A recombinant vector containing human saprovirus nucleic acid fragment and its application

Abstract

The present invention discloses a recombinant vector containing a human saprovirus nucleic acid fragment, which is obtained by inserting an exogenous nucleotide sequence into a prokaryotic expression vector skeleton, wherein the exogenous nucleotide sequence is formed by sequentially connecting the following gene sequences: a SUMO fusion tag DNA sequence, SAP Gene sequence, MS2 bacteriophage-related DNA sequence, human Zaru virus sequence fragment. The human Zaru virus RNA gene sequence in the pseudoviral particles prepared by the present invention contains a stem-loop structure at the end, which is not easily degraded by nucleases, thereby improving the stability of the viral nucleic acid RNA; at the same time, the human Zaru virus conservative RNA gene sequence is wrapped by the bacteriophage MS2 coat protein, which has a high resistance to ribonucleases and deoxyribonucleases, and enhances the long-term stability of the viral nucleic acid RNA. The stability problem of viral RNA quality control products during production and use is solved.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to a recombinant vector containing a human saprovirus nucleic acid fragment and an application thereof. Background Art

[0002] Human Sapovirus (HuSaV), also known as human sapovirus or sapovirus, is a member of the genus Sapovirus in the family Caliciviridae. HuSaV is an important pathogen causing sporadic acute gastroenteritis and related epidemics worldwide, especially for high-risk groups such as infants and immunocompromised patients. As an RNA virus, HuSaV is prone to mutation and persists over time, leading to the continuous emergence of viral variants. At present, HuSaV is divided into at least 19 gene groups (GI-GXIX), of which 4 are susceptible to humans (GI, GII, GIV and GV); they can be further divided into 19 genotypes, of which GI.1, GI.6 and GII.2 are the most common genotypes in children with acute gastroenteritis worldwide.

[0003] Since the promotion and use of rotavirus vaccines, HuSaV has become the second most common pathogen causing acute diarrhea after norovirus. HuSaV is usually transmitted through the "fecal-oral" route through contaminated food and water, and can also be transmitted through direct contact with infected people. Therefore, related epidemics caused by HuSaV often occur in semi-closed environments, such as nursing homes, schools, hospitals, military and cruise ships. HuSaV is milder in pathogenicity than norovirus. The main symptoms after infection are vomiting, diarrhea, and fever. Most patients have mild symptoms, and occasionally a few patients may experience severe symptoms such as dehydration and intestinal cramps. Although the HuSaV epidemic has caused huge social and economic waste, there are still no approved vaccines or antiviral drugs.

[0004] The detection of HuSaV is a key technology in the fields of clinical diagnosis, import and export ports, and food hygiene inspection. The inability to culture HuSaV in cells and the lack of a suitable animal infection model have hampered in-depth research on it. At present, the real-time fluorescence quantitative PCR detection method is the main detection method for HuSaV molecular detection; this method has the advantages of high sensitivity, strong specificity, and rapid detection process. It is especially widely used in the field of food detection with low contamination virus content and high potential transmission importance.

[0005] At present, most HuSaV positive samples, HuSaV RNA nucleic acid samples and HuSaV positive plasmids are used as HuSaV quality control products at home and abroad. However, these HuSaV quality control products have many shortcomings: (1) The virus content between different HuSaV positive samples varies greatly, and cannot be used to evaluate the sensitivity of the detection method. The test results of different laboratories may vary greatly; (2) HuSaV positive samples may have a great biosafety hazard, which may easily infect experimental operators and cause large-scale transmission; (3) Viral RNA samples are not easy to preserve, which will lead to deviations in the test results; (4) Although the chemical synthesis method can improve the stability of RNA positive control, the cost of this technology is relatively high and it is not suitable for widespread promotion and use; (5) Positive plasmids are easy to prepare and purify, but because plasmids are closed-loop double-stranded DNA, they can only be used as PCR controls, and cannot be used to monitor the entire process of viral RNA extraction and reverse transcription.

