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Virus-like particles containing O-type foot-and-mouth disease virus IRES RNA, preparation method and application

A foot-and-mouth disease virus and virus-like technology, applied in the field of virus-like particles, can solve the problems of easy infection, inability to monitor RNA virus detection in the whole process, ribonuclease degradation, etc., and achieve the effects of low cost, favorable promotion and use, and easy preservation.

Inactive Publication Date: 2007-10-24
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a safe, ribonuclease-resistant, Good stability, easy storage, full monitoring of RNA virus extraction, reverse transcription, amplification and

Method used

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  • Virus-like particles containing O-type foot-and-mouth disease virus IRES RNA, preparation method and application
  • Virus-like particles containing O-type foot-and-mouth disease virus IRES RNA, preparation method and application
  • Virus-like particles containing O-type foot-and-mouth disease virus IRES RNA, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the preparation method of the virus-like particle containing O-type foot-and-mouth disease virus IRES RNA

[0042] The preparation method of the virus-like particle containing O-type foot-and-mouth disease virus IRES RNA comprises the following steps:

[0043] (1) Construction of the initial vector pET32a-CP:

[0044] in pMS 2 7. The plasmid is used as a template to amplify the CP fragment. The PCR system is: 10×PCR buffer 2μl, TaqDNA Polymerase 0.2μl (5u / μl), dNTP 0.5μl (10mmol / L), template 1μl, upstream and downstream primers 0.5μl (10μmol / μl) L), add 20 μl of double distilled water. The PCR reaction conditions were: denaturation at 94°C for 5 min, followed by 28 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 2 min, and finally extension at 72°C for 7 min. The amplified product was subjected to agarose gel electrophoresis, and the CP fragment was purified and recovered with a gel extraction kit. The recovered CP fragment and plasmid pET32a were...

Embodiment 2

[0051] Example 2: Double-enzyme digestion identification of RNaseA and DNase I of the expressed product after sonication

[0052] The expression product after sonication was digested with RNaseA and DNase I for single enzyme and double enzyme digestion for 1 hour respectively, as shown in Figure 1: After the expression product was digested with RNaseA and DNase I double enzymes, a fluorescence of about 1500bp can be seen Streaks; the presence of bacterial RNA can be seen after only DNase I digestion; only after RNaseA digestion, bacterial RNA has been degraded. The expression products were placed at room temperature for 20 days and then digested with enzymes. The electrophoresis results showed the presence of the expression products, indicating that the virus-like particles had the characteristics of ribonuclease resistance and good stability.

Embodiment 3

[0053] Example 3: Morphological observation of purified virus-like particles

[0054] The virus-like particles purified by sucrose density gradient centrifugation were stained with uranyl acetate for 5 minutes, and observed under a JM2100 electron microscope. As shown in Figure 2, round particles with a diameter of about 26 nm can be seen, which are expressed virus-like particles (viral like particles, VLPs).

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Abstract

The invention discloses a virus particle and making method and application with O-typed IRES RNA of foot-and-mouth disease virus, which is characterized by the following: the RNA virus extraction, reverse transcription and augumentation and final examination can be full-course monitored, which is easy to reserve to tolerate ribalgilase; the virus particle is a sphere-shaped RNA-protein complex with MS2 bacteriophage coat protein covering the IRES RNA of foot-and-mouth disease virus; the diameter is 24-28nm. The making method comprises the following steps: constructing primary carrier pET32a-CP and final carrier pET32a-CP-IRES; proceeding prokaryotic expression for virus particle; purifying the virus particle.

Description

technical field [0001] The invention relates to a virus-like particle, in particular to a virus-like particle containing O-type foot-and-mouth disease virus IRES RNA which can be used in RNA virus detection and a preparation method thereof. Background technique [0002] RNA virus is a virus with RNA as its genetic material, such as avian influenza virus, foot-and-mouth disease virus and SARS virus. Because of the serious hazards of these RNA viruses, rapid and accurate detection is critical. Commonly used methods for detecting RNA viruses include traditional virus isolation tests, hemagglutination inhibition tests, virus neutralization tests, and enzyme-linked immunosorbent assay (ELISA), as well as molecular biological detection techniques such as nucleic acid-dependent amplification (NASBA) ( Romano J W, Will-iams K G, Shurtliff R N, et al. NASBA technology: isothermal RNA amplification in qualitative and quantitative diagnostics [J]. Immunol. Invest, 1997, 26 (1-2): 15-2...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12Q1/70G01N33/569C12N15/42
Inventor 陈亮窦敏张国广张红心曾雅明
Owner XIAMEN UNIV
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