 gene (S1, S2, S3, S4, S5, S6, S7, S9, S10) mRNA; experimental group 3: transfection of S8-mCherry354 and wild-type BTV1 gene (S1, S2, S3, S4, S5, S6, S7, S9, S10) mRNA; experimental group 4: transfection of wild-type BTV1 gene (S1, S2, S3, S4, S5, S6, S7, S8, S9, S10) mRNA, and a non-transfected cell control group was set up.

[0038] Three days after transfection, cytopathic effects were observed in experimental groups 2 and 4, but not in experimental groups 1 and 3. Samples from all wells were collected and passaged three times. Figure 1 As shown, A represents normal BSR cells; B represents BSR cells inoculated with experimental group 1; C represents BSR cells inoculated with experimental group 2 (i.e., BSR cells infected with BTV1-NS2-354TC); D represents BSR cells inoculated with experimental group 3; and E represents BSR cells infected with wild-type BTV1. The results showed that the S8-TC354 transfected wells in experimental group 2 and the wild-type S8 transfected wells in experimental group 4 showed obvious cytopathic effects, while the S8-TC323 transfected wells in experimental group 1 and the S8-mCherry354 transfected wells in experimental group 3 showed no obvious cytopathic effects, indicating that the recombinant fluorescent virus BTV1-S8-354TC with a TC tag inserted into the C-terminus of NS2 was successfully rescued. However, the insertion of a TC tag after amino acid position 323 and an mCherry tag into the C-terminus of NS2 did not rescue the recombinant fluorescent bluetongue virus.

[0039] The obtained recombinant virus BTV1-NS2-354TC was subjected to three rounds of plaque purification, and RNA was extracted from BSR cells infected with the recombinant virus. Using S8 gene-specific primers, the S8 gene was amplified by RT-PCR and sequenced. It was found that it contained a TC tag gene after base position CDS1062, indicating that the recombinant virus BTV1-NS2-354TC was successfully rescued.

[0040] After infecting BSR cells with the recombinant virus BTV1-NS2-354TC at an MOI of 0.1, the cells were stained with the double arsenic dye FlAsH-EDT2, fixed with tissue fixative, and stained with Hoechst33342 for nuclei. Slides were prepared and observed under a laser confocal microscope. Figure 2 As shown, the recombinant bluetongue virus expressing the TC tag described in the present application was stained with the double arsenic dye FlAsH-EDT2, and multiple clustered green fluorescent spots appeared in the cytoplasm, which were BTV virus inclusion bodies formed by the NS2 protein.

[0041] Viral dsRNA was extracted from BSR cells infected with the recombinant virus BTV1-NS2-354TC and wild-type BTV-1, respectively, and subjected to non-denaturing polyacrylamide gel electrophoresis. Due to the small size of the TC tag, the electrophoresis showed that the electrophoresis patterns of the recombinant virus BTV1-NS2-354TC and wild-type BTV-1 dsRNA were similar (e.g. Figure 3 The above results indicate that the biological characteristics of the recombinant virus BTV1-NS2-354TC are not significantly different from those of the wild-type bluetongue virus, indicating that the rescued recombinant bluetongue virus can be used for fluorescence visualization quantitative determination of virus-infected cells and expression distribution and tracing experiments of NS1 protein.

[0042] Western blot was performed using rabbit anti-BTVNS2 antibody. Figure 4 As shown in the figure, the size of the NS2 protein of wild-type BTV1 and recombinant virus BTV1-NS2-354TC is consistent with the expectation. To further confirm the fusion expression of TC tag on NS2 protein, immunofluorescence identification was performed, using rabbit NS2 antibody and goat anti-rabbit IgG Alexa Fluor 568 antibody as primary and secondary antibodies, respectively, to label the NS2 protein of the recombinant virus. The results are shown in the figure. Figure 5 As shown, the immunofluorescence results of NS2 protein (red) and the double arsenic dye staining results of TC labeling (green) co-localized.