[0006] Artificial preparation of pseudovirus particles is to clone specific gene fragments into expression vectors containing phage genes, and produce RNA fragments wrapped by phage capsid proteins by inducing expression. This RNA quality control product preparation technology is also called armored RNA technology. The E. coli expression system is often used as a recombinant vector to express recombinant proteins. Since this system often forms insoluble and non-functional inclusion bodies when overexpressing exogenous proteins, the recombinant proteins have no biological activity. At present, it has been found that adding fusion tags to recombinant vectors can promote the correct folding of exogenous recombinant proteins and improve the solubility of recombinant proteins, such as maltose binding protein MBP, glutathione S-transferase GST, thioredoxin Trx and small molecule ubiquitin-like modifier protein SUMO. Among them, SUMO tag protein is a fusion tag with a powerful solubility-promoting function, which can greatly improve the stability and solubility of proteins, is resistant to heat and external proteases, and has a small molecular weight and will not affect the function of recombinant proteins. Therefore, SUMO tag proteins are increasingly favored by scientific researchers. SAP protein is a kind of protein composed of hydrophilic and hydrophobic amino acids. When it is fused to the N-terminus of recombinant proteins, it can significantly improve the transcription and translation efficiency of proteins, thereby increasing the expression of proteins. The structure formed based on SAP protein can protect proteins from interference from the external environment, reduce protein degradation and denaturation, and thus prolong the half-life. At present, there is no report on the application of SAP protein to the construction of pseudovirus particles. Linker will connect two or more proteins in the recombinant protein without affecting their independent spatial structure and function. Among them, the flexible linker can effectively separate the different parts of the recombinant protein through its flexibility and stretchability, avoiding mutual interference during the folding process, thereby ensuring that each domain can form its natural conformation and exert the best biological activity. F-Linker is a common flexible linker sequence [(GGGGS)2]. The small size of these amino acids provides flexibility and enables the connected protein functional domains to have a certain mobility, achieving the proper separation of protein functional domains or maintaining the necessary inter-domain interactions. PT-Linker is a special amino acid sequence composed of proline and threonine. PT-Linker has a certain flexibility, which allows the connected proteins or domains to move freely in space to perform their respective functions; it can also improve the expression and stability of recombinant proteins and reduce problems such as misfolding, low yield or impaired biological activity of recombinant proteins due to direct fusion. Summary of the invention

[0007] The object of the present invention is to overcome at least one of the shortcomings of the prior art and to provide a recombinant vector containing a human saproviral nucleic acid fragment and its application.

[0008] The technical solution adopted by the present invention is:

[0009] In the first aspect, the present invention provides a recombinant vector containing a human Zaru virus nucleic acid fragment, which is obtained by inserting an exogenous nucleotide sequence into a prokaryotic expression vector backbone, wherein the exogenous nucleotide sequence is formed by sequentially connecting the following gene sequences: a SUMO fusion tag DNA sequence, a SAP gene sequence, an MS2 phage-related DNA sequence, and a human Zaru virus sequence fragment.

[0010] In some examples, the human viral sequence fragment is followed by a stem-loop structure DNA sequence.

[0011] In some examples, the human Zhaju virus sequence fragments include at least two human Zhaju virus nucleic acid fragments, and the human Zhaju virus nucleic acid fragments are connected by a flexible F-Linker DNA sequence.

[0012] In some examples, the flexible F-Linker DNA sequence is shown as SEQ ID No.3.

[0013] In some examples, the SAP gene sequence and the MS2 phage-related DNA sequence are connected using a flexible PT-Linker DNA sequence.

[0014] In some examples, the flexible PT-Linker DNA sequence is shown as SEQ ID No.6.

[0015] In some examples, the nucleic acid fragment of human Zharu virus is a conserved region of human Zharu virus, and the cDNA fragment corresponding to the nucleic acid fragment of human Zharu virus is shown as SEQ ID No.1.

[0016] In some examples, the MS2 phage-related DNA sequence contains a gene sequence encoding a mature enzyme protein, a lytic protein gene sequence, a coat protein gene sequence and a replicase protein gene sequence, and the MS2 phage-related DNA sequence is shown as SEQ ID No.2.

[0017] In some examples, the SUMO fusion tag DNA sequence is shown as SEQ ID No.4, and the SAP gene sequence is shown as SEQ ID No.5.

[0018] In some examples, the stem-loop structure DNA sequence is shown as SEQ ID No.7.

[0019] In some examples, the prokaryotic expression vector is pET-32a(+), and the exogenous nucleotide sequence is inserted into the prokaryotic expression vector pET-32a(+).

[0020] The above features can be combined arbitrarily unless they conflict with each other.

[0021] In a second aspect, the present invention provides a target for detecting human SARS-CoV, which is obtained by transcribing and translating the recombinant vector described in the first aspect in a cell, followed by separation and purification.

[0022] The beneficial effects of the present invention are:

[0023] (1) Universality: The human Sarcoma virus pseudovirus particles prepared by the present invention have conserved nucleic acid sequences of epidemic strains of the human Sarcoma virus GI genotype, GII genotype, GIV genotype and GV genotype, and thus the human Sarcoma virus pseudovirus particles can be used as positive quality control products for detecting human Sarcoma virus.

[0024] (2) Stability: The human saprovirus RNA gene sequence in the pseudovirus particles prepared by the present invention contains a stem-loop structure at the end, which is not easily degraded by nucleases, thereby improving the stability of the viral nucleic acid RNA; at the same time, the conserved RNA gene sequence of the human saprovirus is encapsulated by the bacteriophage MS2 capsid protein, which has a high resistance to ribonucleases and deoxyribonucleases, thereby enhancing the long-term stability of the viral nucleic acid RNA. This solves the stability problem of viral RNA quality control products during production and use.

