Cloning method of porcine intestinal coronavirus NSP3 toxic protein gene
By using a low-copy number plasmid and anti-toxic gene system in the EPI300 host strain, the toxicity problem of the porcine enteric coronavirus NSP3 gene in conventional systems was solved, and the stable amplification and functional research of the NSP3 gene were achieved, providing key technical support for the development of antiviral drugs.
Patent Information
- Application Number
- CN202510811770.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-10-24
AI Technical Summary
In the existing technology, the porcine enteric coronavirus NSP3 gene exhibits significant host toxicity in conventional prokaryotic expression systems, resulting in slow growth of the recombinant strain and plasmid instability, making it difficult to achieve stable construction.
A low-copy number plasmid combined with the toxicity-resistant gene strain EPI300 was used. The NSP3 gene was inserted into the medium- and low-copy number prokaryotic plasmid pET-28a and the high-copy number eukaryotic plasmid pCAGGS, and then transformed into the EPI300 host strain. The unique expression inhibition mechanism and low copy number characteristics of the NSP3 gene were utilized to achieve stable amplification of the NSP3 gene.
The stable cloning and amplification of the porcine enteric coronavirus NSP3 gene were successfully achieved, and the prokaryotic expression system was optimized, providing a basis for studying its function and developing antiviral drugs, and clarifying the molecular basis and potential targets of NSP3.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a cloning method of a porcine enteric coronavirus NSP3 toxic protein gene. BACKGROUND
[0002] Swine Enteric Coronaviruses (SECs) are a class of viruses that are extremely harmful to the pig industry, mainly including Transmissible gastroenteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV) and Porcine Delta coronavirus (PDCoV). Swine diarrhea-related viruses can infect pigs of all ages, from neonatal pigs, growing pigs, sows, etc., causing significant economic losses to the global livestock industry. Existing studies have shown that swine diarrhea-related viruses have little harm to growing pigs and breeding pigs, but are very harmful to suckling pigs, with a morbidity and mortality rate of 80% to 100% for newborn pigs under 7 days old. However, the protective effect of existing vaccines needs to be improved, and new coronavirus strains have emerged from time to time in recent years. Therefore, it has become an urgent problem to find effective prevention and control strategies to improve the production efficiency of the pig industry.
[0003] TGEV, PEDV, PDCoV and other swine diarrhea coronaviruses mainly infect the small intestinal villus epithelial cells of pigs, causing diarrhea, vomiting, loss of appetite, dehydration and other difficult-to-distinguish clinical signs. The non-structural proteins NSP1 to NSP16 encoded by the genomes of these viruses regulate the synthesis and processing of viral RNA by forming a replication-transcription complex (RTC), and studying these non-structural proteins is crucial for the prevention and control of related diseases. Therefore, constructing eukaryotic expression plasmids of these non-structural proteins is a key step in exploring their functions.
[0004] The present application attempts to construct eukaryotic expression plasmids of non-structural protein genes of swine diarrhea coronaviruses. However, during the construction of eukaryotic expression plasmids of PEDV non-structural protein genes, we found that the NSP3 gene exhibited significant host toxicity in conventional prokaryotic expression systems such as E. coli DH5a, Stbl3, DH10B, etc., which manifested as slow growth of recombinant strains and instability of plasmids. Notably, this phenomenon also exists in TGEV and PEDV and other swine diarrhea coronaviruses. Therefore, it is urgent to find a method to effectively overcome the toxicity of the NSP3 gene in order to achieve stable construction of eukaryotic expression plasmids of PEDV non-structural protein genes. SUMMARY
[0005] To overcome the problems existing in the prior art, the present application innovatively uses a low-copy-number plasmid combined with an anti-toxicity gene strain, successfully achieving stable amplification of the full-length NSP3 gene, and laying a solid foundation for subsequent functional research.
[0006] One of the purposes of the present application is to provide a cloning method of porcine enteric coronavirus NSP3 toxic protein gene.
