Recombinant bluetongue virus with green fluorescent protein carried on NS1 microtube, construction method and application
By inserting green fluorescent protein into specific amino acids of the NS1 protein of the blue tongue virus, recombinant blue tongue virus was solved, and the accuracy and reproducibility of the quantitative detection of blue tongue viruses in the prior art was solved, and efficient visual and quantitative detection of the virus was achieved.
Patent Information
- Application Number
- CN202510083443.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-04-22
AI Technical Summary
The prior art is difficult to effectively detect blue tongue virus (BTV), especially when there are genetic differences between different serotypes and viral isolates, resulting in high cost and low accuracy of the detection method.
By inserting green fluorescent protein between amino acids 534 and 535 of the non-structural protein of the blue tongue virus NS1, a recombinant blue tongue virus BTV1-NS1-534eGFP carrying GFP on the NS1 microtubules was constructed, and visual and quantitative detection of the virus was achieved using genetic engineering methods.
The successfully constructed recombinant blue tongue virus can form viral-like microtubule similar to wild-type viruses. The NS1 protein can display green fluorescence and is used for neutralization experiments, visual quantitative detection and localization research of NS1 protein, improving the accuracy and repeatability of the detection.
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Figure CN119859619A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering, and specifically relates to a recombinant bluetongue virus carrying green fluorescent protein on NS1 microtubules, a construction method and an application thereof. Background Art
[0002] Bluetongue disease is a serious infectious disease caused by bluetongue virus (BTV) infecting sheep, cattle, deer and other ruminants. The World Organization for Animal Health (WOAH) lists it as a notifiable animal disease, and my country lists it as a Category II animal disease. BTV belongs to the Orbivirus genus of the Reoviridae family and is an insect-borne virus transmitted by Culicoides. At least 29 different serotypes have been found, and there is no cross-immunity between different serotypes.
[0003] BTV particles are 20-sided symmetrical and have no envelope. Their genome consists of 10 (S1-S10) segmented linear double-stranded RNAs (dsRNAs). The BTV genome encodes seven structural proteins (VP1-VP7) and four non-structural proteins (NS1, NS2, NS3 / NS3A, NS4); BTV particles have a double-layer capsid, with VP2 and VP5 forming the outer capsid and the inner capsid consisting of VP3 and VP7. BTV forms a viral core particle after removing the outer capsid, and three enzyme proteins VP1, VP4, and VP6 are located inside the core particle; among them, the non-structural protein NS1 protein is encoded by the S6 gene and is the most highly expressed protein among all BTV-encoded proteins, accounting for about 25% of the total viral proteins. NS1 protein can form microtubule structures in the cytoplasm infected by BTV in the form of polymers. These microtubules are about 52nm in diameter and about 1000nm long. This is one of the most significant characteristics of BTV infection of cells. Microtubules appear 2 to 4 hours after BTV infection of cells and exist throughout the infection and replication cycle. The amino acid sequences of NS1 proteins of different serotypes of BTV are highly conserved, with a homology of more than 99%.
[0004] At present, there are qRT-PCR, plaque assay, immunofluorescence, TCID 50 However, since 29 serotypes of BTV have been discovered, and the genes of different virus isolates in each serotype are also different, the qRT-PCR method requires the design and synthesis of specific primers and probes, which is costly and may result in nonspecific amplification. The immunofluorescence method requires the preparation or purchase of virus-specific antibodies, which is prone to nonspecific binding and cannot perform real-time quantification and localization of live viruses in infected cells. Plaque assay and TCID 50The method operation process is cumbersome and the technical requirements are relatively high, which increases the errors caused by experimental operations and has relatively low accuracy and repeatability.
[0005] In recent years, the visualization and quantification of viruses is mainly based on the quantification of recombinant viruses encoding fluorescent proteins (FP), that is, by fusing the gene expressing GFP with the viral protein gene through genetic engineering, the specific proteins of the virus can be fluorescently labeled. The genome of BTV consists of 10 double-stranded RNAs of varying sizes (0.8-3.9kb), and the ability of each gene to accommodate foreign genes is very limited. Since the molecular weight of fluorescent proteins is usually 27ku~37ku, the large-scale expression of fluorescent proteins by the virus will affect the infectivity and replication ability of the recombinant virus.
