A recombinant bluetongue virus with NS1 protein tagged with green fluorescent protein

Abstract

The present invention belongs to the field of genetic engineering, and specifically relates to a recombinant bluetongue virus with NS1 protein labeled with green fluorescent protein. The present invention constructs and obtains NS1 recombinant mutant bluetongue virus BTV‑1S6‑156eGFP by inserting an enhanced green fluorescent protein (eGFP) sequence between amino acids 156 and 157 of the wild-type bluetongue virus nonstructural protein NS1; compared with the wild-type bluetongue virus, the recombinant NS1 protein of the recombinant bluetongue virus can correctly express enhanced green fluorescent protein, but no longer forms a virus-like microtubule structure, and can be used for related research on the failure of NS1 protein to form a virus-like microtubule structure after BTV infects cells, and can also be used for virus neutralization experiments of BTV and visual quantitative detection of virus content, etc.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant bluetongue virus with NS1 protein marked with green fluorescent protein. 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. Microtubules appear 2 to 4 hours after BTV infection and exist throughout the infection and replication cycle. However, the biological function of microtubule formation is not understood at present. The amino acid sequence of NS1 protein of different serotypes of BTV is 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 protein 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, it is difficult to insert fluorescent protein genes into viral genes and successfully express them.

[0006] The present invention unexpectedly discovered that inserting green fluorescent protein after the 156th amino acid of the non-structural protein NS1 of the bluetongue virus does not affect the assembly of the bluetongue virus, and the recombinant virus can be successfully rescued. Compared with the wild-type bluetongue virus, the constructed recombinant bluetongue virus BTV-1S6-156eGFP is not affected in the expression of the recombinant virus NS1 protein and can correctly express enhanced green fluorescent protein, but no longer forms a virus-like microtubule structure. The recombinant bluetongue virus BTV-1S6-156eGFP can be used for related research on the failure of NS1 protein to form a virus-like microtubule structure after BTV infects cells, and can be used for neutralizing antibody determination experiments and visual quantitative detection of BTV. Summary of the invention

[0007] In view of the above technical problems, the present invention unexpectedly found that after inserting green fluorescent protein after the 156th amino acid of the wild-type bluetongue virus non-structural protein NS1, a recombinant bluetongue virus BTV-1S6-156eGFP expressing green fluorescent protein was successfully constructed; compared with the wild-type bluetongue virus, the expression of the recombinant virus NS1 protein is not affected and the enhanced green fluorescent protein can be correctly expressed, but the virus-like microtubule structure is no longer formed, and it can be used for related research on the failure of NS1 protein to form virus-like microtubule structure after BTV infects cells, visual quantitative detection of BTV, virus neutralization experiment of BTV and antiviral drug screening, etc. Specifically including the following contents:

[0008] In a first aspect, the present invention provides a recombinant bluetongue virus labeled with green fluorescent protein, wherein the recombinant bluetongue virus is obtained by inserting green fluorescent protein between amino acids 156 and 157 of nonstructural protein NS1 of wild-type bluetongue virus.

[0009] Preferably, the wild-type bluetongue virus is bluetongue virus serotype 1.

[0010] Preferably, the wild-type bluetongue virus is BTV-1 (GS / 11).

[0011] Preferably, the amino acid sequence of the green fluorescent protein is as shown in SEQ ID NO.1.

[0012] In a second aspect, the present invention provides a method for constructing a recombinant bluetongue virus labeled with green fluorescent protein, the method comprising: inserting green fluorescent protein between amino acids 156 and 157 of the wild-type bluetongue virus nonstructural protein NS1 by genetic engineering means, and obtaining the virus by reverse genetics technology after virus rescue.

[0013] Preferably, the method comprises the following steps:

[0014] (1) inserting the gene sequence of enhanced green fluorescent protein after the 468th 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 gene sequence;

[0015] (2) constructing transcription plasmids of wild-type bluetongue virus genes S1-S5 and S7-S10 respectively and transcribing them into mRNA transcripts in vitro;

[0016] (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 labeled with green fluorescent protein.