[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0008] The cloning method of porcine enteric coronavirus NSP3 toxic protein gene comprises the following steps:
[0009] (1) Extracting genomic DNA or cDNA of porcine enteric coronavirus;
[0010] (2) Taking the genomic DNA or cDNA obtained in step (1) as a template, amplifying the NSP3 gene through PCR;
[0011] (3) Inserting the NSP3 gene into a medium-low copy number prokaryotic plasmid or a high copy number eukaryotic plasmid to obtain a recombinant plasmid;
[0012] (4) Transforming the recombinant plasmid obtained in step (3) into a host strain EPI300 with anti-toxicity characteristics.
[0013] Preferably, the medium-low copy number prokaryotic plasmid is pET-28a, and the high copy number eukaryotic plasmid is pCAGGS.
[0014] The pET-28a has a pBR322 copy replicon, and the T7 lac promoter is strictly regulated, so that the basic expression amount of the plasmid is extremely low when it is not induced; the pCAGGS has a CMV enhancer / chicken beta-actin (CAG) composite promoter with low activity in a prokaryotic system, and in combination with the expression inhibition mechanism specific to the EPI300, a synergistic effect can be formed to maintain the stability of the cloning.
[0015] The complete deletion of the RecA and RecET genes of the EPI300 competent cell blocks the homologous recombination pathway, and the optimization of the host methylation system and the translation regulatory factor can effectively inhibit the leakage expression of the toxic gene.
[0016] Preferably, the EPI300 competent cell carries a pCC1 helper plasmid containing an oriV replication origin and a rop gene.
[0017] Preferably, the porcine enteric coronavirus includes PEDV, TGEV and / or PDCoV.
[0018] Preferably, in step (2), the PCR reaction system comprises: Easy Taq DNA Polymerase 20-30 μL, upstream primer 1.5-3 μL, downstream primer 1.5-3 μL, cDNA template 1.5-3 μL, and ddH2O up to 50 μL; the PCR reaction program comprises: 90-100 ℃ pre-denaturation for 2-5 min; 90-100 ℃ denaturation for 10-20 s, 50-60 ℃ annealing for 10-20 s, 65-80 ℃ extension for 1-3 min, a total of 30-40 cycles; 65-80 ℃ extension for 5-10 min; the sequence of the upstream primer of PEDV NSP3 is shown as SEQ ID NO: 5, and the sequence of the downstream primer is shown as SEQ ID NO: 6; the sequence of the upstream primer of TGEV NSP3 is shown as SEQ ID NO: 31, and the sequence of the downstream primer is shown as SEQ ID NO: 32; the sequence of the upstream primer of PDCoV NSP3 is shown as SEQ ID NO: 33, and the sequence of the downstream primer is shown as SEQ ID NO: 34.
[0019] As a preferred technical solution, in step (2), the PCR reaction system is: Easy Taq DNA Polymerase 25 μL, upstream primer 2 μL, downstream primer 2 μL, cDNA template 2 μL, and ddH2O up to 50 μL.
[0020] As a preferred technical solution, in step (2), the PCR reaction program is: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 56 ℃ annealing for 15 s, 72 ℃ extension for 1-2 min, a total of 35 cycles; 72 ℃ extension for 7 min.
[0021] Preferably, the cDNA sequence of the PEDV is shown in Genebank: AF353511.1.
[0022] Preferably, the cDNA sequence of the TGEV is shown in GenBank: FJ755618.2.
[0023] Preferably, the cDNA sequence of the PDCoV is shown in GenBank: JQ065042.2.
[0024] Preferably, the N-terminal domain of the NSP3 gene has a significant toxicity feature.
[0025] The second object of the present application is to provide a recombinant plasmid for cloning a porcine enteric coronavirus NSP3 toxic protein gene.
[0026] To achieve the above object, the present application adopts the following technical solutions:
[0027] The application discloses a recombinant plasmid for cloning a NSP3 toxic protein gene of a porcine enteric coronavirus, wherein the plasmid is a medium-low copy number prokaryotic plasmid or a high copy number eukaryotic plasmid containing the NSP3 gene; the medium-low copy number prokaryotic plasmid is pET-28a, and the high copy number eukaryotic plasmid is pCAGGS.
