Recombinant blue tongue virus with TC tag fused on NS2 protein and construction method thereof

Abstract

The application belongs to the field of genetic engineering, and particularly relates to a recombinant blue tongue virus which fuses and expresses a tetracysteine (TC) tag on NS2 protein and a construction method. The application inserts a TC tag at the C terminal (after the 354th amino acid) of the non-structural protein NS2 of a wild type blue tongue virus to construct a recombinant blue tongue virus BTV1-NS2-354TC. The recombinant blue tongue virus has no obvious difference from the wild type blue tongue virus in biological characteristics, and the NS2 protein expressed by the recombinant blue tongue virus and carrying the TC tag can be dyed by a double arsenic dye (FlAsH-EDT2), shows green fluorescence under a fluorescence microscope, and can be used for visual quantitative detection of BTV infection and positioning and tracing research of the NS2 protein.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a recombinant bluetongue virus that expresses a TC tag fused to the NS2 protein and its construction method. Background Technology

[0002] Bluetongue disease is a highly contagious and deadly disease of ruminants such as sheep, cattle, and deer, caused by bluetongue virus (BTV). The World Organisation for Animal Health (WOAH) lists it as a notifiable animal disease, and my country classifies it as a Class II animal infectious disease. BTV is a representative member of the Orbivirus genus within the Reoviridae family. It is an arbovirus transmitted by the Culicoides bitingmidges. Currently, 29 different serotypes have been identified, and there is no cross-immunity between serotypes.

[0003] The viral viral volumetric tract (BTV) particle exhibits icosahedral symmetry and lacks an envelope. Its genome consists of 10 segmented linear double-stranded RNAs (dsRNAs) (S1–S10). The BTV genome encodes seven structural proteins (VP1–VP7) and four non-structural proteins (NS1, NS2, NS3 / NS3A, and NS4). The BTV particle has a double capsid: VP2 and VP5 form the outer capsid, while the inner capsid is composed of VP3 and VP7. After shedding the outer capsid, the BTV forms the viral core particle, within which three enzyme proteins—VP1, VP4, and VP6—are located. The four non-structural proteins, NS1, NS2, NS3 / NS3A, and NS4, are primarily involved in viral replication, assembly, maturation, and release. Among these, the non-structural protein NS2 can form viral inclusion bodies in infected cells, serving as the site for viral replication and assembly. It can recruit multiple viral proteins and BTV transcripts. NS2 is also the only BTV protein that undergoes phosphorylation, and NS2 phosphorylation is crucial for protein function during viral assembly and replication. NS2 also acts as a nucleotidase, breaking down NTPs to provide the energy needed for BTV genome packaging and transport.

[0004] In recent years, the visualization and quantification of viruses have mainly relied on the quantification of recombinant viruses encoding fluorescent proteins (FP). This involves fusing the GFP-expressing gene with the viral protein gene using genetic engineering techniques, enabling fluorescent labeling of specific viral proteins. However, this labeling method has significant limitations in the application of virgin viral viruses (BTV). The BTV genome consists of 10 double-stranded RNAs of varying sizes (0.8-3.9 kb), and its capacity to accommodate exogenous genes is very limited. Furthermore, since the molecular weight of fluorescent proteins is typically between 27 kDa and 37 kDa, viral expression of fluorescent proteins can affect the infectivity and replication ability of the labeled virus.

[0005] Previously, 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, which were not significantly different from wild-type bluetongue virus in terms of biological characteristics. However, inserting 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, this invention unexpectedly discovered that inserting a TC tag at the C-terminus of the NS2 protein (after amino acid 354) successfully rescued the bluetongue virus. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention designs a method to insert a TC tag and an mCherry tag at the C-terminus (after amino acid 354) of NS2, and simultaneously insert a TC tag after amino acid 323 of NS2 for virus rescue. This successfully rescued the recombinant virus BTV1-S8-354TC with the TC tag inserted at the C-terminus of NS2. However, inserting a TC tag after amino acid 323 of NS2 and an mCherry tag at the C-terminus failed to rescue the recombinant bluetongue virus. Therefore, this invention unexpectedly discovered that inserting a TC tag sequence at the C-terminus (after amino acid 354) of the wild-type bluetongue virus non-structural protein NS2 successfully constructed the recombinant bluetongue virus BTV1-NS2-354TC. Specifically, this includes the following:

[0007] In a first aspect, the present invention provides a recombinant bluetongue virus that expresses a TC tag fused to the NS2 protein. 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 cysteine ​​residues, 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 the BTV-1 isolate GS / 11.