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

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

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

[0020] In a second aspect, the present invention provides the recombinant bluetongue virus described in any one of the first aspect or the third aspect, having any of the following uses:

[0021] (1) Application in the case where the bluetongue virus NS1 protein does not form virus-like microtubule structures;

[0022] (2) Application in neutralization experiments and visual quantitative detection of bluetongue virus;

[0023] (3) Application in screening of antiviral drugs for bluetongue virus.

[0024] The beneficial effects of the present invention are as follows: (1) the present invention unexpectedly discovered that only by inserting green fluorescent protein between the 156th and 157th amino acids of the wild-type bluetongue virus non-structural protein NS1 can the recombinant bluetongue virus labeled with green fluorescent protein be rescued; and the NS1 protein can display green fluorescence, which can be used for the dynamic expression of the NS1 protein in cells and related research on the failure to form virus-like microtubules; (2) the recombinant bluetongue virus changes the virus-like microtubule structure formed by the original wild-type bluetongue virus NS1 protein; (3) the recombinant bluetongue virus can also be used for the visual quantitative detection of BTV, the virus neutralization experiment of BTV and the screening of antiviral drugs, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Western blot analysis results of recombinant bluetongue virus;

[0026] Figure 2 Laser confocal microscope observation of fluorescence results of recombinant bluetongue virus; BSR cells were infected with BTV-1S6-156eGFP recombinant virus, blue is the cell nucleus stained with Hoechst 33342, green is NS1 protein, and red is anti-Tubulin antibody labeled cell microtubules;

[0027] Figure 3 Observation under a fluorescence microscope: After BSR cells were infected with the BTV-1S6-156eGFP recombinant virus, obvious green fluorescence was visible under a fluorescence microscope.

[0028] Figure 4 Transmission electron microscopy observation; yellow arrows indicate virus particles, and red arrows indicate virus-like microtubules; compared with the wild type (left), no virus-like microtubules were observed around the nucleus after BSR cells were infected with BTV-1S6-156eGFP recombinant virus (right). DETAILED DESCRIPTION

[0029] 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.

[0030] The following examples take blue tongue virus serotype 1 BTV-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 gene after position 468 of the S6 gene to construct an S6-eGFP mRNA containing an eGFP fluorescent protein gene sequence: using a wild-type S6 gene transcription plasmid as a template, a S6 gene transcription plasmid containing a T7 promoter and green fluorescent protein is constructed by PCR gene site-directed mutagenesis technology, and after enzyme linearization, in vitro transcription is performed using a T7 polymerase transcription kit to generate an S6-eGFP mRNA containing an eGFP fluorescent protein gene sequence;

[0031] The transcription plasmids of the other nine wild-type genes (S1-S5, S7-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. Four days after transfection, obvious cytopathic effect (CPE) appeared. The diseased cells were collected and purified by plaque three times to obtain a NS1 recombinant mutant virus. After RT-PCR amplification of S6-eGFP, sequencing was performed to confirm that the sequence was correct and named BTV-1S6-156eGFP. BSR cells were infected with the recombinant virus and the cell nuclei were stained with Hoechst33342. Confocal microscopy revealed that the NS1 protein in cells infected with the recombinant virus showed bright green fluorescence. The specific implementation method is as follows:

[0032] Example 1 Construction of recombinant bluetongue virus labeled with green fluorescent protein

[0033] 1. Construction method

[0034] The green fluorescent protein gene was inserted after the 468th base of the CDS sequence of the BTV-1 S6 gene. A mutation primer with a sequence encoding the green fluorescent protein gene (shown in SEQ ID NO.2) was designed, and a wild-type S6 gene transcription plasmid was used as a template to construct an NS1 gene transcription plasmid containing a T7 promoter and a green fluorescent protein gene by PCR gene fusion mutation technology. After restriction enzyme linearization, in vitro transcription was performed using a T7 polymerase transcription kit to generate S6-eGFP mRNA containing green fluorescent protein. At the same time, the BTV-1 wild-type gene S1-S5, S7-S10 transcription plasmids were restriction enzyme linearized and in vitro transcribed into mRNA transcripts of each gene.