[0028] A third object of the present application is to provide an anti-toxic host strain.
[0029] To achieve the above object, the present application adopts the following technical solution:
[0030] An anti-toxic host strain comprises the aforementioned recombinant plasmid.
[0031] Preferably, the host strain is EPI300.
[0032] A fourth object of the present application is to provide a method for studying the function of the NSP3 toxic protein gene of the porcine enteric coronavirus.
[0033] To achieve the above object, the present application adopts the following technical solution:
[0034] The method for studying the function of the NSP3 toxic protein gene of the porcine enteric coronavirus comprises the aforementioned cloning method.
[0035] Preferably, the study is used for developing an anti-virus drug.
[0036] A fifth object of the present application is to provide an application of a composition in the preparation of a product for cloning the NSP3 toxic protein gene of the porcine enteric coronavirus.
[0037] To achieve the above object, the present application adopts the following technical solution:
[0038] The application of the composition in the preparation of the product for cloning the NSP3 toxic protein gene of the porcine enteric coronavirus comprises a plasmid system and an anti-toxic host strain; the plasmid system comprises a medium-low copy number prokaryotic plasmid and / or a high copy number eukaryotic plasmid; the medium-low copy number prokaryotic plasmid is pET-28a, the high copy number eukaryotic plasmid is pCAGGS, and the host strain is EPI300.
[0039] The present application has the following beneficial effects:
[0040] 1. The present application successfully realizes stable cloning and amplification of the toxic gene by integrating the medium-low copy plasmid system pET-28a and the anti-toxic host strain EPI300 in a synergistic strategy. Through the strategy, the present application not only optimizes the prokaryotic expression system of the NSP3 protein in SECs, but also lays an experimental foundation for in-depth study of the molecular mechanism of the interaction between the NSP3 protein and the host, and provides a potential target for developing a new inhibitor for the virus replication complex.
[0041] 2. In the process of constructing the recombinant plasmid of the non-structural protein NSP3 of porcine epidemic diarrhea virus, the present invention found that its expression product showed a significant growth inhibitory effect on the Escherichia coli host, suggesting that the NSP3 gene has prokaryotic toxicity characteristics. Further studies have shown that the NSP3 genes of TGEV and PDCoV, which belong to the genus Porcine Diarrhea Coronavirus, also face the same problem. It is preliminarily speculated that the porcine enteric coronavirus NSP3 is toxic to commonly used cloning competent cells. Further functional screening of truncated mutants confirmed that the toxic effect of NSP3 is closely related to the nucleic acid binding activity of its N-terminal domain (aa 1-1068). The present invention not only clarifies the molecular basis of the prokaryotic toxicity of PEDVNSP3, but also establishes a universal cloning technology system for highly toxic viral genes, providing key technical support for the functional analysis of coronavirus non-structural proteins and the development of antiviral targets. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] Figure 1 The result map for PEDV nonstructural protein cloning is constructed, where: Figure 1 -a is the result map of PEDVNSP1 clone construction, Figure 1 -b is the result map of PEDVNSP2 clone construction, Figure 1 -c is the result map for PEDVNSP3 clone construction, Figure 1 -d is the result map for PEDVNSP4 clone construction, Figure 1 -e builds the result graph for PEDVNSP5 clones, Figure 1 -f is the result map for PEDVNSP6 clone construction, Figure 1 -g is the result map of PEDV NSP7 clone construction, Figure 1 -h is the result map of PEDVNSP8 clone construction, Figure 1 -i builds the result map for PEDVNSP9 clone, Figure 1 -j builds the result map for PEDV NSP10 clone, Figure 1 -k is the result map of PEDV NSP12 clone construction, Figure 1 -l is the result map of PEDV NSP13 clone construction, Figure 1 -m is the result map for PEDVNSP14 clone construction, Figure 1 -n builds the result map for PEDVNSP15 clone, Figure 1 -o Build result map for PEDV NSP16 clone, Figure 1 -p is the result map of PEDV (1-4674nt) cloning construction;