[0025] (3) High concentration: In order to obtain high-concentration, soluble human saprovirus pseudoviral particles, the present invention inserts a SUMO tag sequence and a SAP gene sequence into a recombinant vector, thereby promoting the production of high-concentration and soluble human saprovirus pseudoviral particles; and the prepared pseudoviral particles contain a conserved nucleic acid fragment of the human saprovirus dimer, which serves as a target for detecting human saprovirus, so that the human saprovirus pseudoviral particles contain more target nucleic acid sequences for detecting human saprovirus.

[0026] (4) Simulation: The RNA-protein complex structure of the human Ssavirus pseudovirus particles is similar to that of the natural virus. The pseudovirus particles can be equated with viral materials and can be used as a quality control tool for the entire process of human Ssavirus detection to ensure the authenticity of the detected virus.

[0027] (5) Safety: The pseudovirus particles of human influenza virus have a shell similar to that of the virus and encapsulate viral nucleic acid. They are not contagious and have no ability to self-replicate. Therefore, the pseudovirus particles of human influenza virus are not contagious and will not cause harm to experimenters or pollute the environment.

[0028] (6) Easy purification: The prepared human SARS-CoV pseudovirus is expressed in E. coli BL21 cells and can be quickly released into the supernatant after the E. coli BL21 cells are lysed, which can improve the purification efficiency of the pseudovirus particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1It is a schematic diagram of the composition of the recombinant vector pET-32a(+)-SSP-MS2-HuSaV in a specific implementation manner.

[0030] Figure 2 It is a schematic diagram of the composition of the recombinant vector pET-32a(+)-MS2-HuSaV in a specific implementation manner.

[0031] Figure 3 The expression products of the recombinant plasmid in E.coli BL21 cells were analyzed by SDS-PAGE. M: Protein Marker; 1: pET-32a(+)-MS2-HuSaV recombinant bacteria without inducer; 2: pET-32a(+)-MS2-HuSaV recombinant bacteria after induction; 3: pET-32a(+)-MS2-HuSaV recombinant bacteria after preliminary purification; M: Protein Marker; 4: pET-32a(+)-SSP-MS2-HuSaV recombinant bacteria without inducer; 5: pET-32a(+)-SSP-MS2-HuSaV recombinant bacteria after induction; 6: pET-32a(+)-SSP-MS2-HuSaV recombinant bacteria after preliminary purification.

[0032] Figure 4 This is a picture of human SARS-CoV pseudovirion particles observed under transmission electron microscopy.

[0033] Figure 5 The figure shows the nuclease and deoxyribonuclease resistance results of human sabivirus pseudoviral nucleic acid, where M: DNALadder 2000; 1: RT-PCR amplification results of pseudoviral particles without human sabivirus nucleic acid fragments after deoxynuclease treatment; 2: PCR amplification results of human sabivirus pseudoviral particles after deoxynuclease treatment; 3: RT-PCR amplification results of pseudoviral particles after deoxynuclease treatment; 4: RT-PCR amplification results of pseudoviral particles without human sabivirus nucleic acid fragments after ribonuclease treatment; 5: RT-PCR amplification results of pseudoviral particles after 10μL ribonuclease treatment; 6: RT-PCR amplification results of pseudoviral particles after 20μL ribonuclease treatment.

[0034] Figure 6 Schematic diagram of the amplification curve of real-time fluorescence RT-PCR detection of human Sarcoma virus pseudoviral particles. DETAILED DESCRIPTION

[0035] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0036] Example 1: Construction of recombinant plasmid pET-32a(+)-SSP-MS2-HuSaV

[0037] 1. Construction of recombinant plasmid pET-32a(+)-MS2

[0038] 1.1. Design of primers for RT-PCR amplification of MS2 phage-related genes

[0039] RT-PCR amplification primers were designed for the MS2 phage (purchased from the American Type Culture Collection ATCC 15597-B1) genome sequence encoding the mature enzyme protein gene sequence, the lytic protein gene sequence, the coat protein gene sequence and the replicase protein gene sequence as shown in SEQ ID No. 2, namely the upstream primer MS2 F:G GATATC GGGTGGGACCCCTT (the underline indicates the introduced restriction site EcoR V), as shown in the sequence table SEQ ID No. 8, downstream primer MS2 R: CGC GGATCC AGTAAGCAATTGCTGTA (the underline indicates the introduced restriction site BamH I), as shown in the sequence listing SEQ ID No. 9.