[0006] NS1 tubules have potential in vaccine delivery, treatment and nanotechnology. The present invention previously found that after inserting a green fluorescent protein tag between the 156th and 157th amino acids of the wild-type bluetongue virus nonstructural protein NS1, the rescued recombinant bluetongue virus BTV1-NS1-156eGFP can correctly express enhanced green fluorescent protein, but no longer forms a virus-like microtubule structure, affecting the assembly of the virus-like microtubule structure. Although the existing literature discloses the rescue of the recombinant bluetongue virus BTV1-NS1-552eGFP after inserting a green fluorescent protein tag at the C-terminus of the wild-type bluetongue virus nonstructural protein NS1, compared with the bluetongue virus BTV1-NS1-534eGFP in the present invention, the NS1 in the form of non-microtubule-like monomers in the recombinant bluetongue virus BTV1-NS1-552eGFP infected cells is significantly reduced. Summary of the invention
[0007] In response to the above technical problems, the present invention unexpectedly found that after inserting green fluorescent protein between amino acids 534 and 535 of the non-structural protein NS1 of the wild-type bluetongue virus, a recombinant bluetongue virus BTV1-NS1-534eGFP expressing green fluorescent protein was successfully constructed; the recombinant bluetongue virus can form virus-like microtubules similar to those of the wild-type bluetongue virus (BTV1-WT), and there is no obvious difference in the growth characteristics of BTV-WT. The NS1 protein can show green fluorescence and can be used for neutralization experiments of BTV, visual quantitative detection, and localization and tracing studies of NS1 protein. And compared with the recombinant bluetongue virus BTV1-NS1-552eGFP, the abundance of NS1 monomers in the recombinant bluetongue virus BTV1-NS1-534eGFP is higher without destroying the formation of virus-like microtubules. This provides a basis for studying the function of NS1 microtubule structure in viral replication and provides an example for controlling the conversion of the proportion of NS1 monomer-microtubule form.
[0008] Specifically include the following:
[0009] In a first aspect, the present invention provides a recombinant bluetongue virus carrying GFP on NS1 microtubules, wherein the recombinant bluetongue virus is obtained by inserting green fluorescent protein between amino acids 534 and 535 of the nonstructural protein NS1 of the wild-type bluetongue virus and then rescuing the virus using reverse genetics technology.
[0010] Preferably, the wild-type bluetongue virus is bluetongue virus serotype 1.
[0011] Preferably, the wild-type bluetongue virus is the BTV-1GS / 11 strain.
[0012] Preferably, the amino acid sequence of the non-structural protein NS1 is as shown in SEQ ID NO.1.
[0013] Preferably, the amino acid sequence of the green fluorescent protein is as shown in SQE ID NO.3.
[0014] In a second aspect, the present invention provides a method for constructing a recombinant bluetongue virus carrying GFP on NS1 microtubules, the method comprising: inserting green fluorescent protein between the 534th and 535th amino acids of the wild-type bluetongue virus nonstructural protein NS1 by genetic engineering means.
[0015] Preferably, the method comprises the following steps:
[0016] (1) inserting the gene sequence of enhanced green fluorescent protein after the 1602th base of the CDS sequence of the S6 gene of the wild-type bluetongue virus to construct an S6 gene transcription plasmid containing the eGFP fluorescent protein gene sequence;
[0017] (2) constructing transcription plasmids of wild-type bluetongue virus genes S1-S5 and S7-S10 respectively and transcribing them into mRNA transcripts in vitro;
[0018] (3) Co-transfecting cells with the S6 gene transcription plasmid containing the eGFP fluorescent protein gene sequence described in step (1) and the mRNA transcript described in step (2) to screen and obtain a recombinant bluetongue virus expressing green fluorescent protein.
[0019] Preferably, the wild-type bluetongue virus is bluetongue virus serotype 1.
[0020] Preferably, the wild-type bluetongue virus is the BTV-1GS / 11 strain.
[0021] Preferably, the CDS sequence of the wild-type bluetongue virus S6 gene is as shown in SEQ ID NO.2.
[0022] Preferably, the gene sequence of the enhanced green fluorescent protein is as shown in SEQ ID NO.4.
[0023] In a third aspect, the present invention provides a recombinant bluetongue virus carrying GFP on NS1 microtubules obtained by the method described in the second aspect.
[0024] In a fourth aspect, the present invention provides a recombinant bluetongue virus carrying GFP on NS1 microtubules as described in the first aspect or the third aspect, having any of the following uses:
[0025] (1) Application in the study of dynamic expression, localization and tracing of bluetongue virus NS1 protein;
[0026] (2) Application in neutralization experiments and visual quantitative detection of bluetongue virus.