[0043] Figure 2 The result map was constructed for the TGEV / PDCoVNSP3 clone, where Figure 2-a is the result map of TGEV NSP3 clone construction, Figure 2 -b is the result map of PDCoV NSP3 clone construction;
[0044] Figure 3 is the result map of NSP3 gene cloning into pCAGGS vector in EPI300 competent cells;
[0045] Figure 4 is the result map of NSP3 gene cloning into pET-28a vector in EPI300 competent cells;
[0046] Figure 5 is the result map of plasmid copy number comparison of pcDNA3.1, pCAGGS and pET28a in EPI300 competent cells;
[0047] Figure 6 is the result map of DH5α single colony data detection, wherein, Figure 6 -a is the result map of control group single colony detection; Figure 6 -b is the result map of experimental group (NSP3) single colony detection; Figure 6 -c is the result map of DH5α single colony number statistics of experimental group and control group;
[0048] Figure 7 is the result map of DH5α bacterial liquid culture for 12h, wherein, Figure 7 -a is the picture of DH5α bacterial liquid culture for 12h; Figure 7 -b is the result map of OD600 value detection of DH5α bacterial liquid culture for 12h;
[0049] Figure 8 is the result map of DH10B bacterial liquid culture for 12h, wherein, Figure 8 -a is the picture of DH10B bacterial liquid culture for 12h; Figure 8 -b is the result map of DH10B bacterial liquid OD600 value detection;
[0050] Figure 9 is the result map of STBL3 bacterial liquid culture for 12h, wherein, Figure 9 -a is the picture of STBL3 bacterial liquid culture for 12h; Figure 9 -b is the result map of STBL3 bacterial liquid OD600 value detection;
[0051] Figure 10 is the result map of PEDV NSP3 truncated recombinant plasmid colony PCR identification, wherein, Figure 10 -a is the structure mode map of PEDV NSP3; Figure 10 -b is the schematic diagram of PEDV NSP3 truncation; Figure 10-c is the construction map of NSP3-1, a truncated gene of NSP3, cloned in DH5a; Figure 10 -d is the construction map of NSP3-2, a truncated gene of NSP3, cloned in DH5a; Figure 10 -e is the construction map of NSP3-3, a truncated gene of NSP3, cloned in DH5a; Figure 10 -f is the detection result map of a single colony of PEDV NSP3 truncated recombinant plasmid transformed into DH5a; Figure 10 -g is a picture of the bacterial liquid culture of PEDV NSP3 truncated recombinant plasmid transformed into DH5a for 12 hours. DETAILED DESCRIPTION
[0052] The technical solutions of the present application will be further clearly and completely described in combination with specific examples. Obviously, the described examples are only some of the examples of the present application, but not all. Therefore, all other examples obtained by those skilled in the art based on the examples in the present application without creative labor are within the protection scope of the present application.
[0053] In the embodiments of the present application, the cloning method of the NSP3 toxic protein gene of porcine enteric coronavirus includes the following steps:
[0054] (1) Extract the genomic DNA or cDNA of porcine enteric coronavirus. The cDNA sequence of PEDV is seen in Genebank: AF353511.1, the cDNA sequence of TGEV is seen in GenBank: FJ755618.2, and the cDNA sequence of PDCoV is seen in GenBank: JQ065042.2.
[0055] (2) Amplify the NSP3 gene by PCR using the genomic DNA or cDNA obtained in step (1) as a template. The PCR reaction system includes: Easy Taq DNA Polymerase 25 μL, 2 μL of each of the upstream and downstream primers, 2 μL of the cDNA template, and ddH2O to make up 50 μL; the sequences of the upstream and downstream primers are seen in Table 1; the PCR reaction program includes: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1-2 min, a total of 35 cycles; and 72℃ extension for 7 min.
[0056] (3) Insert the NSP3 gene into the medium-low copy number prokaryotic plasmid pET-28a or the high copy number eukaryotic plasmid pCAGGS to obtain a recombinant plasmid.
[0057] (4) Transform the recombinant plasmid obtained in step (3) into the host strain EPI300 with anti-toxicity characteristics.