[0040] 1.2. Construction of recombinant plasmid pET-32a(+)-MS2

[0041] The TRIzol method was used to extract the whole genome RNA of MS2 phage, and the MS2 phage related genes were amplified by RT-PCR, as shown in the sequence table SEQ ID No. 2. The MS2 phage related gene products amplified by RT-PCR and the prokaryotic expression vector pET-32a (+) were digested with restriction endonucleases EcoR V and BamH I (both preserved in this laboratory), and the double enzyme digestion reaction system is shown in Table 1.

[0042] Table 1 Double enzyme digestion reaction system

[0043]

[0044] Incubate at 37°C for 2 h; recover the double-digested products and connect them using T4 DNA Ligase ligase kit. The reaction system is shown in Table 2.

[0045] Table 2 DNA enzyme ligation reaction system

[0046]

[0047] The ligation was carried out at 16°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, plated, and a single colony was picked and sequenced for identification. The correct recombinant plasmid was named pET-32a(+)-MS2.

[0048] 2. Construction of recombinant plasmid pET-32a(+)-SSP-MS2

[0049] 2.1. Construction of recombinant plasmid pET-32a(+)-S-MS2

[0050] The synthetic SUMO fusion tag DNA sequence is shown in the sequence table SEQ ID No. 4. The synthetic SUMO fusion tag DNA sequence and the pET-32a(+)-MS2 recombinant plasmid were digested with BgIII and KpnI (both preserved in this laboratory), respectively. The double enzyme digestion reaction system is shown in Table 3.

[0051] Table 3 Double enzyme digestion reaction system

[0052]

[0053] Incubate at 37°C for 2 h; recover the double-digested products and connect them using T4 DNA Ligase ligase kit. The reaction system is shown in Table 4.

[0054] Table 4 DNA enzyme ligation reaction system

[0055]

[0056]

[0057] The ligation was carried out at 16°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, plated, and a single colony was picked and sequenced. The correct recombinant plasmid was named pET-32a(+)-S-MS2

[0058] 2.2. Construction of recombinant plasmid pET-32a(+)-SS-MS2

[0059] The artificially synthesized SAP gene sequence is shown in the sequence table SEQ ID No. 5. The synthetic SAP gene sequence and the pET-32a(+)-S-MS2 recombinant plasmid were digested with Kpn I and Nco I (both preserved in this laboratory), respectively. The double enzyme digestion reaction system is shown in Table 5.

[0060] Table 5 Double enzyme digestion reaction system

[0061]

[0062] Incubate at 37°C for 2 h; recover the double-digested products and connect them using T4 DNA Ligase ligase kit. The reaction system is shown in Table 6.

[0063] Table 6 DNA enzyme ligation reaction system

[0064]

[0065] The ligation was carried out at 16°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, plated, and a single colony was picked and sequenced for identification. The correct recombinant plasmid was named pET-32a(+)-SS-MS2.

[0066] 2.3. Construction of recombinant plasmid pET-32a(+)-SSP-MS2

[0067] The synthetic flexible PT-Linker DNA sequence is shown in the sequence table as SEQ ID No. 6. The synthetic flexible PT-Linker DNA sequence and the pET-32a(+)-SS-MS2 recombinant plasmid were digested with Nco I and EcoR V (both stored in this laboratory), respectively. The double enzyme digestion reaction system is shown in Table 7.

[0068] Table 7 Double enzyme digestion reaction system

[0069]

[0070] Incubate at 37°C for 2 h; recover the double-digested products and connect them using T4 DNA Ligase ligase kit. The reaction system is shown in Table 8.

[0071] Table 8 DNA enzyme ligation reaction system

[0072]

[0073] The ligation was carried out at 16°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, plated, and a single colony was picked and sequenced for identification. The correct recombinant plasmid was named pET-32a(+)-SSP-MS2.

[0074] 3. Construction of recombinant plasmid pET-32a(+)-SSP-MS2-LS

[0075] 3.1. Construction of recombinant plasmid pET-32a(+)-SSP-MS2-L

[0076] The synthetic flexible F-Linker DNA sequence is shown in the sequence table SEQ ID No. 3. The synthetic flexible F-Linker DNA sequence and the pET-32a(+)-SSP-MS2 recombinant plasmid were digested with EcoR I and Sac I (both preserved in this laboratory), respectively. The double enzyme digestion reaction system is shown in Table 9.

[0077] Table 9 Double enzyme digestion reaction system

[0078]

[0079] Incubate at 37°C for 2 h; recover the double-digested products and connect them using T4 DNA Ligase ligase kit. The reaction system is shown in Table 10.

[0080] Table 10 DNA enzyme ligation reaction system

[0081]

[0082] The ligation was carried out at 16°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, plated, and a single colony was picked and sequenced for identification. The correct recombinant plasmid was named pET-32a(+)-SSP-MS2-L.

