Construction method and application of a red fluorescent protein-labeled recombinant blue tongue virus

Abstract

The application belongs to the field of genetic engineering, and particularly relates to a construction method and application of a red fluorescent protein labeled recombinant blue tongue virus. The application inserts a red fluorescent protein label sequence (mScarlet3) between 158th and 159th amino acids of a wild type blue tongue virus NS3 protein, and obtains a NS3 labeled recombinant blue tongue virus BTV-1 / NS3-158mScarlet3. Compared with the wild type blue tongue virus, the recombinant NS3 protein can correctly express the red fluorescent protein, but does not affect the replication of the virus, and can be used for expression positioning and dynamic tracking of the NS3 protein in the BTV infected cell, interaction between the NS3 protein and the host protein, BTV neutralization test, antiviral drug screening and gene delivery and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the construction method and application of a recombinant bluetongue virus labeled with red fluorescent protein. Background Technology

[0002] Bluetongue disease is a serious and highly contagious disease caused by bluetongue virus (BTV) infecting ruminants such as sheep, cattle, and deer. The World Organisation for Animal Health (WOAH) lists it as a notifiable animal disease, and my country classifies it as a Class II animal disease. BTV belongs to the Reoviridae family (…). Reoviridae Orbiviruses are a genus of viruses produced by Culicoides midges. Culicoides The vector-borne viruses that spread through the virus have at least 29 different serotypes, and there is no cross-immunity between different serotypes.

[0003] The BTV particle exhibits icosahedral symmetry, lacks an envelope, and 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 possesses 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.

[0004] Currently, methods for quantitative detection of BTV include qRT-PCR, plaque assay, immunofluorescence, and TCID. 50 Methods such as qRT-PCR and TCID are available. However, since 29 serotypes of BTV have been identified, and the genes of different viral isolates within each serotype differ, qRT-PCR requires the design and synthesis of specific primers and probes, resulting in high costs and the potential for non-specific amplification. Immunofluorescence requires the preparation or purchase of virus-specific antibodies, is prone to non-specific binding, and cannot perform real-time quantification and localization of live viruses in infected cells. Plaque assays and TCID are also available. 50 The method is cumbersome to operate and has high technical requirements, which increases the error generated by the experimental operation and results in low accuracy and repeatability.

[0005] Fluorescent protein-labeled recombinant viruses can be visualized and quantitatively detected by measuring the intensity of their fluorescence. The genome of BTV consists of 10 double-stranded RNAs of varying sizes (0.8-3.9 kb), and each gene has a very limited capacity to accommodate foreign genes. Since the molecular weight of fluorescent proteins is usually between 27 kDa and 37 kDa, inserting fluorescent protein genes into viral genes and successfully expressing them is quite difficult.

[0006] This invention analyzed the possible insertion sites of the BTV NS3 protein and designed an mScarlet3 tag sequence insertion site without disrupting the function of the NS3 protein, thus constructing a recombinant bluetongue virus. Compared with wild-type bluetongue virus, the recombinant bluetongue virus can correctly express red fluorescent protein without affecting viral replication. It can be used for subcellular localization and tracing of the NS3 protein in BTV-infected cells, NS3 protein-host protein interaction, BTV neutralization experiments, antiviral drug screening, and gene delivery studies. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention successfully constructed a recombinant bluetongue virus (BTV-1 / NS3-158mScarlet3) expressing red fluorescent protein by inserting a red fluorescent protein after amino acid 158 of the wild-type bluetongue virus non-structural protein NS3. Compared to the wild-type bluetongue virus, the recombinant bluetongue virus correctly expresses red fluorescent protein without affecting viral replication. Specifically, it includes the following: In a first aspect, the present invention provides a recombinant bluetongue virus with NS3 protein labeled with red fluorescent protein, wherein the recombinant bluetongue virus is obtained by inserting a red fluorescent protein sequence between amino acids 158 and 159 of the wild-type bluetongue virus NS3 protein.

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

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

[0010] Preferably, the amino acid sequence of the red fluorescent protein is as shown in SQE ID NO.1.

[0011] Secondly, the present invention provides a method for constructing a recombinant bluetongue virus labeled with red fluorescent protein. The method involves inserting red fluorescent protein between amino acids 158 and 159 of the wild-type bluetongue virus NS3 protein using genetic engineering techniques, and then obtaining the virus through reverse genetics technology.

[0012] Preferably, the method includes the following steps: (1) Insert the gene sequence of red fluorescent protein after the 474th base of the CDS sequence of wild-type bluetongue virus S10 gene to construct an S10 gene transcription plasmid containing the red fluorescent protein gene sequence. (2) Construct transcription plasmids for wild-type bluetongue virus genes S1-S9 respectively, and transcribe them into mRNA transcripts in vitro; (3) The S10 gene transcription plasmid containing the red fluorescent protein gene sequence described in step (1) and the mRNA transcript described in step (2) are co-transfected into cells, and recombinant bluetongue virus labeled with fluorescent protein is obtained by screening.

