Recombinant blue tongue virus capable of visualizing virus inclusion bodies and method for constructing the same

Abstract

This invention belongs to the field of genetic engineering. By introducing a TC tag between amino acids 199 and 200 of the wild-type bluetongue virus non-structural protein NS2, a recombinant virus was rescued. Analysis of viral plaques and growth curves revealed that the recombinant virus formed plaques of similar size to the wild-type virus, and their growth curves showed no significant difference. Immunofluorescence and FlasH-EDT2 staining of NS2 showed that the fluorescence of NS2 labeled with anti-NS2 antibody overlapped with that labeled with FlasH-EDT2. Three-dimensional imaging of FlasH-EDT2-labeled NS2 revealed that the viral inclusion bodies exhibited fluorescence around the periphery but no fluorescence inside, displaying an overall "core-shell" hierarchical structure. This recombinant bluetongue virus can be used for tracing and localizing NS2 protein and viral inclusion bodies in living cells, and can also be applied to BTV neutralization assays, antiviral drug screening, and other related research.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a recombinant bluetongue virus with visualized viral inclusion bodies 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 biting midge. Currently, 29 different serotypes have been identified, and there is no cross-immunity between serotypes.

[0003] The 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, 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, BTV forms the viral core particle, within which three enzyme proteins, VP1, VP4, and VP6, reside. The four non-structural proteins, NS1, NS2, NS3 / NS3A, and NS4, are primarily involved in viral replication, assembly, maturation, and release.

[0004] The non-structural protein NS2 is the main scaffold protein of viral inclusion bodies (VIBs) in BTV and plays a central role in the viral replication cycle. It is highly hydrophilic and charged, can form multimeric complexes, and is the only BTV protein that undergoes phosphorylation and possesses a Ca²⁺ molecule. 2+ The binding site is capable of recruiting core proteins and possesses NTPase hydrolytic activity and non-specific binding ability to ssRNA. NS2 is responsible for both recruiting viral ssRNA and participating in the assembly of viral core particles. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention, by evaluating the conservation and structural characteristics of the NS2 protein sequence, inserts a tetracysteine ​​(TC) tag between amino acids 199 and 200, and between amino acids 323 and 324, respectively, to construct a recombinant virus. Introducing the TC tag between amino acids 199 and 200 of the NS2 protein successfully yields a recombinant virus, and the rescued recombinant virus exhibits growth characteristics identical to the wild type, making it suitable for live-cell tracing and localization of the NS2 protein. Specifically, it includes the following: In a first aspect, the present invention provides a recombinant bluetongue virus with visualized viral inclusion bodies, wherein the recombinant bluetongue virus is obtained by inserting a TC tag between amino acids 199 and 200 of the non-structural protein NS2 of wild-type bluetongue virus; the amino acid sequence of the TC tag is CCPGCC.

[0006] Preferably, the wild-type bluetongue virus is bluetongue virus serotype 1.

[0007] Preferably, the wild-type bluetongue virus is the BTV-1 isolate GS / 11.

[0008] Preferably, the amino acid sequence of the NS2 protein is shown in SEQ ID NO.1.

[0009] Secondly, the present invention provides a method for constructing recombinant bluetongue virus, wherein the method is as follows: a TC tag is inserted between amino acids 199 and 200 of the non-structural protein NS2 of wild-type bluetongue virus by means of genetic engineering technology.

[0010] Preferably, the wild-type bluetongue virus is bluetongue virus serotype 1.

[0011] Preferably, the wild-type bluetongue virus is the BTV-1 isolate GS / 11.

[0012] Preferably, the method includes the following steps: (1) Insert a TC-tagged gene sequence after the 597th base of the wild-type bluetongue virus S8 gene CDS sequence 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 S8 gene CDS sequence is shown in SEQ ID NO.2; (2) Construct transcription plasmids of wild-type bluetongue virus genes S1~S7, S9 and 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, S9 and S10 mRNA transcripts described in step (2) were co-transfected into cells, and recombinant bluetongue virus was obtained by screening.

[0013] Preferably, 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 597th 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.

[0014] Thirdly, the present invention provides a recombinant bluetongue virus constructed by the method described in the second aspect above.