[0043] After 12 hours of infection of BSR cells with the recombinant virus BTV1-NS2-354TC, the cells were stained with the double arsenic dye FlAsH-EDT2. The cells were then placed in a spinning disk confocal live cell workstation for continuous imaging analysis. The ambient temperature was set at 37°C and the CO2 concentration was 5%. The images were taken every 20 minutes for 4 hours. Some of the results are shown below. Figure 6 As shown, the position and size changes of viral inclusion bodies (green focus) formed by the recombinant NS2 protein in BSR cells can be observed, indicating that the recombinant virus BTV1-NS2-354TC can be used for the localization and tracing study of NS2 protein in infected cells.

[0044] In summary, the recombinant bluetongue virus expressing the TC tag described in the present application can be used for the visual quantitative detection of BTV infection, as well as the localization of the NS1 protein and the tracing study of BTV viral inclusion bodies.

[0045] Although the present invention takes BTV-1 (GS / 11) as an example, based on the high conservation of NS2 amino acid sequences between BTV-1 and other serotypes (homology of more than 99%), the scheme of the present invention is also applicable to other serotypes of bluetongue virus.

Claims

1. A recombinant bluetongue virus expressing a TC tag fused to the NS2 protein, characterized in that: The recombinant bluetongue virus is obtained by inserting a TC tag after the 354th amino acid of the non-structural protein NS2 of the wild-type bluetongue virus; the TC tag is a hairpin structure composed of four cysteines, and its amino acid sequence is CCPGCC.

2. The recombinant bluetongue virus according to claim 1, wherein The wild-type bluetongue virus is bluetongue virus serotype 1.

3. The recombinant bluetongue virus according to claim 2, wherein The wild-type bluetongue virus is BTV-1 isolate GS / 11.

4. A method for constructing a recombinant bluetongue virus, characterized in that: The method comprises the following steps: using genetic engineering technology to fuse and express a TC tag after the 354th amino acid of the non-structural protein NS2 of the wild-type bluetongue virus.

5. The construction method according to claim 4, wherein: The method comprises the following steps: (1) Inserting the TC tag gene sequence after base 1062 of the CDS sequence of the wild-type bluetongue virus S8 gene to construct a bluetongue virus S8 gene transcription plasmid containing the TC tag; in vitro transcription was performed to generate S8 mRNA transcripts containing the TC tag; (2) constructing transcription plasmids for wild-type bluetongue virus genes S1-S7 and S9-S10, respectively, and transcribing them into mRNA transcripts in vitro; (3) Co-transfecting cells with the S8 mRNA containing the TC tag described in step (1) and the S1-S7 and S9-S10 mRNA transcripts described in step (2) to screen and obtain recombinant bluetongue virus.

6. The construction method according to claim 5, wherein: The method for constructing the bluetongue virus S8 gene transcription plasmid containing the TC tag in step (1) is as follows: Using the wild-type bluetongue virus S8 gene as a template, the TC tag gene sequence was inserted after the 1062th base of the wild-type bluetongue virus S8 gene CDS sequence by PCR gene site-directed mutagenesis technology to construct a bluetongue virus S8 gene transcription plasmid containing the TC tag; the gene sequence of the TC tag is shown in SEQ ID NO.

2.

7. The construction method according to claim 6, wherein: The wild-type bluetongue virus is bluetongue virus serotype 1.

8. The construction method according to claim 7, wherein: The wild-type bluetongue virus is BTV-1 isolate GS / 11.

9. The recombinant bluetongue virus constructed and obtained by the method according to any one of claims 4 to 8.

10. The recombinant bluetongue virus according to any one of claims 1 to 3 or claim 9 has any of the following uses: (1) Application in visual quantitative detection of bluetongue virus; (2) Application in the study of dynamic expression, localization and tracing of bluetongue virus NS2 protein.

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