CN110791526B - Mutant foot-and-mouth disease virus infectious clone and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of viruses in biotechnology, in particular to an infectious clone of a mutant foot-and-mouth disease virus, and also relates to preparation and application of the infectious clone. The site-directed mutagenesis technology is used for mutating the lysine at the 51 st, 54 th, 110 th and 159 th positions of the 3C protein into arginine. The SUMO modification site of the 3C protein of the constructed mutant infectious clone FMDV IC-K5154110159R is mutated, and the infectious clone can be proliferated, so that a powerful tool is provided for detecting and researching FMDV infection and SUMO modification at a virus level.
Description
Technical Field
The invention relates to the technical field of viruses in biotechnology, in particular to an infectious clone of a mutant foot-and-mouth disease virus, and also relates to preparation and application of the infectious clone.
Background
Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease of cloven-hoofed animals caused by FMDV (Foot-and-mouth disease virus). The outbreak of the disease causes great loss to the animal husbandry production and people's life, and is listed as the first infectious disease of A-class animals by the international veterinary medical office. FMDV belongs to the family of picornaviridae and the genus of foot and mouth disease virus, has a genome of single-stranded positive strand RNA with a full length of about 8500bp, and consists of a 5 'end non-coding region (UTR), a large Open Reading Frame (ORF) and a 3' end non-coding region (UTR). The ORF encodes a large polyprotein, which is cleaved by a virally encoded protease (L)pro2A and 3Cpro) Through a series of cleavages, precursor and mature structural and non-structural proteins are formed.
Small Ubiquitin-like Modifier (SUMO) Small Ubiquitin-like modifications are a post-translational modification with a similar enzymatic process to ubiquitination modifications discovered in recent years. SUMO modification has a variety of biological functions, such as maintaining genome stability, regulating transcription factor activity, mediating interactions between proteins, regulating the localization or stability of proteins within cells, participating in signal transduction and DNA damage repair, and the like. For viruses, many viral proteins can be modified by SUMO, and play an important role in regulation during viral infection.
There are currently no reports on the role of SUMO modification in FMDV infection.
Disclosure of Invention
In order to further identify the SUMO modification sites of the 3C protein at the virus level and the influence of the SUMO modification of the 3C protein on virus proliferation and the like, it is necessary to construct an FMDV 3C protein SUMO modification site mutant infectious clone which is an important tool in the process of researching the interaction between FMDV and a host at the molecular level and disclosing the anti-virus mechanism of an organism.
The invention provides a mutant foot-and-mouth disease virus infectious clone, and also relates to preparation and application of the infectious clone.
The invention is obtained by the following steps:
an FMDV infectious clone with a 3C protein SUMO modification site mutation is obtained by the following steps:
dividing a base sequence into A, B, C three segments according to the self-carried enzyme cleavage site of an O-type FMDV Tibet/CHA/99 strain (GenBank accession number: AJ539138), obtaining A, B, C three segments through RT-PCR amplification, sequentially cloning A, B, C three segments into a pBluescript II SK + vector to construct a plasmid pSK-AmBC with the full-length cDNA of the O-type FMDV Tibet/CHA/99 strain, and mutating the 51 st, 54 th, 110 th and 159 th lysines of a 3C protein into arginine through a site-directed mutagenesis technology to construct FMDV IC K5154110159R. The PacI enzyme linearized pSK-AmBC fragment is used as a template to be transcribed in vitro and then is transfected into BHK-21 cells to obtain the PacI enzyme linearized pSK-AmBC gene.
The infectious clone is preferably obtained by RT-PCR amplification of A, B, C three fragments, wherein 3C gene is located in C fragment, constructed pT-BC is used as template, 3C K5154R-F/R, 3C K110R-F/R and 3C K159R-F/R are used as primers, and QuikChange XL site-dir is usedSelected mutagenetics kit the mutation site was introduced into pT-BC in sequence to construct pT-BC K5154110159R, and then the mutant fragment was cloned from pT-BC K5154110159R into pSK-Am using NotI and PacI to construct pSK-AmBC K5154110159R. Plasmid pSK-AmBC was linearized with PacI enzyme and the linearized and purified fragment was used as template by mMESSAGE
T7 Ultra Kit according to the instructions for in vitro transcription, using DEPC water will be obtained by RNA precipitation heavy suspension concentration determination after standby or placed at-70 degrees C storage. BHK-21 cells were plated in 6-well plates and 5. mu.g of RNA was transfected into BHK-21 cells with 10. mu.L of DMRIE-C when the cell density reached 80%. After 4h, wash once and replace with fresh nutrient solution. At 48h post-transfection, supernatants were collected and blinded for 5 passages on PK-15 cells.