[0027] The invention has the following beneficial effects: after the green fluorescent protein is inserted between the amino acids 534 and 535 of the non-structural protein NS1 of the bluetongue virus, the assembly of the bluetongue virus is not affected and the virus can be successfully rescued; compared with the recombinant bluetongue virus BTV1-NS1-156eGFP, the constructed recombinant bluetongue virus BTV1-NS1-534eGFP can form virus-like microtubules similar to the wild-type bluetongue virus (BTV1-WT), and has no obvious difference from the growth characteristics of BTV1-WT; compared with the recombinant bluetongue virus BTV1-NS1-552eGFP, the constructed recombinant bluetongue virus BTV1-NS1-534eGFP has more NS1 in monomer form, and has potential in vaccine delivery, treatment and nanotechnology; and the NS1 protein can display green fluorescence, and can be used for neutralization experiments, visual quantitative detection and real-time positioning and tracing research of BTV, and can also be applied to neutralization experiments and visual quantitative detection of BTV, providing tools and technical platforms for studying the function of BTV NS1 and intuitive visual quantitative detection of viruses. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Observation results under laser confocal microscopy after BSR cells were infected with BTV1-NS1-534eGFP recombinant virus;
[0029] Figure 2 Virus genome dsRNA non-denaturing polyacrylamide gel electrophoresis results;
[0030] Figure 3 Western blot analysis results of recombinant bluetongue virus;
[0031] Figure 4Laser confocal microscope observation of fluorescence of recombinant bluetongue virus; BSR cells were infected with BTV1-NS1-534eGFP recombinant virus, green is NS1-534eGFP protein, red is BTV NS1 protein labeled with rabbit anti-NS1 antibody;
[0032] Figure 5 Transmission electron microscopy observation of the virus microtubule structure results; red arrows indicate virus particles, blue arrows indicate virus-like microtubules, and yellow arrows indicate virus inclusion bodies;
[0033] Figure 6 The fluorescence results of laser confocal imaging of recombinant bluetongue viruses BTV1-NS1-534eGFP, BTV1-NS1-156eGFP and BTV1-NS1-552eGFP under the same shooting parameters;
[0034] Figure 7 Fluorescence background values of recombinant bluetongue viruses BTV1-NS1-534eGFP, BTV1-NS1-156eGFP and BTV1-NS1-552eGFP in cells. DETAILED DESCRIPTION
[0035] 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 exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.
[0036] The following examples take bluetongue virus serotype 1 (GS / 11) as an example. The present invention uses bioinformatics software in combination with published BTV-1NS1 (S6 gene encoding) related literature. The present invention inserts a green fluorescent protein expression gene after the 1602 base of the S6 gene to construct S6-534eGFP mRNA containing the eGFP fluorescent protein gene sequence: using the wild-type S6 gene transcription plasmid as a template, constructing an S6 gene transcription plasmid containing a T7 promoter and green fluorescent protein by PCR gene site-directed mutagenesis technology, and performing in vitro transcription with a T7 polymerase transcription kit after enzyme linearization to generate S6-534eGFP mRNA containing the eGFP fluorescent protein gene sequence;
[0037] The transcription plasmids of the other nine wild-type genes (S1 to S10) of BTV-1 were linearized by restriction enzyme digestion, transcribed in vitro into mRNA transcripts of each gene, and co-transfected into BSR cells. Obvious cytopathic effect (CPE) appeared 3 days after transfection. The diseased cells were collected and purified through 3 rounds of plaque purification to obtain a NS1 recombinant mutant virus named BTV1-NS1-534eGFP. After the recombinant bluetongue virus BTV1-NS1-534eGFP infected BSR cells, the cells were fixed with 4% paraformaldehyde, the cell nuclei were stained with Hoechst 33342, and the cells were observed with a laser confocal microscope. The results are shown in Figure 1 As shown, NS1 protein showed green fluorescence in BSR cells infected with the recombinant bluetongue virus BTV1-NS1-534eGFP.