[0058] Example 1. Study on the toxicity of porcine enteric coronavirus NSP3 gene to the clonal competent cells
[0059] The NSP1 to NSP10 and NSP12 to NSP16 genes were amplified by PCR using the cDNA of PEDV vaccine strain CV777 (Genebank: AF353511.1) as a template, and the PCR reaction system included: Easy Taq DNA Polymerase 25 μL, 2 μL of each of the upstream and downstream primers, 2 μL of the cDNA template, and ddH2O to make up 50 μL; the primer sequences are shown in Table 1. The PCR reaction program included: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 1-2 min, for a total of 35 cycles; 72 °C extension for 7 min. The PCR products were detected by electrophoresis using 1.0% agarose gel, and the PCR positive products were recovered by gel cutting. The pcDNA3.1 was digested by selecting EcoRI and XhoI enzyme cutting sites, and the enzyme cutting reaction system was: 10×NEBuffer 5 μL, EcoRI 1 μL, XhoI 1 μL, DNA 1 μg, and ddH2O to make up 50 μL. The enzyme cutting reaction program was: 37 °C for 15 min; 80 °C for 20 min. The enzyme cutting products were recovered by gel cutting. The recovered and purified fragments were connected to the linearized vector using the homologous recombination method. The connection products were transformed into DH5α competent cells, which were cultured at 37 °C for 12 h, and 8 single colonies were randomly selected for colony PCR identification using the pcDNA3.1 vector universal primers (the upstream primer sequence is SEQ ID NO: 35, and the downstream primer sequence is SEQ ID NO: 36). The single colonies identified as positive were sent to the GenScript Biotech Co., Ltd. for sequencing, and the PEDV gene sequence published on NCBI was referred to.
[0060] Table 1. Primer sequence table
[0061]
[0062]
[0063] The results of the nucleic acid gel electrophoresis are shown in Figure 1 -a ~ Figure 1 -o, and the positive cloning rate of all the non-structural protein genes except NSP3 was more than 75%, and NSP3 failed to produce any positive clones, which are shown in detail in Figure 1- c. Sequencing analysis further confirmed the successful construction of all PEDV non-structural protein gene recombinant plasmids except NSP3. Considering that NSP3 is the largest gene encoding non-structural proteins in PEDV, in order to exclude the low cloning efficiency caused by the length of the gene, a PEDV sequence (1-4674 nt) similar in length to NSP3 was selected and inserted into the same vector for transformation experiments, and the results showed that the positive rate of cloning of this sequence was 87.5%, as shown in Table 2. Figure 1 - p. This result suggests that NSP3 may contain sequences that are toxic to E. coli.
[0064] Further studies found that the NSP3 genes of TGEV (Genebank: FJ755618.2) and PDCoV (Genebank: JQ065042.2) strains were also unable to be cloned into the pcDNA3.1 vector, as shown in Table 3. Figure 2 This indicates that the NSP3 gene of porcine enteric coronavirus is toxic to commonly used cloning competent cells, and this finding provides an important clue for further research on how to avoid this toxicity to facilitate successful cloning.
[0065] Example 2. Cloning of NSP3 gene into pET-28a and pCAGGS vectors in EPI300 competent cells
[0066] In order to successfully construct a recombinant plasmid containing the PEDV NSP3 gene, the present application transformed the recombination system into EPI300 competent cells carrying an anti-toxicity gene, and performed colony PCR identification using universal vector primers (upstream primer sequence as SEQ ID NO: 35, downstream primer sequence as SEQ ID NO: 36) to evaluate the positive cloning rate. Nucleic acid gel electrophoresis results showed that bands consistent with the expected size of the NSP3 gene fragment were observed in both pCAGGS / EPI300 and pET28a / EPI300 combinations, with positive cloning rates of 37.5% and 87.5%, respectively, as shown in Table 4. Figure 3 and Figure 4 Further sequencing analysis confirmed the successful construction of NSP3 recombinant plasmids in EPI300 competent cells.