Application of the mutant foot-and-mouth disease virus infectious clone in interferon signal channel inhibition.
Application of the mutant foot-and-mouth disease virus infectious clone in apoptosis induction.
Application of the mutant foot-and-mouth disease virus infectious clone in the aspect of 3C protein stability.
The application of the mutant foot-and-mouth disease virus infectious clone in improving the virus titer is provided.
The invention has the beneficial effects that:
(1) the SUMO modification site of the 3C protein of the constructed mutant infectious clone FMDV IC-K5154110159R is mutated, and the infectious clone can be proliferated, so that a powerful tool is provided for detecting and researching FMDV infection and SUMO modification at a virus level;
(2) by measuring TCID50And a one-step growth curve shows that the virus titer of the infectious clone FMDV IC-K5154110159R constructed by the invention is 107.5TCID50is/mL and is obviously higher than the wild type infectious clone (P) at 24h, 36h and 48h<0.01)；
(3) Mutant infectious clone FMDV IC-K5154110159R produced stronger CPE lesions; the half-life of the mutant infectious clone FMDV IC-K5154110159R 3C protein is 3.5-4h, which is obviously higher than 2.6h (P <0.01) of the wild type infectious clone FMDV IC-WT3C protein; mutant infectious clone FMDV IC-K5154110159R caused stronger cleavage of PARP-1 (P <0.01) and apoptosis (P < 0.01); mutant infectious clone FMDV IC-K5154110159R caused more degradation of KPNA 1; the mutant infectious clone FMDV IC-K5154110159R has more remarkable inhibition on ISRE promoter activity.
Description of the drawings:
FIG. 1 shows the electrophoresis of amplified fragments of FMDV Tibet/CHA/99 strain O, FMDV A and B, C, in which lanes 1, 2 and 3 are A, B, C fragment RT-PCR products; m is DL 5,000DNA Marker,
FIG. 2 is a double restriction enzyme identification chart of the recombinant plasmid pSK-AmBC of the present invention, wherein lane 1 is the double restriction enzyme result; m is DL10,000DNA Marker,
FIG. 3 shows the comparison of amino acid sequences of FMDV IC-K5154110159R infectious clone and wild type infectious clone IC-WT virus 3C protein, in which lysine at position 51, 54, 110 and 159 is mutated to arginine (indicated by asterisk in the figure),
FIG. 4 shows SUMO modification of the 3C protein of FMDV IC-K5154110159R infectious clone and wild type infectious clone IC-WT virus, wherein the arrow indicates the SUMO modification band of 3C protein,
FIG. 5 is a graph of CPE produced by FMDV IC-K5154110159R infectious clone versus wild-type infectious clone IC-WT virus,
FIG. 6 is a one-step growth curve of FMDV IC-K5154110159R infectious clone versus wild-type infectious clone IC-WT virus,
FIG. 7 shows the comparison of the stability of the infectious clone of FMDV IC-K5154110159R with the wild type infectious clone IC-WT virus 3C protein,
FIG. 8 shows the apoptosis of FMDV IC-K5154110159R infectious clone and wild type infectious clone IC-WT virus,
FIG. 9 shows the inhibition of interferon signaling pathway by FMDV IC-K5154110159R infectious clone versus wild-type infectious clone IC-WT virus.
Detailed Description
The invention is further illustrated by the following specific examples:
example 1
1. Construction of FMDV IC-K5154110159R infectious clone
1.1 primer design
Primers for amplifying A, B, C three fragment sequences were designed based on the sequence of type O FMDV Tibet/CHA/99 strain (GenBank accession number: AJ539138) as follows:
FIG. 1A, B, C fragment amplification primers
The restriction sites are underlined; the T7 promoter is shown in bold
1.2 construction and identification of full-length cDNA plasmid containing O-type FMDV Tibet/CHA/99 strain
(1) Viral total RNA was extracted using TRIzol reagent, and SuperScript was used as a reverse transcriptase from InvitrogenTMIII Reverse transcription of Reverse Transcriptase according to the instructions, followed by high fidelity enzyme Pfu UltraTMII Fusion HS DNA polymers and designed primers FMDV-A-Fwd/Rev, FMDV-B-Fwd/Rev and FMDV-C-Fwd/Rev PCR amplified A, B, C fragments, the results are shown in FIG. 1.