[0083] 3.2. Construction of recombinant plasmid pET-32a(+)-SSP-MS2-LS

[0084] The artificially synthesized stem-loop structure DNA sequence is shown in the sequence table SEQ ID No. 7. The synthetic stem-loop structure DNA sequence and the pET-32a(+)-SSP-MS2-L recombinant plasmid were digested with Sal I and Xho I (both preserved in this laboratory), respectively. The double enzyme digestion reaction system is shown in Table 11.

[0085] Table 11 Double enzyme digestion reaction system

[0086]

[0087] Incubate at 37°C for 2 h; recover the double-digested products and connect them using T4 DNA Ligase ligase kit. The reaction system is shown in Table 12.

[0088] Table 12 DNA enzyme ligation reaction system

[0089]

[0090] The ligation was carried out at 16°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, plated, and a single colony was picked and sequenced for identification. The correct recombinant plasmid was named pET-32a(+)-SSP-MS2-LS.

[0091] 4. Construction of recombinant plasmid pET-32a(+)-SSP-MS2-HuSaV

[0092] 4.1. Synthesis of conserved gene sequences of human Zarvirus

[0093] The conserved nucleic acid sequences of the GI genotype, GII genotype, GIV genotype and GV genotype epidemic strains of human szabvirus were analyzed, and DNA fragments including cDNA fragments corresponding to the conserved nucleic acid fragments were obtained by artificial synthesis, as shown in SEQ ID No. 1, and restriction sites BamH I / EcoR I and SacI / Sal I were inserted at both ends of two identical synthetic conserved nucleic acid sequences, respectively.

[0094] 4.2. Construction of recombinant plasmid pET-32a(+)-SSP-MS2-HuSaV

[0095] The two identical artificially synthesized conserved nucleic acid sequences of human Ssavirus and the recombinant plasmid pET-32a(+)-SSP-MS2-LS were digested with restriction endonucleases BamH I and EcoR I, respectively, and with Sac I and Sal I (all preserved in this laboratory). The double enzyme digestion reaction system is shown in Table 13.

[0096] Table 13 Double enzyme digestion reaction system

[0097]

[0098] Incubate at 37°C for 2 h; recover the double-digested products and ligate them using T4 DNA Ligase ligase kit. The reaction system is shown in Table 14.

[0099] Table 14 DNA enzyme ligation reaction system

[0100]

[0101] The ligation was carried out at 16°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, plated, and a single colony was picked and sequenced for identification. The correct recombinant plasmid was named pET-32a(+)-SSP-MS2-HuSaV. Its structural diagram is shown in the figure. Figure 1 shown.

[0102] 5. Construction of recombinant plasmid pET-32a(+)-MS2-HuSaV

[0103] For the conserved nucleic acid sequences of the GI genotype, GII genotype, GIV genotype and GV genotype epidemic strains of human Szabovirus, DNA fragments including cDNA fragments corresponding to the conserved nucleic acid fragments were obtained by artificial synthesis, as shown in SEQ ID No. 1, and restriction sites BamH I and EcoR I were inserted at both ends of the conserved nucleic acid sequence; the conserved gene sequence of human Szabovirus and the recombinant plasmid pET-32a(+)-MS2 constructed above were respectively digested with restriction endonucleases BamH I and EcoR I (both preserved in this laboratory), and the double enzyme digestion reaction system is shown in Table 15.

[0104] Table 15 Double enzyme digestion reaction system

[0105]

[0106] Incubate at 37°C for 2 h; recover the double-digested products and ligate them using T4 DNA Ligase ligase kit. The reaction system is shown in Table 16.

[0107] Table 16 DNA enzyme ligation reaction system

[0108]

[0109] The ligation was carried out at 16°C for 30 minutes. The ligation product was then transformed into E. coli DH5α competent cells, plated, and a single colony was picked and sequenced for identification. The correct recombinant plasmid was named pET-32a(+)-MS2-HuSatV. Its structural diagram is shown in the figure. Figure 2 shown.

[0110] Example 2: Preparation of human SARS-CoV pseudoviral particles

[0111] 1. The recombinant plasmids pET-32a(+)-SSP-MS2-HuSaV and pET-32a(+)-MS2-HuSaV were transformed into E. coli BL21 competent cells, respectively, to obtain positive colonies containing the recombinant plasmids pET-32a(+)-SSP-MS2-HuSaV and pET-32a(+)-MS2-HuSaV, and inoculated into LB liquid culture medium for culture.

[0112] 2. Inoculate the above cultured bacterial solution with Kan at a ratio of 1:100. + LB liquid medium (containing 0.2% glucose) was added and the OD 600 When the concentration reached 0.4, the inducer IPTG was added to a final concentration of 0.5 mmol / L to induce expression; after culturing for 7 hours, the bacteria were collected by centrifugation.