[0038] The genome of the recombinant bluetongue virus BTV1-NS1-534eGFP was extracted and the RT-PCR amplification product was sequenced, and it was found that the eGFP gene was present at the position corresponding to the S6 gene. The viral genome dsRNA was extracted from the infected cells and subjected to non-denaturing polyacrylamide gel electrophoresis. The results are as follows Figure 2 As shown in Figure 1, the migration speed of the S6 gene of the recombinant bluetongue virus BTV1-NS1-534eGFP was slower than that of BTV1-WT, confirming that the modified S6 (S6-534eGFP) was integrated into the viral genome. Figure 3 As shown in Figure 1, the NS1 protein of the recombinant bluetongue virus BTV1-NS1-534eGFP is 27Kda larger than the NS1 protein of BTV-1, which is in line with the expected size. The fluorescence results of the recombinant bluetongue virus observed by laser confocal microscopy are shown in Figure 1. Figure 4 As shown, the rabbit polyclonal antibody of NS1 protein was used as the primary antibody and goat anti-rabbit 568 was used as the secondary antibody to stain the NS1 protein of the recombinant virus. It was found that the red fluorescence of the fluorescent immunolabeled NS1 protein and the green fluorescence of eGFP were co-localized, indicating that the green fluorescence in the recombinant bluetongue virus BTV1-NS1-534eGFP was indeed labeled on NS1.
[0039] At the same time, the eGFP tag was inserted between the 156th and 157th amino acids of the NS1 protein to rescue the recombinant bluetongue virus BTV1-NS1-156eGFP; BTV1-WT, BTV1-NS1-156eGFP and BTV1-NS1-534eGFP were infected with BSR cells, and ultrathin sections were prepared and observed by transmission electron microscopy; the results are shown in Figure 5 As shown, the recombinant bluetongue virus BTV1-NS1-534eGFP can form virus-like microtubules similar to BTV1-WT, while the recombinant bluetongue virus BTV1-NS1-156eGFP did not form virus-like microtubules.
[0040] Under the same shooting parameters of laser confocal microscope, the recombinant bluetongue virus BTV1-NS1-534eGFP has a different green fluorescence background from the recombinant bluetongue virus BTV1-NS1-156eGFP and BTV1-NS1-552eGFP ( Figure 6 At the same time, under the same shooting parameters, the fluorescence background of 50 cells was counted for each recombinant virus. The results showed that the fluorescence background value of the recombinant bluetongue virus BTV1-NS1-534eGFP in cells was significantly different from that of BTV1-NS1-156eGFP and BTV1-NS1-552eGFP ( Figure 7 This indicates that the insertion of eGFP after amino acid 534 did not disrupt the formation of viral-like microtubules, but compared with the recombinant bluetongue virus BTV1-NS1-552eGFP, the NS1 in the recombinant bluetongue virus BTV1-NS1-534eGFP existed more in monomeric form, which has potential in vaccine delivery, therapy and nanotechnology; it also confirms the possibility of controlling the conversion of the proportion of NS1 monomer-microtubule form; at the same time, it also provides a basis for studying the function of NS1 microtubule structure in viral replication.
[0041] In summary, the recombinant bluetongue virus BTV1-NS1-534eGFP can be used for the visual quantitative detection of BTV, virus neutralization experiments and antiviral drug screening of BTV, the migration and aggregation study of NS1 protein after cells are infected with BTV virus, the function of NS1 protein in forming virus-like microtubules, and the interaction study between NS1 protein and host proteins.
[0042] The specific implementation is as follows:
[0043] Example 1 Construction of recombinant bluetongue virus BTV1-NS1-534eGFP
[0044] 1. Construction method
[0045] Insert the green fluorescent protein (SEQ ID NO.3) gene after the 1602th base of the CDS sequence of the BTV-1 S6 gene. Design a mutant primer with a sequence encoding the green fluorescent protein gene (SEQ ID NO.4), use the wild-type S6 gene transcription plasmid as a template, and construct the NS1 gene transcription plasmid containing the T7 promoter and the green fluorescent protein gene by PCR gene fusion mutation technology. After restriction enzyme linearization, perform in vitro transcription with a T7 polymerase transcription kit to generate S6-534eGFP mRNA containing green fluorescent protein. At the same time, perform restriction enzyme linearization on the transcription plasmids of the other 9 wild-type genes (S1 to S10) of BTV-1, and transcribe them in vitro into mRNA transcripts of each gene.
[0046] S6-534eGFP mRNA was co-transfected with wild-type S1-S5, S7-S10 mRNA into BSR cells, and the transfected cells were observed continuously for 3-5 days.
[0047] The RNA of cells infected with the recombinant virus was extracted, and RT-PCR amplification was performed using primers specific for the S6-534eGFP gene fragment, and sequencing was performed to verify whether the insertion position of the green fluorescent protein was correct. At the same time, the dsRNA of the recombinant virus was extracted for genome electrophoresis analysis.