(2) The A fragment containing the T7 promoter was double-digested with AscI and NotI and cloned into pBluescript II SK + vector to construct pSK-A.
(3) pSK-Am was constructed by introducing NheI into pSK-A using the FMDV-Am-Fwd/Rev primer through the QuikChange XL site-directed mutagenesis kit.
(4) The B and C fragments are sequentially connected into a TaKaRapMD18-T vector to construct pT-BC, then the B and C fragments are cloned from the pT-BC into pSK-Am by using NotI and PacI to construct pSK-AmBC, plasmid pSK-AmBC containing full-length cDNA of O type FMDV Tibet/CHA/99 strain is obtained, and the result of double enzyme digestion identification is shown in figure 2. The positive plasmids were sequenced by Shanghai Biotech service, Inc. The inserted gene sequence and deduced amino acid sequence were analyzed using Vector NTI and DNASAR software.
1.3 construction of infectious clones
(1) The plasmid pSK-AmBC was linearized with the PacI enzyme, and the linearized and purified slide was usedSegment as template by mMESSAGE
T7 Ultra Kit according to the instructions for in vitro transcription, using DEPC water will be obtained by RNA precipitation heavy suspension concentration determination after standby or placed at-70 degrees C storage.
(2) BHK-21 cells were plated in 6-well plates and 5. mu.g of RNA was transfected into BHK-21 cells with 10. mu.L of DMRIE-C when the cell density reached 80%. After 4h, wash once and replace with fresh nutrient solution. After 48h post-transfection, lesions appeared, supernatants were collected and blinded on PK-15 cells for 5 passages.
1.4 construction of mutant infectious clones
The 3C gene is located in a C fragment, the constructed pT-BC is used as a template, 3C K5154R-F/R, 3C K110R-F/R and 3C K159R-F/R are used as primers, mutation sites are sequentially introduced into the pT-BC through a QuikChange XL site-directed mutagenesis kit to construct pT-BC K5154110159R, and then the mutation fragment is cloned into pSK-Am from pT-BC K5154110159R by using NotI and PacI to construct pSK-AmBC K5154110159R so as to rescue the virus. The comparison of the amino acid sequences of the 3C protein of the mutant infectious clone with that of the wild-type infectious clone is shown in FIG. 3.
TABLE 2 mutant primers
| | Sequence | |
| 3C K5154R- | TTTTCGCAGAGAGATACGACAGAATCATGTTGGA | |
| 3C K5154R- | TCCAACATGATTCTGTCGTATCTCTCTGCGAAAA | |
| 3C K110R- | GATGTGGCAAGAATGAGAAAAGGCACCCCCGTC | |
| 3C K110R- | GACGGGGGTGCCTTTTCTCATTCTTGCCACATC | |
| 3C K159R- | TACAAAGCCGCCACCAGAGCGGGTTACTGTGGA | |
| 3C K159R-R | TCCACAGTAACCCGCTCTGGTGGCGGCTTTGTA |
2. SUMO modification of infectious clone 3C protein
BHK-21 cells are paved into a 6cm cell culture dish, when the cell density reaches 70% -80%, pXJ-His-Ubc9 and pXJ-Flag-SUMO-1 are Co-transfected, the mutant infectious clone FMDV IC-K5154110159R and the wild type infectious clone FMDV IC-WT are infected after 24 hours, the cells are collected at 0 hour and 10 hours, the SUMO modification condition of the 3C protein is identified through Co-IP and Western blotting detection, and the result is shown as an arrow in figure 4, and the SUMO modification band of the mutant infectious clone FMDV IC-K5154110159R 3C protein disappears.