[0010] Secondly, the present invention provides a method for constructing recombinant bluetongue virus, wherein the method is as follows: a TC tag is inserted after the 354th amino acid of the non-structural protein NS2 of wild-type bluetongue virus by means of genetic engineering technology.

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

[0012] (1) Insert the TC-tagged gene sequence after the 1062nd base 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; and generate an S8 mRNA transcript containing the TC tag in vitro.

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

[0014] (3) The S8 mRNA containing the TC tag described in step (1) and the S1-S7 and S9-S10 mRNA transcripts described in step (2) were co-transfected into cells, and recombinant bluetongue virus was obtained by screening.

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

[0016] Using the wild-type bluetongue virus S8 gene as a template, a TC-tagged gene sequence was inserted after the 1062nd base of the CDS sequence of the wild-type bluetongue virus S8 gene by PCR site-directed mutagenesis, thereby constructing 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 the BTV-1 isolate GS / 11.

[0019] Thirdly, the present invention provides a recombinant bluetongue virus constructed by the method described in the second aspect above.

[0020] Fourthly, the present invention provides the recombinant bluetongue virus described in the first or third aspect above as 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 beneficial effects of this invention are as follows: Initially, TC tags were inserted after amino acids 156 and 493 of the NS1 protein, respectively, to construct recombinant bluetongue viruses BTV-1S6-156TC and BTV-1S6-493TC, which have biological characteristics indistinguishable from wild-type bluetongue virus. However, inserting 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, inserting an mCherry tag at the C-terminus of the NS2 protein (after amino acid 354) and a TC tag after amino acid 323 of the NS2 protein failed to rescue the bluetongue virus. Therefore, this invention unexpectedly discovered that only by inserting a TC tag sequence at the C-terminus (after amino acid 354) of the wild-type bluetongue virus non-structural protein NS2 could the recombinant bluetongue virus BTV1-NS2-354TC be rescued. The biological characteristics of the recombinant bluetongue virus are not significantly different from those of the wild-type bluetongue virus, and the recombinant bluetongue virus expressing the TC tag can be stained with arsenic diarsenic dye (FlAsH-EDT2). The TC-labeled NS2 protein can show fluorescence, which can be used for the visual quantitative detection of BTV infection and the localization and tracing of NS2 protein. Attached Figure Description

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

[0025] Figure 2 The staining results of NS2 protein and cell nucleus by BSR cells after infection with BTV1-NS2-354TC recombinant virus using arsenic dyes FlAsH-EDT2 and Hoechst33342, respectively;

[0026] Figure 3 Results of non-denaturing polyacrylamide gel electrophoresis of dsRNA from wild-type BTV1 and BTV1-NS2-354TC recombinant viruses.

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

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

[0029] Figure 6 Twelve hours after BSR cells were infected with BTV1-NS2-354TC recombinant virus, images were continuously taken for 4 hours in a confocal live cell workstation. Green fluorescence represents recombinant NS2 protein labeled with the arsenic dye FlAsH-EDT2, and blue fluorescence represents cell nuclei labeled with Hoechst33342 dye. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0031] The following examples use bluetongue virus serotype 1 (BTV-1, GS / 11) as an example. This invention utilizes bioinformatics software combined with published literature on BTV1-NS2 (encoded by the S8 gene) to design two tetracysteine ​​(TC) tag sequence insertion sites (amino acid sequence CCPGCC) and one mCherry tag insertion site on the S8 gene. It was found that inserting the TC tag after amino acid position 354 (C-terminus) of the NS2 protein successfully rescued the bluetongue virus. 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] TC tags (TGTTGTCCCGGGTGTTGT) are inserted at the C-terminus of the BTV-1NS2 protein (after amino acid 354), corresponding to the 1062nd and 323rd bases of the S8 gene CDS sequence, and the 969th base of the S8 gene CDS sequence, respectively. An mCherry tag is inserted at the C-terminus of the BTV-1NS2 protein (after amino acid 354), corresponding to the 1062nd base of the S8 gene CDS sequence.