[0048] The BSR cells were infected with the correctly sequenced recombinant virus containing green fluorescent protein, and the cell nuclei were stained with Hoechst33342. The fluorescent staining of the recombinant virus NS1 protein was observed by confocal microscopy.
[0049] 2. Results Analysis
[0050] Three days after transfection, the transfection wells containing S6-534eGFP mRNA showed obvious cytopathic effect (CPE), indicating that the insertion of green fluorescent protein between amino acids 534-535 of the nonstructural protein NS1 of bluetongue virus did not affect the rescue of the recombinant virus. After the recombinant bluetongue virus BTV1-NS1-534eGFP infected BSR cells, the cells were fixed with 4% paraformaldehyde, the cell nuclei were stained with Hoechst33342, and the cells were observed with a laser confocal microscope. The results are shown in Figure 1 As shown, the NS1 protein showed green fluorescence in BSR cells infected with the recombinant bluetongue virus BTV1-NS1-534eGFP. That is, the insertion of green fluorescent protein between amino acids 534-535 of the non-structural protein NS1 of the bluetongue virus as described in the present application can successfully rescue the recombinant bluetongue virus. The transfected well cells with CPE were collected and subjected to three rounds of plaque purification to obtain the recombinant bluetongue virus BTV1-NS1-534eGFP.
[0051] The genome of the recombinant bluetongue virus BTV1-NS1-534eGFP was extracted and the RT-PCR amplification product was sequenced, and it was found that the eGFP gene was present at the position corresponding to the S6 gene. The viral genome dsRNA was extracted from the infected cells and subjected to non-denaturing polyacrylamide gel electrophoresis. The results are as follows Figure 2 As shown, compared with the wild-type bluetongue virus (BTV1-WT), the migration speed of the S6 gene of the recombinant bluetongue virus BTV1-NS1-534eGFP was slowed down, confirming that the modified S6 (S6-534eGFP) was integrated into the viral genome.
[0052] Weston-blot results are as follows Figure 3As shown, the NS1 protein of the recombinant bluetongue virus BTV1-NS1-534eGFP is 27Kda larger than the NS1 protein of BTV-1, which is consistent with the expected size.
[0053] The fluorescence results of laser confocal microscopy of recombinant bluetongue virus are as follows: Figure 4 As shown, the rabbit polyclonal antibody of NS1 protein was used as the primary antibody and goat anti-rabbit 568 was used as the secondary antibody to stain the NS1 protein of the recombinant virus. It was found that the red fluorescence of the immunolabeled NS1 protein and the green fluorescence of eGFP were co-localized, indicating that the green fluorescence in the recombinant bluetongue virus BTV1-NS1-534eGFP was indeed labeled on NS1.
[0054] Example 2 Structural verification of recombinant bluetongue virus BTV1-NS1-534eGFP
[0055] Using the same method as described in Example 1, an eGFP tag was inserted between amino acids 156 and 157 of the NS1 protein for rescue, thereby constructing a recombinant bluetongue virus BTV1-NS1-156eGFP; an eGFP tag was inserted after amino acid 552 of the NS1 protein for rescue, thereby constructing a recombinant bluetongue virus BTV1-NS1-552eGFP.
[0056] After BSR cells were infected with BTV1-WT, BTV1-NS1-156eGFP and BTV1-NS1-534eGFP, ultrathin sections were prepared and the microtubule structure of the virus was observed using transmission electron microscopy.
[0057] The results are as follows Figure 5 As shown, compared with the wild-type bluetongue virus (BTV1-WT), after the recombinant bluetongue virus BTV1-NS1-156eGFP infected BSR cells, no viral-like microtubules were observed around the nucleus; while the recombinant bluetongue virus BTV1-NS1-534eGFP was the same as the wild-type bluetongue virus (BTV1-WT), and viral-like microtubules could be observed. This shows that the present application can rescue a recombinant bluetongue virus with similar growth characteristics to the wild-type bluetongue virus after inserting green fluorescent protein between the 534th and 535th amino acids of the structural protein NS1 of the wild-type bluetongue virus (BTV1-WT).
[0058] Under the same shooting parameters, laser confocal fluorescence photography was performed on the recombinant bluetongue viruses BTV1-NS1-534eGFP, BTV1-NS1-156eGFP and BTV1-NS1-552eGFP. Figure 6 As shown, the recombinant bluetongue virus BTV1-NS1-534eGFP has a different green fluorescence background from BTV1-NS1-156eGFP and BTV1-NS1-552eGFP.