[0035] S6-eGFP 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.

[0036] The RNA of cells infected with the recombinant virus was extracted, and RT-PCR amplification was performed using specific primers for the S6-eGFP 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.

[0037] 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 labeling of the recombinant virus NS1 protein was observed by confocal microscopy.

[0038] 2. Results Analysis

[0039] Three days after transfection, the transfected wells containing S6-eGFP mRNA showed obvious cytopathic effect (CPE), indicating that the insertion of green fluorescent protein between amino acids 156 and 157 of the nonstructural protein NS1 of bluetongue virus did not affect the rescue of the recombinant virus (results are shown in Figure 1 As shown), that is, the insertion of green fluorescent protein after position 156 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 BTV-1S6-156eGFP.

[0040] The total RNA of cells infected with the recombinant bluetongue virus BTV-1S6-156eGFP was extracted and amplified by RT-PCR using S6-eGFP gene-specific primers. The PCR products were obtained and sequenced. The results showed that the rescued recombinant bluetongue virus correctly inserted green fluorescent protein between amino acids 156 and 157 of the NS1 protein.

[0041] By electrophoresis of the double-stranded RNA genome of the recombinant bluetongue virus, it was found that the recombinant bluetongue virus with green fluorescent protein was significantly different from the wild-type bluetongue virus BTV-1, indicating that the rescued recombinant bluetongue virus contains the eGFP tag gene, which can be used for expression distribution and tracing experiments of NS1 protein after virus infection of cells, as well as fluorescent visualization quantitative measurements such as virus neutralization experiments.

[0042] BSR cells were infected with BTV-1S6-156eGFP and fixed with 4% paraformaldehyde. TM Tubulin was stained with 561 secondary antibody, and the cell nucleus was stained with Hoechst. Confocal microscopy revealed that the NS1 protein in cells infected with the recombinant virus showed fluorescence (results as shown in Figure 2 shown).

[0043] It was found by fluorescence microscopy that the NS1 protein in cells infected with the recombinant bluetongue virus expressing green fluorescent protein described in the present application can show bright green fluorescence (the results are shown in FIG. Figure 3 shown).

[0044] Transmission electron microscopy revealed that, compared with wild-type BTV, no virus-like microtubules were observed around the nucleus of BSR cells infected with the recombinant virus BTV-1S6-156eGFP (results shown in Figure 4 shown).

[0045] In summary, the above-mentioned recombinant bluetongue virus expressing green fluorescent protein can be used for related research on the failure of NS1 protein to form virus-like microtubules after BTV infection, and can be used for BTV neutralization experiments and visual quantitative detection.

[0046] Although the present invention takes BTV-1 (GS / 11) as an example, based on the high conservation of 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 with NS1 protein labeled with green fluorescent protein, characterized in that: The recombinant bluetongue virus is obtained by inserting a green fluorescent protein sequence between the 156th and 157th amino acids of the wild-type bluetongue virus non-structural protein NS1; the wild-type bluetongue virus is BTV-1 (GS / 11); the amino acid sequence of the green fluorescent protein is shown in SQE ID NO.

1.

2. A method for constructing a recombinant bluetongue virus labeled with green fluorescent protein, characterized in that: The method comprises: inserting green fluorescent protein between amino acids 156 and 157 of non-structural protein NS1 of wild-type bluetongue virus by genetic engineering, and rescuing the virus by reverse genetic technology; the wild-type bluetongue virus is BTV-1 (GS / 11); and the amino acid sequence of the green fluorescent protein is shown in SQE ID NO.

1.

3. The construction method according to claim 2, characterized in that: The method comprises the following steps: (1) inserting the gene sequence of enhanced green fluorescent protein after the 468th 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 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 labeled with green fluorescent protein.

4. The recombinant bluetongue virus constructed and obtained by the method according to any one of claims 2 to 3.

5. Use of the recombinant bluetongue virus according to any one of claim 1 or claim 4 in screening of bluetongue virus antiviral drugs.

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