3.3 determination of the growth characteristics of the infectious FMDV clones mutated at the SUMO modification sites of the C protein
3.13 determination of titer of FMDV infectious clone mutated at SUMO modification site of C protein
BHK-21 cells were plated in 96-well plates for viral TCID at cell densities up to 90%50The measurement of (1). The mutant infectious clone FMDV IC-K5154110159R and the wild type infectious clone FMDV IC-WT were separately performed from 10-1To 10- 12Diluting by 10 times. The 96-well plate BHK-21 cell supernatant was discarded and virus was added to the 96-well plate for each gradient, four replicates for each gradient. The pathological changes were observed every day, and when they stabilized, they were determined in triplicate according to the Reed-Muench methodToxic TCID50. The titer of FMDV infectious clone virus with the mutation of the SUMO modification site of the 3C protein is 107.5TCID50mL, viral titer of wild-type infectious clone 106.8TCID50/mL。
3.23C protein SUMO modification site mutated FMDV infectious clone producing CPE instances
BHK-21 cells were plated in 6-well plates and cells were infected with mutant infectious clone FMDV IC-K5154110159R and wild-type infectious clone FMDV IC-WT at MOI of 0.01, respectively, when the cell density reached 90%, and CPE lesions were observed at any time. 48h CPE lesion results As shown in FIG. 5, mutant infectious clone FMDV IC-K5154110159R produced stronger CPE lesions compared to wild type infectious clone FMDV IC-WT.
3.33C protein SUMO modified site mutated FMDV infectious clone one-step growth Curve mapping
Spreading BHK-21 cells into 12-well plate, infecting the mutant infectious clone FMDV IC-K5154110159R and wild type infectious clone FMDV IC-WT with MOI of 0.01 when cell density reaches 90%, collecting virus supernatant at 12h, 24h, 36h, and 48h, and determining TCID of each collected virus supernatant50According to TCID of different time points50The virus one-step growth curve is drawn, and the result is shown in figure 6, and the virus titer of the mutant infectious clone FMDV IC-K5154110159R is obviously higher than that of the wild type infectious clone FMDV IC-WT at 24h, 36h and 48 h.
4. Determination of related functions of FMDV infectious clone with mutation of SUMO modification site of 3C protein
4.1 infectious clone 3C protein stability assay
PK-15 cells were plated on 10 35mm cell culture dishes, when the cell density reached 85%, the cells were infected with the mutant infectious clone FMDV IC-K5154110159R and the wild-type infectious clone FMDV IC-WT at an MOI of 0.01, after 36 hours, 200. mu.g/mL of Cycloheximide (CHX) was added, and the cells were harvested at 0 hours, 1.5 hours, 3 hours, 4.5 hours, and 6 hours, respectively, to determine the half-life of the 3C protein. The results are shown in FIG. 7, and the half-life of the mutant infectious clone FMDV IC-K5154110159R 3C protein is about 3.7h, which is significantly higher than 2.6h (P <0.01) of the wild type infectious clone FMDV IC-WT3C protein.
4.23 apoptosis of FMDV infectious clones with SUMO modification site mutation of the C protein
The PK-15 cells are paved into 6 cell culture dishes with the cell density of 35mm, when the cell density reaches 85%, the mutant infectious clone FMDV IC-K5154110159R and the wild infectious clone FMDV IC-WT are infected by the MOI of 0.01 respectively, the cells are collected for 0h, 10h and 15h, Western blotting is used for detecting the cutting of apoptosis marker PARP-1, and an apoptosis detection kit is used for detecting the apoptosis caused by infectious clones. The results are shown in FIG. 8, where mutant infectious clone FMDV IC-K5154110159R caused stronger cleavage of PARP-1 (P <0.01) and apoptosis (P <0.01) compared to wild type infectious clone FMDV IC-WT.
4.33 inhibition of Interferon Signaling pathway by FMDV infectious clones mutated at the SUMO modification site of the C protein
In the earlier stage, the subject group finds that FMDV 3C protein mainly inhibits I-type interferon signal pathway by degrading KPNA1, and on the basis, the conditions of KPNA1 cutting and ISRE promoter activity inhibition under the condition of infection of mutant infectious clone FMDV IC-K5154110159R and wild infectious clone FMDV IC-WT are mainly detected.
PK-15 cells were plated on 3 35mm cell culture dishes, when the cell density reached 85%, the cells were infected with the mutant infectious clone FMDV IC-K5154110159R and the wild-type infectious clone FMDV IC-WT at an MOI of 0.01, and after 24 hours, the cells were harvested, and the degradation of KPNA1 was detected by Western blotting using KPNA1 antibody (Santa Cruz Biotechnology), with the result that mutant infectious clone FMDV-K5154110159R caused more KPNA1 degradation as shown in A in FIG. 9.