[0113] 3. Take 8 mL of bacterial lysis buffer (30 mmol / L Tris-HCl, pH 8.5, 4 mmol / L EDTA, 80 mmol / L NaCl, 1.0% Triton X-100, lysozyme 50 mg / L) to suspend the bacteria and incubate at 37°C for 30 min; then ultrasonically disrupt the cells and collect the supernatant by centrifugation.

[0114] 4. Add RNase and DNase I to the supernatant described in step 3 to a final concentration of 2 mg / L, incubate at 37°C for 1 h, and collect the supernatant by centrifugation.

[0115] 5. Collect the supernatant as described in step 4 and transfer it to an ultrafiltration centrifuge tube (10kD), and centrifuge it at 4°C, 4,000r / min for 1h; aspirate all the concentrated liquid from the collection tube of the ultrafiltration tube to obtain purified pseudovirus particles, take 10μL as a sample for SDS-PAGE electrophoresis, and store the rest at -80°C for later use.

[0116] 6. Add a small amount of PBS solution to the pseudovirus particle solution obtained above and mix well. Add protein loading buffer [0.5 mmol / L Tris-HCl (pH 6.8) 2.5 mL, DTT 0.42 g, SDS 0.5 g, bromophenol blue 0.025 g, glycerol 2.8 mL, deionized water to 5 mL] in a ratio of 1:4 and mix well. Boil in boiling water for 10 min and cool to room temperature for use. Perform SDS-PAGE gel electrophoresis in 5% SDS-PAGE concentrating gel and 12% SDS-PAGE separation gel, stain with Coomassie brilliant blue staining solution, and then decolorize with decolorizing solution until the protein bands are clearly visible. Figure 3 The SDS-PAGE electrophoresis diagram showed that a relatively obvious target band appeared at about 13 kDa after induction, indicating that the recombinant bacteria can normally express pseudovirion protein; and the protein expression level of the pET-32a(+)-SSP-MS2-HuSaV strain was significantly higher than that of the pET-32a(+)-MS2-HuSaV strain, so the expression of pseudovirion protein by pET-32a(+)-SSP-MS2-HuSaV is better than the classic strategy for constructing pseudovirion.

[0117] Example 3: Characterization of human SARS-CoV pseudovirion particles

[0118] 1. As Figure 4 As shown, the above pseudovirus particle stock solution was negatively stained with 2% phosphotungstic acid, and then the pseudovirus particles were observed under a transmission electron microscope, as shown in FIG. Figure 3 As shown, virus-like particles with dense arrangement and consistent size and shape can be observed.

[0119] 2. Analysis of ribonuclease resistance of human Zaru virus pseudoviral particles. The RNA in the prepared human Zaru virus pseudoviral particles was extracted by TRIzol method. The specific steps are as follows: take 50 μL of the human Zaru virus pseudoviral particle stock solution prepared in Example 2 and place it in an RNase-free EP tube, add 1 mL of TRIzol solution, shake for 30 seconds and let it stand at room temperature for 5 minutes; add 0.2 mL of chloroform, shake for 30 seconds and let it stand at room temperature for 5 minutes, and centrifuge at 12,000 r / min for 15 minutes; collect the upper aqueous phase solution and place it in a new RNase-free EP tube, add an equal volume of isopropanol solution, let it stand at room temperature for 30 minutes, and centrifuge at 12,000 r / min for 15 minutes; add 1 mL of 75% ethanol to the precipitate for washing, and centrifuge at 8,000 r / min for 15 minutes; discard the supernatant, and the RNA in the precipitate evaporates the ethanol at room temperature; add 20 μL of RNase-free ddH2O to dissolve the RNA precipitate and immediately store it at -80°C for later use.

[0120] In order to determine whether the pseudovirus particles prepared in Example 2 contain human saprovirus RNA fragments, the RNA extracted as above was used as a template, and the upstream primer HuSaV 1F was designed, as shown in SEQ ID No. 10 in the sequence table, and the downstream primer HuSaV1245R was designed, as shown in SEQ ID No. 11 in the sequence table. This primer set can amplify the cDNA fragment corresponding to the human saprovirus nucleic acid fragment. Deionized water was used as a template as a negative control, and an RT-PCR kit (such as One-Step RT-PCR Kit) was used for detection, and the reaction system is shown in Table 15 below.

[0121] Table 17 RT-PCR reaction system

[0122]

[0123] Amplification conditions: reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 5 min; denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 20 s, 35 cycles; post-extension at 72°C for 10 min. RT-PCR amplification products were analyzed by 1.2% agarose gel electrophoresis.

