Virus-like particle containing novel coronavirus nucleic acid as well as preparation method and application of virus-like particle

A coronavirus and virus-like technology, which is applied in the field of virus-like particles containing novel coronavirus nucleic acid and its preparation, can solve the problems of less purification amount, long time, and small sample amount, so as to improve the purification speed and yield , good stability, and the effect of improving the speed of digestion

Pending Publication Date: 2020-12-04
河南省生物工程技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the virus-like particles obtained by the PEG precipitation method are not high in purity and take a long time; when gel filtration chromatography purifies virus-like particles, it needs to be crude first, and finally use molecular sieves, but the sample volume is small and the time is long; Although the gradient centrifugation method has high purity, it takes a long time and cannot be prepared in large quantities, requiring an ultracentrifuge; the ultrafiltration method cannot remove large molecular impurity proteins, the purity is not high, the amount of purification is small each time, and the loss is large

Method used

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  • Virus-like particle containing novel coronavirus nucleic acid as well as preparation method and application of virus-like particle
  • Virus-like particle containing novel coronavirus nucleic acid as well as preparation method and application of virus-like particle
  • Virus-like particle containing novel coronavirus nucleic acid as well as preparation method and application of virus-like particle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of pET28a / MS2 / S recombinant vector

[0035] 1 Preparation of pET28a / MS2 vector

[0036] Using phage MS2 RNA as a template, use random primers (N6) and the first primer pair to carry out reverse transcription and amplification to obtain the first PCR product including MS2 phage coat protein (CP) and PAC sites; reverse transcription and PCR The one-step amplification system is: 5 μl 10×One-Step Buffer, 1 μl 50×dNTP Mix, 0.5 μl Recombinant RNase Inhibitor (40 units / μl), 25 μl Thermostabilizing Reagent, 10 μl GC-Melt, 1 μl Random(N6) Primer, 1 μl μl 50×Titanium Taq RT Enzyme Mix, 1μl the first primer pair (45μM each), 1μl MS2RNA, 2.5μl RNase-Free H 2 O, total volume: 50 μl. The reverse transcription and PCR amplification procedures are: reverse transcription at 50°C for 1 h, pre-deformation at 94°C for 5 m, 30 cycles: denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 68°C for 1 min, and extension at 68°C for 2 min; After the a...

Embodiment 2

[0049] Example 2 Preparation of MS2 virus-like particles containing novel coronavirus nucleic acid

[0050] Transform the pET28a / MS2 / S recombinant plasmid into Escherichia coli expression strain BL21(DE3), and the BL21(DE3) / pET28a / MS2 / S engineering strain was recovered and cultured at 37°C to OD 600 After the value is about 0.8, the temperature of the shaker is lowered to 16°C, and the culture is continued for 1h, and the final concentration of 0.2mmol / L isopropylthio-β-D-galactoside (IPTG) is added, and at 16°C, 200r / min induced expression for 20h. Centrifuge at 8000r / min for 10min to collect cultured bacterial cells, discard the supernatant, resuspend and wash the bacterial pellet with 20mM Tris-HCL (pH=8.0), centrifuge again at 8000r / min for 10min, discard the supernatant, and use the lysate for bacterial pellet Resuspend, after low-temperature sonication, centrifuge at 12000r / min for 10min, collect the supernatant, and discard the precipitate.

[0051] Precipitation of c...

Embodiment 3

[0055] Example 3 Physical Characterization and Identification of Virus-Like Particles Containing New Crown Nucleic Acid MS2

[0056] 1. Physical characterization of virus-like particles:

[0057] The virus-like particle solution purified in Example 2 was diluted with 20mM Tris-HCL (pH=8.0, containing 150mM NaCl) to a concentration of about 0.5mg / ml, filtered through a 0.22μm filter membrane, and placed in an ultra-clean bench. 2% phosphotungstic acid negative staining, natural drying, under the condition of 100kV accelerating voltage, through JEM-1400 (Japan) transmission electron microscope, observe and measure the particle size. At the same time, the particle size and distribution of the particles were measured with a Brookhaven instrument potentiometric and particle size analyzer (90Plus PALS in the United States).

[0058] Virus-like particles are observed by transmission electron microscope (as shown in the accompanying drawing of the description) figure 1 As shown): Th...

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Abstract

The invention belongs to the field of applied molecular biology, and particularly relates to a virus-like particle containing novel coronavirus nucleic acid as well as a preparation method and application of the virus-like particle. The virus-like particle containing the novel coronavirus nucleic acid contains MS2 bacteriophage coat protein and a recombinant gene S sequence wrapped in the coat protein, wherein the S sequence consists of a novel coronavirus ORF1ab gene segment, a human beta-Globin gene segment, a novel coronavirus N gene segment and an E gene segment which are connected in sequence; and the S sequence has a nucleotide sequence as shown in SEQ ID NO.5. The virus-like particle can be used for whole-course quality control of nucleic acid extraction, amplification and detectionof the novel coronavirus, and can be popularized and applied as a positive control and internal standard quality control product of the novel coronavirus in a kit.

Description

technical field [0001] The invention belongs to the field of applied molecular biology, and in particular relates to a virus-like particle containing novel coronavirus nucleic acid and its preparation method and application. Background technique [0002] Novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) in humans with fever, fatigue, dry cough and dyspnea, Severe cases are complicated by infectious diseases of acute respiratory distress syndrome. It is highly contagious and can infect people of all ages, especially the elderly with underlying diseases can cause higher morbidity and mortality. [0003] At present, many researchers at home and abroad have established various molecular biology diagnostic methods for pathogens, mainly including the detection of antigens and nucleic acids. However, in the actual detection of nucleic acids, e...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70C07K1/36C07K1/30C07K1/16C12Q1/70C12Q1/686C12R1/19C12R1/93
CPCC07K14/005C07K14/805C07K2319/00C12N15/70C12N2770/20022C12N2795/00022C12N2795/00023C12N2795/00051C12Q1/686C12Q1/701C12Q2563/107
Inventor 王云龙王国强李振勇李玉林孙新城王继创王敏程蕾王运从
Owner 河南省生物工程技术研究中心
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