[0034] A TC-tagged NS2 gene transcription plasmid was constructed using PCR point mutagenesis technology: site-directed mutagenesis primers encoding the TC tag sequence were designed. Using the wild-type S8 gene transcription plasmid as a template, PCR was performed using high-fidelity DNA polymerase. The PCR product was purified by gel extraction and the template plasmid was eliminated using DpnI enzyme. The treated PCR product was transformed into competent *E. coli* Trans5α cells and cultured overnight on LB agar plates (containing ampicillin). After single colonies were picked and expanded, plasmids were extracted and sequenced for verification, yielding in vitro transcription plasmids of the S8 gene containing the TC tag sequence (S8-TC323, S8-TC354).

[0035] Homologous recombination was used to construct an NS2 gene transcription plasmid containing the mCherry tag: Primers for the insert fragment and linearized vector were designed on the Novizumi primer design website (https: / / crm.vazyme.com / cetool / singlefragment.html). Using the plasmid containing the mCherry tag and the wild-type S8 gene transcription plasmid as templates, high-fidelity DNA polymerase was used for PCR. After gel purification of the PCR products, homologous recombination was performed on the purified PCR products. The homologous recombination products were transformed into competent *E. coli* Trans5α and cultured overnight on LB agar plates (containing ampicillin). Single colonies were picked, expanded, and sequenced for verification, yielding the S8 gene transcription plasmid (S8-mCherry354) containing the mCherry tag protein gene sequence.

[0036] S8-TC323, S8-TC354, S8-mCherry354 and wild-type BTV in vitro transcription plasmids (S1-S10) were linearized by enzyme digestion and then transcribed in vitro to obtain mRNA transcripts.

[0037] BSR cells were cultured in 12-well plates until the cell confluence reached 70%–90%. S8-TC323, S8-TC354 or S8-mCherry354 mRNA were co-transfected into BSR cells with the mRNAs of the other nine wild-type genes (S1, S2, S3, S4, S5, S6, S7, S9, S10). The specific groupings are as follows: Experimental group 1: transfected with S8-TC323 and wild-type BTV1 gene (S1, S2, S3, S4, S5, S6, S7, S9, S10) mRNA; Experimental group 2: transfected with S8-TC354 and wild-type BTV1 gene (S1, S2, S3, S4, S5, S6, S7, S9, S10) mRNA; Experimental group 3: transfected with S8-mCherry354 and wild-type BTV1 gene (S1, S2, S3, S4, S5, S6, S7, S9, S10) mRNA; Experimental group 4: transfected with wild-type BTV1 gene (S1, S2, S3, S4, S5, S6, S7, S8, S9, S10) mRNA, with an untransfected cell control group.

[0038] Three days after transfection, cytopathic effects appeared in experimental groups 2 and 4, while no cytopathic effects were observed in experimental groups 1 and 3. Samples from all wells were collected and passaged three times each. Results are as follows: 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 cytopathic effects were evident in the transfection wells of S8-TC354 in experimental group 2 and the wild-type S8 transfection well in experimental group 4, while no significant cytopathic effects were observed in S8-TC323 in experimental group 1 and S8-mCherry354 in experimental group 3. This indicates that the recombinant fluorescent virus BTV1-S8-354TC with a TC tag inserted at the C-terminus of NS2 was successfully rescued. However, the recombinant fluorescent blue tongue virus was not rescued when a TC tag was inserted after amino acid 323 of NS2 or when an mCherry tag was inserted at the C-terminus.

[0039] The obtained recombinant virus BTV1-NS2-354TC was purified by plaque 3 rounds. 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 its sequence was determined. It was found that the S8 gene contained a TC tag gene after the CDS1062 base, indicating that the recombinant virus BTV1-NS2-354TC was successfully rescued.