[0059] Under the same shooting parameters, the fluorescence background of 50 cells of each recombinant bluetongue virus was counted and the background value was calculated. Figure 7 As shown, the fluorescence background value of the recombinant bluetongue virus BTV1-NS1-534eGFP in cells is significantly higher than that of the recombinant bluetongue virus BTV1-NS1-552eGFP. This shows that the insertion of eGFP after the 534th amino acid did not destroy the formation of viral-like microtubules, but compared with BTV1-NS1-552eGFP, NS1 in BTV1-NS1-534eGFP exists more in the form of monomers, which has potential in vaccine delivery, treatment and nanotechnology. The emergence of recombinant bluetongue virus BTV1-NS1-534eGFP confirms the possibility of controlling the conversion of the proportion of NS1 monomer-microtubule forms. At the same time, it also provides a material basis for studying the function of NS1 microtubule structure in viral replication.
[0060] In summary, the insertion of green fluorescent protein between amino acids 534 and 535 of the non-structural protein NS1 of the bluetongue virus in the present application does not affect the assembly of the bluetongue virus and can be successfully rescued. The recombinant bluetongue virus BTV1-NS1-534eGFP constructed can form virus-like microtubules similar to those of the wild-type bluetongue virus (BTV1-WT), and has no obvious difference in growth characteristics from the wild-type bluetongue virus. In addition, the NS1 protein can display green fluorescence and can be used for neutralization experiments, visualized quantitative detection, and real-time localization and tracing studies of NS1 protein of BTV. It can also be applied to neutralization experiments and visualized quantitative detection of BTV, providing tools and technical platforms for studying the function of BTV NS1 and intuitive visualized quantitative detection of viruses.
[0061] Although the present invention takes BTV-1 (GS / 11) as an example, based on the high conservation of the amino acid sequences of BTV-1 and other serotypes of NS1 (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 carrying GFP on NS1 microtubules, characterized in that: The recombinant bluetongue virus is obtained by inserting green fluorescent protein between the 534th and 535th amino acids of the non-structural protein NS1 of the wild-type bluetongue virus and then rescuing the virus using reverse genetic technology.
2. The recombinant bluetongue virus carrying GFP on NS1 microtubules according to claim 1, characterized in that: The wild-type bluetongue virus is the BTV-1GS / 11 strain.
3. The recombinant bluetongue virus carrying GFP on NS1 microtubules according to claim 2, characterized in that: The amino acid sequence of the non-structural protein NS1 is shown in SEQ ID NO.
1.
4. The recombinant bluetongue virus carrying GFP on NS1 microtubules according to claim 3, characterized in that: The amino acid sequence of the green fluorescent protein is shown in SQE ID NO.
3.
5. A method for constructing a recombinant bluetongue virus carrying GFP on NS1 microtubules, characterized in that: The method comprises the following steps: inserting green fluorescent protein between the 534th and 535th amino acids of the non-structural protein NS1 of the wild-type bluetongue virus by means of genetic engineering to obtain the green fluorescent protein.
6. The construction method according to claim 5, characterized in that: The method comprises the following steps: (1) inserting the gene sequence of enhanced green fluorescent protein after the 1602th base of the CDS sequence of the S6 gene of the wild-type bluetongue virus to construct an S6 gene transcription plasmid containing the eGFP fluorescent protein gene sequence; (2) constructing transcription plasmids of wild-type bluetongue virus genes S1-S5 and S7-S10 respectively and transcribing them into mRNA transcripts in vitro; (3) Co-transfecting cells with the S6 gene transcription plasmid containing the eGFP fluorescent protein gene sequence described in step (1) and the mRNA transcript described in step (2) to screen and obtain recombinant bluetongue virus carrying GFP on NS1 microtubules.
7. The construction method according to claim 6, characterized in that: The wild-type bluetongue virus is the BTV-1GS / 11 strain.
8. The construction method according to claim 7, characterized in that: The CDS sequence of the wild-type bluetongue virus S6 gene is shown in SEQ ID NO.
2.
9. The recombinant bluetongue virus carrying GFP on the NS1 microtubule obtained by the method of any one of claims 5 to 8.
10. The recombinant bluetongue virus carrying GFP on NS1 microtubules according to any one of claims 1 to 4 or claim 9 has any of the following uses: (1) Application in the study of dynamic expression, localization and tracing of bluetongue virus NS1 protein; (2) Application in neutralization experiments and visual quantitative detection of bluetongue virus.