[0067] Notably, the EPI300 strain can significantly improve the cloning stability of toxic genes through a triple regulatory mechanism: first, the complete deletion of RecA and RecET genes blocks the homologous recombination pathway; second, the trfA gene confers the copy number regulation ability of the oriV replicon plasmid, maintaining the plasmid copy number at a low level of 5-10 per cell under non-induced conditions; third, the optimization of the host methylation system and translation regulatory factors can effectively inhibit the leaky expression of toxic genes. Among the applied plasmids, pET28a has a low-copy replicon in pBR322, although its replication process is not regulated by trfA, but the natural low-copy characteristics are compatible with the low metabolic pressure requirements of EPI300. Moreover, the T7lac promoter is strictly regulated, so the basal expression of the plasmid is very low without induction. The low activity of the CMV enhancer / chicken beta-actin (CAG) composite promoter in the prokaryotic system of the pCAGGS vector, combined with the methylation modification and recombination defect mechanisms of EPI300, can still maintain the cloning stability. The high-copy replication mechanism of the ColE1 / pUC type replicon in the pcDNA3.1 vector may cause the metabolic products to exceed the host tolerance threshold. Based on this, we speculate that the cloning of the NSP3 gene in the pCAGGS and pET28a vectors may benefit from the relatively low copy number environment, while the failure of the pcDNA3.1 vector to successfully implement the recombination construction of the NSP3 gene may be due to its own high copy number characteristics.
[0068] To verify this hypothesis, the present application compared the plasmid copy number of pcDNA3.1, pCAGGS and pET28a in EPI300 competent cells. The experimental results are shown in Figure 5 As shown, the copy number of pcDNA3.1 / EPI300 was significantly higher than that of pCAGGS / EPI300 (P=0.0124) and pET28a / EPI300 (P<0.0001), suggesting that the high and low of plasmid copy number has an important influence on the successful cloning of NSP3 gene in EPI300 cells. This finding provides a theoretical basis for further understanding the role of vector selection in the process of recombination plasmid construction.
[0069] Example 3. Detection of the effect of pET28a-NSP3 on the growth of E. coli
[0070] To verify whether NSP3 is difficult to clone into an expression vector due to bacterial toxicity, we transformed the pET28a-NSP3 recombination plasmid into E. coli DH5α, DH10B or STBL3, and the bacterial solution was simultaneously expanded and plated, and the pET28a-F1 recombination plasmid was transformed into E. coli DH5α as a control. The results of the toxicity verification of NSP3-pET-28a on competent cells are shown in Figures 6-9 As shown, the number of single bacteria of DH5α is as follows Figure 6As shown, the number of single bacterial colonies in the experimental group was significantly less than that in the control group (P=0.0009). After 12 h of culture of the DH5a bacterial liquid, the OD600 value of the experimental group was significantly lower than that of the control group (P<0.0001). Similarly, the OD600 values of the DH10B and STBL3 bacterial liquids were significantly lower than those of the control groups (P<0.0001), as shown in Table 2. Figure 7 As shown, the experimental group was significantly clearer than the control group, and the OD600 value was significantly lower than that of the control group (P<0.0001). Similarly, the OD600 values of the DH10B and STBL3 bacterial liquids were significantly lower than those of the control groups (P<0.0001), as shown in Table 2. Figure 8 and Figure 9 .
[0071] Example 4. Finding of the toxic region of NSP3 gene
[0072] PEDV NSP3 amino acid sequence domain analysis showed that the NSP3 protein was mainly located in the endoplasmic reticulum membrane and was a multi-transmembrane protein, and its structural model diagram was as shown in Figure 10 -a, 1-1068 aa was the N-terminal region towards the endoplasmic reticulum region, 1068-1257 aa was the multi-transmembrane region, and 1257-1614 aa was the C-terminal region towards the endoplasmic reticulum region. Therefore, the three regions were cut off and named as NSP3-1, NSP3-2 and NSP3-3, as shown in Figure 10 -b; they were cloned into the pcDNA3.1 vector, and colony PCR was performed using the universal primer (the upstream primer sequence was as shown in SEQ ID NO: 35, and the downstream primer sequence was as shown in SEQ ID NO: 36) to evaluate the positive rate of cloning. The results are shown in Figure 10 -c, Figure 10 -d, Figure 10 -e, the positive rate of NSP3-1-pcDNA3.1 was 75%, while the positive rates of NSP3-2 and NSP3-3-pcDNA3.1 were both 0%, indicating that the toxicity of NSP3 might be concentrated in its N-terminal region. To further verify the above finding, the three truncated fragments were again cloned into the pET28a vector and transformed into EPI300 competent cells to obtain positive clones, and the plasmids were extracted and transferred to DH5a competent cells. The experimental results are shown in Figure 10 -f and Figure 10 -g, the positive transformation efficiency of NSP3-1-pET28a was significantly higher than that of NSP3-2 and NSP3-3-pET28a, further confirming that the toxicity of NSP3 mainly originated from its N-terminal region.