Spreading PK-15 cells into 3 35mm cell culture dishes, co-transfecting PK-21 cells with ISRE reporter gene plasmid (pISRE-Luc) and internal reference plasmid pRL-TK when the cell density reaches 75%, infecting the cells with mutant infectious clone FMDV IC-K5154110159R and wild infectious clone FMDV IC-WT with MOI of 0.01 after 12h, and infecting the cells with the wild infectious clone FMDV IC-WT after 12h
The results of cell lysis assays collected by the dual luciferase assay kit instructions are shown in FIG. 9B, where mutant infectious clone FMDV IC-K5154110159R has more significant inhibition of ISRE promoter activity than wild-type infectious clone FMDV IC-WT.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (6)
1. An O-type FMDV Tibet/CHA/99 strain, GenBank accession no: AJ539138 mutant foot-and-mouth disease virus infectious clone, characterized in that the 51 st, 54 th, 110 th and 159 th lysines of 3C protein are mutated into arginine.
2. A method for preparing the infectious clone of mutant foot-and-mouth disease virus according to claim 1, characterized by comprising the steps of:
according to GenBank accession No.: AJ539138, O type FMDV Tibet/CHA/99 strain carry enzyme cutting sites to divide the base sequence into A, B, C three segments, obtain A, B, C three segments through RT-PCR amplification, clone A, B, C three segments into pBluescript II SK + carrier in turn to construct plasmid pSK-AmBC with full length cDNA of O type FMDV Tibet/CHA/99 strain, then mutate lysine 51, 54, 110, 159 of 3C protein into arginine through site directed mutagenesis technology to construct FMDV IC K5154110159R; the PacI enzyme linearized pSK-AmBC fragment is used as a template to be transcribed in vitro and then is transfected into BHK-21 cells to obtain the PacI enzyme linearized pSK-AmBC gene.
3. Use of the mutant foot-and-mouth disease virus infectious clone of claim 1 or the mutant foot-and-mouth disease virus infectious clone obtained by the preparation method of claim 2 in inhibition of ISRE promoter activity.
4. The use of the mutant foot-and-mouth disease virus infectious clone of claim 1 or the mutant foot-and-mouth disease virus infectious clone obtained by the preparation method of claim 2 in KPNA1 degradation.
5. The use of the mutant foot-and-mouth disease virus infectious clone of claim 1 or the mutant foot-and-mouth disease virus infectious clone obtained by the preparation method of claim 2 for prolonging the half-life of 3C protein.
6. Use of the mutant foot-and-mouth disease virus infectious clone of claim 1 or the mutant foot-and-mouth disease virus infectious clone obtained by the preparation method of claim 2 for increasing virus titer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343110A (en) * | 2013-07-06 | 2013-10-09 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Construction method of recombinant coatpox virus used for expressing foot and mouth disease virus empty capsid |
| CN110129348A (en) * | 2019-06-03 | 2019-08-16 | 诺华生物科技（武汉）有限责任公司 | Efficiently recombinant DNA carrier, application and the vaccine of preparation foot and mouth disease virus sample particle |
| US10385319B2 (en) * | 2016-09-08 | 2019-08-20 | The Governement of the United States of America, as represented by the Secretary of Homeland Security | Modified foot-and-mouth disease virus 3C proteases, compositions and methods thereof |
-
2019
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343110A (en) * | 2013-07-06 | 2013-10-09 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Construction method of recombinant coatpox virus used for expressing foot and mouth disease virus empty capsid |
| US10385319B2 (en) * | 2016-09-08 | 2019-08-20 | The Governement of the United States of America, as represented by the Secretary of Homeland Security | Modified foot-and-mouth disease virus 3C proteases, compositions and methods thereof |
| CN110129348A (en) * | 2019-06-03 | 2019-08-16 | 诺华生物科技（武汉）有限责任公司 | Efficiently recombinant DNA carrier, application and the vaccine of preparation foot and mouth disease virus sample particle |
Non-Patent Citations (1)
| Title |
|---|
| 猪繁殖与呼吸综合征病毒对 I 型干扰素下游信号通路影响的研究;王蓉;《中国博士学位论文全文数据库 农业科技辑》;20140515(第5期);第D050-39页 * |
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