[0015] Fourthly, the present invention provides a recombinant bluetongue virus as described in the first or third aspect above, having any of the following uses: (1) Application in the visual quantitative detection of bluetongue virus; (2) Application in the study of dynamic expression, localization and tracing of bluetongue virus NS2 protein; (3) Application in bluetongue virus and in experiments; (4) Application in screening anti-BTV drugs.

[0016] The beneficial effects of this invention are as follows: First, by evaluating the conservation and structural characteristics of the NS2 protein sequence, a tetracysteine ​​(TC) tag is inserted between amino acids 199 and 200, and between amino acids 323 and 324, respectively, to construct a recombinant virus. Analysis of viral plaques and growth curves revealed that the recombinant virus and the wild-type virus formed plaques of similar size and showed no significant difference in growth curves. Immunofluorescence and FLASH-EDT2 staining of NS2 showed that the fluorescence of the anti-NS2 antibody labeling overlapped with that of the FLASH-EDT2 labeling. 3D imaging of NS2 labeled with FLASH-EDT2 revealed that some VIBs (visible intracellular blocks) were non-fluorescent, exhibiting an overall "core-shell" hierarchical structure. This invention can be used for live-cell tracing and localization of the NS2 protein. Attached Figure Description

[0017] Figure 1 dsRNA detection results.

[0018] Figure 2 NS2 protein expression results.

[0019] Figure 3 Results of viral plaque and growth curve analysis.

[0020] Figure 4 Immunofluorescence labeling and TC tag labeling results of NS2.

[0021] Figure 5 Distribution of NS2 in VIBs. Detailed Implementation

[0022] 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.

[0023] 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 (between amino acids 199 and 200, and between amino acids 323 and 324) on the S8 gene. It was found that only the insertion of the TC tag between amino acids 199 and 200 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, and the gene sequence of the TC tag is shown in SEQ ID NO.3. Example 1: Construction of Recombinant Bluetongue Virus

[0024] This invention first assesses the conservation and structural characteristics of the NS2 protein sequence. Then, we insert tetracysteine ​​(TC) tags between amino acids 199 and 200, and between amino acids 323 and 324, of the NS2 protein to construct recombinant viruses. Introducing the TC tag between amino acids 199 and 200 of the NS2 protein successfully yields recombinant viruses, and the rescued recombinant viruses exhibit growth characteristics identical to the wild type, making them suitable for live-cell tracing and localization of the NS2 protein. Details are as follows: TC tags (TGTTGTCCCGGGTGTTGT) were inserted between amino acids 199 and 200 (after base 597 of the S8 gene CDS sequence) and between amino acids 323 and 324 (after base 969 of the S8 gene CDS sequence), respectively, encoding the BTV-1 NS2 protein.

[0025] Point mutation primers were designed based on the BTV S8 gene sequence. Using the S8 in vitro transcription plasmid as a template, site-directed mutagenesis PCR was performed using high-fidelity DNA polymerase to introduce TC tag sequences at the corresponding sites. The amplified DNA product was treated with DPN I enzyme and purified from the target band by gel electrophoresis. The DNA product was transformed into competent Trans 5α cells and plated on LB agar plates containing ampicillin for overnight culture. Single colonies were picked and inoculated into LB liquid medium containing ampicillin for culture. Colony PCR was performed using S8 identification primers, and sequencing was performed by Qingke Biotechnology Co., Ltd. Colony cultured with the expected sequencing results to extract recombinant plasmids. Restriction endonucleases were used to linearize the recombinant plasmid and 10 wild-type BTV in vitro transcription plasmids, and mutant S8 mRNA and 10 wild-type mRNAs (S1~S10) were generated using a T7 polymerase in vitro transcription kit. First, BHK-21 cells were co-transfected with seven wild-type mRNAs (S1, S3, S4, S6, S7, S8, and S9). Sixteen hours later, all ten wild-type mRNAs were transfected to rescue the wild-type virus. The wild-type S8 mRNA was then replaced with a mutant to rescue the recombinant virus. The recombinant virus with a TC tag inserted between amino acids 323 and 324 of the NS2 protein did not show cytopathic effects, while the recombinant virus with a TC tag inserted between amino acids 199 and 200 of the NS2 protein did show cytopathic effects. All samples from the wells were collected and continuously passaged in BHK-21 cells. These results indicate that the recombinant fluorescent virus BTV1-NS2-199TC with a TC tag inserted between amino acids 199 and 200 of NS2 was successfully rescued, while the recombinant fluorescent blue tongue virus was not rescued with a TC tag inserted after amino acid 323 of NS2.