Claims
1. A method for cloning the NSP3 virulence protein gene of porcine enteric coronavirus, characterized in that, The method comprises the following steps: (1) extracting genomic DNA or cDNA of porcine enteric coronavirus; (2) amplifying the NSP3 gene by PCR using the genomic DNA or cDNA obtained in step (1) as a template; (3) inserting the NSP3 gene into a medium-low copy number prokaryotic plasmid or a high copy number eukaryotic plasmid to obtain a recombinant plasmid; (4) transforming the recombinant plasmid obtained in step (3) into a host strain EPI300 with anti-toxicity characteristics.
2. The cloning method of claim 1, wherein, The medium-low copy number prokaryotic plasmid is pET-28a, and the high copy number eukaryotic plasmid is pCAGGS.
3. The cloning method of claim 1, wherein, The porcine enteric coronavirus includes PEDV, TGEV and / or PDCoV.
4. The cloning method of claim 1, wherein, In step (2), the PCR reaction system comprises: EasyTaqDNA Polymerase 20-30 μL, upstream primer 1.5-3 μL, downstream primer 1.5-3 μL, cDNA template 1.5-3 μL, and ddH2O 50 μL; the PCR reaction program comprises: 90-100 °C pre-denaturation for 2-5 min; 90-100 °C denaturation for 10-20 s, 50-60 °C annealing for 10-20 s, 65-80 °C extension for 1-3 min, a total of 30-40 cycles; 65-80 °C extension for 5-10 min; the upstream primer sequence of PEDV NSP3 is shown as SEQ ID NO: 5, and the downstream primer sequence is shown as SEQ ID NO: 6; the upstream primer sequence of TGEV NSP3 is shown as SEQ ID NO: 31, and the downstream primer sequence is shown as SEQ ID NO: 32; the upstream primer sequence of PDCoV NSP3 is shown as SEQ ID NO: 33, and the downstream primer sequence is shown as SEQ ID NO:
34.
5. The cloning method of claim 1, wherein, The N-terminal domain of the NSP3 gene has significant toxicity characteristics.
6. A recombinant plasmid for cloning the NSP3 virulence protein gene of porcine enteric coronavirus, characterized in that, The plasmid is a medium-low copy number prokaryotic plasmid or a high copy number eukaryotic plasmid containing the NSP3 gene; the medium-low copy number prokaryotic plasmid is pET-28a, and the high copy number eukaryotic plasmid is pCAGGS.
7. An anti-virulence host strain, characterized in that, The recombinant plasmid of claim 6 is included.
8. The toxicity resistant host strain of claim 7, wherein, The host strain is EPI300.
9. A method for studying the function of the NSP3 virulence protein gene of porcine enteric coronavirus, characterized by, The cloning method of any one of claims 1-5 is included.
10. Use of a composition for the manufacture of a product for cloning of NSP3 virulence protein gene of porcine enteric coronavirus, characterized in that, The composition comprises a plasmid system and an anti-toxicity host strain; the plasmid system comprises a medium-low copy number prokaryotic plasmid and / or a high copy number eukaryotic plasmid; the medium-low copy number prokaryotic plasmid is pET-28a, the high copy number eukaryotic plasmid is pCAGGS, and the host strain is EPI300.