[0124] 3. Analysis of the DNA resistance of human saprovirus pseudovirions. PCR and RT-PCR methods were used to detect whether the human saprovirus pseudovirions prepared in Example 2 contained residual recombinant plasmid DNA. The above-mentioned human saprovirus pseudovirion stock solution was used as a PCR amplification template and the RNA in the pseudovirion prepared by the TRIzol method was extracted as a RT-PCR amplification template, respectively. The upstream primer HuSaV 1F was designed, as shown in SEQ ID No. 10, and the downstream primer HuSaV 1245R was designed, as shown in SEQ ID No. 11. This primer set can amplify the cDNA fragment corresponding to the nucleic acid sequence in the human saprovirus pseudovirion. Deionized water was used as a template as a negative control, and PCR amplification enzymes (such as 2×Taq Master Mix) and RT-PCR kits (such as One-Step RT-PCR Kit) was used for detection, and the reaction system is shown in Table 18 below.

[0125] Table 18 PCR reaction system and RT-PCR reaction system

[0126]

[0127] PCR amplification reaction conditions: 95℃ pre-denaturation for 10min; 94℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 20s, 35 cycles; 72℃ post-extension for 10min. PCR products were analyzed by 1.2% agarose gel electrophoresis. RT-PCR amplification reaction conditions: 42℃ reverse transcription for 5min; 95℃ pre-denaturation for 5min; 94℃ denaturation for 20s, 58℃ annealing for 30s, 72℃ extension for 20s, 35 cycles; 72℃ post-extension for 10min. RT-PCR products were analyzed by 1.2% agarose gel electrophoresis.

[0128] like Figure 5 The results showed that the MS2 phage capsid encapsidated the human Sarcoma virus RNA gene and the human Sarcoma virus pseudovirus particles were not doped with plasmid DNA; at the same time, it was found that the human Sarcoma virus pseudovirus particles were resistant to degradation by ribonucleases of different concentrations, indicating that the prepared pseudovirus particles can resist external ribonucleases and have good stability.

[0129] Example 4: Evaluation of human SARS-CoV pseudovirion quality control

[0130] 1. According to the TRIzol method in Example 3, RNA was extracted from the human Zaru virus pseudovirus particles assembled by the pET-32a(+)-SSP-MS2-HuSaV strain and the pET-32a(+)-MS2-HuSaV strain, and the primer and probe sequences in the entry-exit inspection and quarantine industry standard "SN / T 5325.4-2020 Quantitative Detection of Foodborne Viruses in Exported Foods by Digital PCR Method Zaru Virus" were analyzed to prepare the human Zaru virus pseudovirus particles in Example 2 as quality control products for detecting human Zaru virus. The RNA in the pseudovirion particles of the two human SARS-CoVs is used as the research object. The RNA extracted from the two different sources is diluted 10 times as a template. The upstream primer HuSaV 1F is designed as shown in SEQ ID No. 10 in the sequence table. The upstream primer HuSaV 124F is shown in SEQ ID No. 12 in the sequence table. The upstream primer HuSaV 5F is shown in SEQ ID No. 13 in the sequence table. The downstream primer HuSaV1245R is shown in SEQ ID No. 11 in the sequence table. The probe sequence HuSaV 124TP is shown in SEQ ID No. 14 in the sequence table. The fluorescent group VIC is added to the 5' end of the probe sequence. The quenching group BHQ1 is introduced to the 3' end of the probe sequence. The probe sequence HuSaV 5TP is shown in SEQ ID No. 15 in the sequence table. The fluorescent group VIC is added to the 5' end of the probe sequence. The quenching group BHQ1 is introduced to the 3' end of the probe sequence. Real-time fluorescence quantitative RT-PCR detection is performed. For example, using a real-time fluorescence quantitative RT-PCR kit (such as Multiplex Probe One Step qRT-PCR Kit (UDG) was used for detection, and deionized water was used as a template as a negative control. The reaction system is shown in Table 19 below.

[0131] Table 19 Reaction system of real-time fluorescence quantitative RT-PCR detection method

[0132]

[0133] Reaction conditions: reverse transcription at 50°C for 10 min; pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, denaturation at 60°C for 20 s, and 40 cycles; fluorescence signals were collected in each cycle.

[0134] Figure 6 The amplification curve results showed that the prepared human Sarcoma virus pseudovirus particles can be used as a positive quality control for detecting human Sarcoma virus and can evaluate the entire detection process. The prepared human Sarcoma virus pseudovirus particle stock solution and its gradient dilution (10 -1 ~10 -4) showed obvious exponential amplification, while there was no obvious amplification when deionized water was used as a template as a negative control, indicating that the prepared pseudovirus particles contained human sasavirus nucleic acid RNA fragments. In addition, the CT value of viral nucleic acid RNA in the human sasavirus pseudovirus particles formed by the pET-32a(+)-SSP-MS2-HuSaV strain was lower than that in the human sasavirus pseudovirus particles formed by the pET-32a(+)-MS2-HuSaV strain, indicating that the content of viral nucleic acid RNA in the human sasavirus pseudovirus particles formed by the pET-32a(+)-SSP-MS2-HuSaV strain was higher, which is helpful as a positive quality control for detecting human sasavirus.