[0026] After infecting BHK-21 cells with the rescued recombinant virus, all RNA was extracted from the infected cells, identified by RT-PCR using specific S8 primers, and sequenced. Similarly, after extracting all RNA from the infected cells, dsRNA was purified, and the BTV genome size was analyzed by non-denaturing polyacrylamide gel electrophoresis. The genome fragment size was not significantly different from that of the wild type. Figure 1 (As shown).

[0027] Cells were infected with recombinant virus and wild-type virus, and the BTV NS2 protein was detected by immunoblotting. The results showed that the protein was expressed normally. Figure 2 (As shown).

[0028] The obtained recombinant virus BTV1-NS2-199TC underwent three rounds of plaque purification and growth curve analysis. 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. The presence of a TC tag gene after CDS 597 was found, indicating successful rescue of the recombinant virus BTV1-NS2-199TC. Furthermore, the plaque size formed by the recombinant virus was similar to that of the wild-type virus, and the growth curves showed no significant difference. Figure 3 (As shown).

[0029] BSR cells were infected with recombinant virus BTV1-NS2-199TC at MOI=0.1 using immunofluorescence and FLASH-EDT2 staining to label NS2. Cells were then stained with arsenic disulfide, 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 4 As shown, the recombinant bluetongue virus expressing the TC tag described in this application was stained with arsenic diarsenic dye (FlAsH-EDT2), and the fluorescence of the anti-NS2 antibody labeling was observed to overlap with that of the FlAsH-EDT2 labeling. Three-dimensional imaging of NS2 labeled with FlAsH-EDT2 revealed that the VIBs showed fluorescence around their periphery, but no fluorescence inside, exhibiting an overall "core-shell" hierarchical structure. Figure 5 (As shown).

[0030] The above results indicate that the biological characteristics of the recombinant virus BTV1-NS2-199TC 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 NS2 protein.

[0031] 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 NS2 protein and the tracing of BTV virus inclusion bodies.

[0032] 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 with visualized viral inclusion bodies, characterized in that, The recombinant bluetongue virus was obtained by inserting a TC tag between amino acids 199 and 200 of the non-structural protein NS2 of wild-type bluetongue virus; the amino acid sequence of the TC tag is CCPGCC.

2. The recombinant blue tongue virus of claim 1, wherein, The wild-type bluetongue virus is bluetongue virus serotype 1.

3. The recombinant bluetongue virus as described in claim 2, characterized in that, The wild-type bluetongue virus was the BTV-1 isolate GS / 11.

4. The recombinant bluetongue virus as described in claim 3, characterized in that, The amino acid sequence of the NS2 protein is shown in SEQ ID NO.

1.

5. A method for constructing a recombinant bluetongue virus, characterized in that, The method involves inserting a TC tag between amino acids 199 and 200 of the wild-type bluetongue virus non-structural protein NS2 using genetic engineering techniques.

6. The construction method as described in claim 5, characterized in that, The wild-type bluetongue virus was the BTV-1 isolate GS / 11.

7. The construction method as described in claim 6, characterized in that, The method includes the following steps: (1) Insert a TC-tagged gene sequence after the 597th base of the wild-type bluetongue virus S8 gene CDS sequence 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 S8 gene CDS sequence is shown in SEQ ID NO.2; (2) Construct transcription plasmids of wild-type bluetongue virus genes S1~S7, S9 and 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, S9 and S10 mRNA transcripts described in step (2) were co-transfected into cells, and recombinant bluetongue virus was obtained by screening.

8. The construction method as described in claim 7, 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 597th 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.

9. The recombinant bluetongue virus obtained by the method described in any one of claims 5-8.

10. The recombinant bluetongue virus according to any one of claims 1-4 or claim 9 has any of the following uses: (1) Application in the visual quantitative detection of bluetongue virus; (2) Application in the study of dynamic expression, localization and tracing of bluetongue virus NS2 protein; (3) Application in bluetongue virus and in experiments; (4) Application in screening anti-BTV drugs.

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