[0013] Preferably, the wild-type bluetongue virus is bluetongue virus serotype 1.

[0014] Preferably, the wild-type bluetongue virus is BTV-1 (GS / 11).

[0015] Thirdly, the present invention provides a recombinant bluetongue virus constructed by the method described in the second aspect above.

[0016] Fourthly, the present invention provides the recombinant bluetongue virus described in the first or third aspect above as having any of the following uses: (1) Application in the localization and tracing of NS3 protein in bluetongue virus; (2) Application in the study of the interaction between bluetongue virus NS3 protein and host protein; (3) Application in bluetongue virus and in experiments; (4) Application in screening anti-BTV drugs; (5) Application of BTV as a vector for gene delivery.

[0017] The beneficial effects of this invention are as follows: This invention studies the sequence of the non-structural protein NS3 of wild-type bluetongue virus, designs an mScarlet3 tag sequence insertion site without destroying the function of the NS3 protein, and successfully constructs a recombinant bluetongue virus; compared with wild-type bluetongue virus, the recombinant bluetongue virus can correctly express red fluorescent protein without affecting viral replication, and can be used for research such as expression localization and tracing of NS3 protein after cell infection with BTV, interaction between NS3 protein and host protein, neutralization assay of BTV, screening of antiviral drugs, and gene delivery. Attached Figure Description

[0018] Figure 1 Purification results of recombinant bluetongue virus plaques; A: Plaques formed by BHK-21 cells under bright field; B: Red fluorescent plaques; C: Bright field and fluorescence overlay image.

[0019] Figure 2 Results of immunoblotting of cell lysates infected with wild-type bluetongue virus BTV-1 / WT and recombinant bluetongue virus BTV-1 / NS3-158mScarlet3 using anti-NS3 antibody.

[0020] Figure 3Results of polyacrylamide gel electrophoresis (PAGE) analysis of wild-type bluetongue virus BTV-1 / WT and recombinant bluetongue virus BTV-1 / NS3-158mScarlet3 dsRNA.

[0021] Figure 4 Comparison of plaque morphology between wild-type bluetongue virus BTV-1 / WT and recombinant bluetongue virus BTV-1 / NS3-158mScarlet3 on BHK-21 cells. 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 BTV-1 NS3 (encoded by the S10 gene). This invention inserts a red fluorescent protein gene after position 474 of the S10 gene CDS to construct S10-mScarlet3 mRNA containing a red fluorescent protein tag gene sequence: Using a wild-type S10 gene transcription plasmid as a template, a S10 gene transcription plasmid containing the T7 promoter and red fluorescent protein is constructed using PCR site-directed mutagenesis. After linearization by enzyme digestion, in vitro transcription is performed using a T7 polymerase transcription kit to generate S10-mScarlet3 mRNA containing the red fluorescent protein (mScarlet3 tag) gene sequence.

[0024] The transcriptional plasmids of the other nine wild-type genes (S1-S9) of BTV-1 were linearized by enzyme digestion and transcribed in vitro into mRNA transcripts for each gene. These transcripts were then co-transfected into BHK-21 cells, resulting in significant cytopathic effects (CPE) three days after transfection. Cells were collected and purified through three rounds of plaque phage, yielding a recombinant mutant NS3 virus, named BTV-1 / NS3-158mScarlet3. When BTV-1 / NS3-158mScarlet3 was seeded into BHK-21 cells, significant red fluorescence was observed under a fluorescence microscope after 36 hours.

[0025] The amino acid sequence of the NS3 protein is shown in SEQ ID NO.1, and the CDS sequence of the S10 gene is shown in SEQ ID NO.2; the amino acid sequence of the red fluorescent protein is shown in SEQ ID NO.3, and the gene sequence is shown in SEQ ID NO.4.

[0026] This invention also attempts to construct recombinant bluetongue virus by inserting red fluorescent protein at other sites. Inserting red fluorescent protein after amino acid 47 of the NS3 protein failed to rescue the recombinant bluetongue virus. Only inserting red fluorescent protein after amino acid 158, as described in this application, successfully rescued the corresponding recombinant bluetongue virus, and it could be passaged for more than 10 generations. Specific implementation methods are as follows. Example 1 Construction of recombinant bluetongue virus labeled with red fluorescent protein

[0027] 1. Construction Method A red fluorescent protein gene was inserted after the 474th base of the CDS sequence of the BTV-1 S10 gene. Mutation primers encoding the red fluorescent protein gene sequence (SEQ ID NO.2) were designed. Using the wild-type S10 gene transcription plasmid as a template, an NS3 gene transcription plasmid containing the T7 promoter and the red fluorescent protein gene was constructed using PCR gene fusion mutation technology. After linearization by enzyme digestion, in vitro transcription was performed using a T7 polymerase transcription kit to generate S10-mScarlet3 mRNA containing red fluorescent protein. Simultaneously, the BTV-1 wild-type gene S1~S9 transcription plasmids were linearized by enzyme digestion and transcribed in vitro into mRNA transcripts for each gene.