[0040] BSR cells were infected with the recombinant virus BTV1-NS2-354TC at an MOI of 0.1. Cells were then stained with the arsenic-based dye FlAsH-EDT2, fixed with tissue fixative, and the nuclei were stained with Hoechst 33342. Slides were prepared and observed under a laser confocal microscope. Results are as follows: Figure 2 As shown, the recombinant bluetongue virus expressing the TC tag described in this application was stained with the arsenic dye FlAsH-EDT2, and multiple clusters of green fluorescent spots appeared in the cytoplasm. These are BTV virus inclusion bodies formed by the NS2 protein.

[0041] Viral dsRNA was extracted from BSR cells infected with 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 electrophoretic patterns of recombinant virus BTV1-NS2-354TC and wild-type BTV-1 dsRNA were similar (e.g., Figure 3 (As shown in the figure). 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, suggesting 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 analysis was performed using rabbit anti-BTVNS2 antibody, and the results are as follows: Figure 4 As shown, the NS2 protein sizes of wild-type BTV1 and recombinant virus BTV1-NS2-354TC are consistent with expectations. To further confirm the TC tag fusion expression on the NS2 protein, immunofluorescence identification was performed. Rabbit-derived NS2 antibody and goat anti-rabbit IgG Alexa Fluor 568 antibody were used as primary and secondary antibodies, respectively, to label the NS2 protein of the recombinant virus. The results are as follows. Figure 5 As shown, the immunofluorescence (red) results of NS2 protein and the double arsenic dye staining results of TC tag (green) co-localize.

[0043] BSR cells were infected with the recombinant virus BTV1-NS2-354TC for 12 hours, followed by staining with the arsenic dye FlAsH-EDT2. The cells were then placed in a rotating confocal live-cell workstation for continuous imaging analysis. The ambient temperature was set at 37°C and the CO2 concentration at 5%. Images were taken every 20 minutes for 4 hours. Some results are shown below. Figure 6 As shown, the location and size changes of viral inclusion bodies (green focus) formed by 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 of NS2 protein in infected cells.

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

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

Claims

1. A recombinant bluetongue virus strain fused to the NS2 protein expressing a TC tag, characterized in that, The recombinant bluetongue virus was obtained by inserting a TC tag after the 354th amino acid of the non-structural protein NS2 of wild-type bluetongue virus; the amino acid sequence of the TC tag is CCPGCC; the wild-type bluetongue virus is the BTV-1 isolate GS / 11; the amino acid sequence of the NS2 protein is shown in SEQ ID NO.

1.

2. A method for constructing a recombinant bluetongue virus, characterized in that, The method is as follows: a TC tag is obtained by fusing expression after amino acid 354 of the non-structural protein NS2 of wild-type bluetongue virus using genetic engineering technology; the wild-type bluetongue virus is BTV-1 isolate GS / 11; the amino acid sequence of the TC tag is CCPGCC; the amino acid sequence of the NS2 protein is shown in SEQ ID NO.

1.

3. The construction method as described in claim 2, characterized in that, The method includes the following steps: (1) Insert a TC-tagged gene sequence after the 1062nd base 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; transcribe in vitro to generate an S8 mRNA transcript containing the TC tag; the CDS sequence of the S8 gene is shown in SEQ ID NO.2; (2) Construct transcription plasmids for wild-type bluetongue virus genes S1-S7 and S9-S10 respectively, and transcribe them into mRNA transcripts in vitro; (3) The S8 mRNA containing the TC tag described in step (1) and the S1-S7 and S9-S10 mRNA transcripts described in step (2) were co-transfected into cells, and recombinant blue tongue virus was obtained by screening.

4. The construction method as described in claim 3, characterized in that, 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, a TC-tagged gene sequence was inserted after the 1062nd base of the CDS sequence of the wild-type bluetongue virus S8 gene by PCR site-directed mutagenesis, thereby constructing a bluetongue virus S8 gene transcription plasmid containing the TC tag; the gene sequence of the TC tag is shown in SEQ ID NO.

3.

5. The recombinant bluetongue virus obtained by the method described in any one of claims 2-4.

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