[0135] Since the human Zaru virus nucleic acid fragments contained in the above pseudovirus particles can be used as the RNA gene fragment quality control products corresponding to the target sequence of human Zaru virus in the entry-exit inspection and quarantine industry standard of my country "SN / T 5325.4-2020 Digital PCR Method for Quantitative Detection of Foodborne Viruses in Exported Foods Part 4 Zaru Virus" and the entry-exit inspection and quarantine industry standard of my country "SN / T 2531-2010 Zaru virus detection method in shellfish and water samples - ordinary RT-PCR method and real-time fluorescence RT-PCR method", they can be used in conjunction with the above standards. Thus, the bottlenecks that seriously restrict the development of human Zaru virus detection, such as "there are detection methods but no standard samples", "the research and development of standard samples does not correspond to the standard method, resulting in narrow application", and "the poor matching between standard samples and detection methods", have been changed.

[0136] The above is a detailed description of a specific embodiment of the present invention through the examples, but the protection scope of the present invention is not limited thereto. Under the premise of achieving the purpose of the present invention, those skilled in the art can make various changes and modifications to the technical solution of the present invention. For example, in addition to the RNA fragments corresponding to the human Zaru virus detection target sequence determined in the Chinese entry-exit inspection and quarantine industry standard "SN / T5325.4-2020 Digital PCR Method for Quantitative Detection of Foodborne Viruses in Exported Foods Part 4 Zaru Virus" and the Chinese entry-exit inspection and quarantine industry standard "SN / T 2531-2010 Ordinary RT-PCR Method and Real-time Fluorescence RT-PCR Method for Zaru Virus Detection in Shellfish and Water Samples", the nucleic acid fragments contained in the pseudovirus particles of the present invention can also be other nucleic acid fragments of the human Zaru virus detection target. In addition, in addition to the pET-32a (+) prokaryotic expression vector mentioned in the present invention, other prokaryotic expression vectors can also be used for transcription and / or translation expression of the conserved sequence of the human Zaru virus described in the present invention.

Claims

1. A recombinant vector containing a human saprovirus nucleic acid fragment, obtained by inserting an exogenous nucleotide sequence into a prokaryotic expression vector backbone, characterized in that: The exogenous nucleotide sequence is formed by sequentially connecting the following gene sequences: SUMO fusion tag DNA sequence, SAP Gene sequence, MS2 bacteriophage-related DNA sequence, human SARS-CoV sequence fragment.

2. The recombinant vector according to claim 1, characterized in that The human saprolegnia virus sequence fragment is also followed by a stem-loop structure DNA sequence.

3. The recombinant vector according to claim 1 or 2, characterized in that The human ZA virus sequence fragments include at least two human ZA virus nucleic acid fragments, and the human ZA virus nucleic acid fragments are connected by a flexible F-Linker DNA sequence; and / or Said SAP The gene sequence and MS2 phage-related DNA sequence were connected using a flexible PT-Linker DNA sequence.

4. The recombinant vector according to claim 3, characterized in that The flexible F-Linker DNA sequence is shown as SEQ ID No. 3 and / or the flexible PT-Linker DNA sequence is shown as SEQ ID No.

6.

5. The recombinant vector according to claim 3, characterized in that The nucleic acid fragment of human Zharu virus is a conserved region of human Zharu virus, and the cDNA fragment corresponding to the nucleic acid fragment of human Zharu virus is shown as SEQ ID No.

1.

6. The recombinant vector according to claim 1, characterized in that The MS2 phage-related DNA sequence contains a gene sequence encoding a mature enzyme protein, a lytic protein gene sequence, a coat protein gene sequence and a replicase protein gene sequence, and the MS2 phage-related DNA sequence is shown in SEQ ID No.

2.

7. The recombinant vector according to claim 1, characterized in that The SUMO fusion tag DNA sequence is shown in SEQ ID No. 4 and / or SAP The gene sequence is shown in SEQ ID No.

5.

8. The recombinant vector according to claim 2, characterized in that The stem-loop structure DNA sequence is shown as SEQ ID No.

7.

9. The recombinant vector according to claim 1, characterized in that The prokaryotic expression vector is pET-32a(+), and the exogenous nucleotide sequence is inserted into the prokaryotic expression vector pET-32a(+).

10. A target for detecting human SARS-CoV-2, characterized in that: The recombinant vector according to any one of claims 1 to 9 is transcribed and translated in cells, and then separated and purified to obtain the product.

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