[0028] S10-mScarlet3 mRNA was co-transfected with wild-type S1-S9 mRNA into BHK-21 cells, and the transfected cells were continuously observed. Obvious cytopathic effects (CPE) appeared 3 days after transfection. Cells were collected and purified through three rounds of plaque phage analysis to obtain a recombinant mutant NS3 virus, named BTV-1 / NS3-158mScarlet3. When BTV-1 / NS3-158mScarlet3 was seeded into BHK-21 cells, obvious red fluorescence (e.g., 56 μg / mScarlet3) was observed under a fluorescence microscope after 36 hours. Figure 1 (As shown).

[0029] 2. Genome electrophoresis analysis Western blot analysis of cells infected with recombinant virus BTV-1 / NS3-158mScarlet3 revealed that the NS3 protein of recombinant bluetongue virus BTV-1 / NS3-158mScarlet3 was 26 kDa larger than that of BTV-1, consistent with the expected size. Figure 2 (As shown).

[0030] 3. Polyacrylamide gel electrophoresis BHK-21 cell suspensions infected with wild-type bluetongue virus (BTV-1 / WT) and recombinant bluetongue virus BTV-1 / NS3-158mScarlet3 were collected, and viral dsRNA was extracted. Genomic changes following the insertion of the mScarlet3 tag into the BTV S10 fragment were detected by polyacrylamide gel electrophoresis (PAGE). Results are as follows: Figure 3 As shown, the size of the S10 fragment changes from 822 bp to 1524 bp due to the insertion of the mScarlet3 tag.

[0031] 4. Phagocytosis analysis Phagocytosis analysis revealed that the phagocytosis plaques formed by the recombinant bluetongue virus BTV-1 / NS3-158mScarlet3 were similar in size to those of the wild-type bluetongue virus BTV-1 (e.g., ...). Figure 4 (As shown). The results indicate that inserting the mScarlet3 fluorescent tag after amino acid 158 of the NS3 protein does not affect the replication of recombinant bluetongue virus.

[0032] In summary, the recombinant bluetongue virus expressing red fluorescent protein described above can be used for research on the expression, localization, and tracing of NS3 protein in BTV-infected cells, the interaction between NS3 protein and host proteins, BTV neutralization experiments, antiviral drug screening, and gene delivery.

[0033] 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 NS3 (homology of more than 99%).

Claims

1. A recombinant bluetongue virus strain labeled with red fluorescent protein NS3 protein, characterized in that, The recombinant bluetongue virus was obtained by inserting a red fluorescent protein sequence between amino acids 158 and 159 of the wild-type bluetongue virus NS3 protein.

2. The recombinant bluetongue virus as described in claim 1, characterized in that, 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 is BTV-1 (GS / 11).

4. The recombinant bluetongue virus as described in claim 1, characterized in that, The amino acid sequence of the red fluorescent protein is shown in SQE ID NO.

1.

5. A method for constructing recombinant bluetongue virus labeled with red fluorescent protein, characterized in that, The method involves inserting a red fluorescent protein between amino acids 158 and 159 of the wild-type bluetongue virus NS3 protein using genetic engineering techniques, and then rescuing the virus using reverse genetics.

6. The construction method as described in claim 5, characterized in that, The method includes the following steps: (1) Insert the gene sequence of red fluorescent protein after the 474th base of the CDS sequence of wild-type bluetongue virus S10 gene to construct an S10 gene transcription plasmid containing the red fluorescent protein gene sequence. (2) Construct transcription plasmids for wild-type bluetongue virus genes S1-S9 respectively, and transcribe them into mRNA transcripts in vitro; (3) The S10 gene transcription plasmid containing the red fluorescent protein gene sequence described in step (1) and the mRNA transcript described in step (2) are co-transfected into cells to screen and obtain recombinant blue tongue virus labeled with red fluorescent protein.

7. The construction method as described in claim 6, characterized in that, The wild-type bluetongue virus is bluetongue virus serotype 1.

8. The construction method as described in claim 7, characterized in that, The wild-type bluetongue virus is BTV-1 (GS / 11).

9. The recombinant bluetongue virus obtained by the method described in any one of claims 4-8.

10. The recombinant bluetongue virus according to any one of claims 1-3 or claim 9 has any of the following uses: (1) Application in the localization and tracing of NS3 protein in bluetongue virus; (2) Application in the study of the interaction between bluetongue virus NS3 protein and host protein; (3) Application in bluetongue virus and in experiments; (4) Application in screening anti-BTV drugs; (5) Application of BTV as a vector for gene delivery.

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