A simple, low-cost method and system for producing DNA standards based on ms2 virus-like particles

Abstract

The application provides a simple, low-cost and stable nucleic acid standard preparation method and system based on MS2 virus-like particles (VLP). A His-tagged MS2 capsid protein expression plasmid is constructed, transformed into an E. coli competent cell, induced to express by arabinose, and then purified by nickel ion affinity chromatography to obtain MS2-VLP, and the CP protein is obtained by lysis. After the exogenous DNA containing a pac site is amplified by PCR, the CP protein obtained by lysis is mixed, and the self-assembly characteristics are used to realize DNA wrapping. The nucleic acid standard prepared by the method has excellent stability, nuclease resistance, high temperature resistance and freeze-thaw resistance, can effectively simulate the existing form of nucleic acid in a clinical sample, solves the problem that naked nucleic acid is easy to degrade, and provides a reliable control tool for clinical nucleic acid detection.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method and system for preparing nucleic acid standards by encapsulating exogenous DNA with MS2 virus-like particles (VLPs), which is suitable for quality control in clinical nucleic acid testing. Background Technology

[0002] In the field of clinical diagnostics, nucleic acid detection technology plays a crucial role in the diagnosis and monitoring of infectious diseases, tumors, and genetic diseases due to its high sensitivity and specificity. However, traditional nucleic acid standards, including plasmids, naked DNA, or in vitro transcribed RNA, have significant drawbacks. These standards are easily degraded by nucleases in clinical sample matrices and cannot simulate the processing of real samples, which limits their application in detection validation and proficiency testing.

[0003] Virus-like particles (VLPs), as a novel carrier, are increasingly becoming an ideal choice for nucleic acid standards due to their safety and stability. MS2-VLPs, particularly those derived from MS2 bacteriophage, exhibit special advantages due to their unique structure and function. MS2-VLPs consist of an icosahedral structure composed of 90 capsid protein (CP) dimers, approximately 30 nm in diameter, and are non-infectious. Their assembly depends on the interaction between the CP protein and the pac site (a 19 nt RNA stem-loop structure), enabling MS2-VLPs to efficiently encapsulate exogenous nucleic acids and protect them from environmental influences.

[0004] Despite the advantages mentioned above, the simple and low-cost production of these particles, and ensuring their effective encapsulation of exogenous DNA and stability, remain challenges in practical applications. Furthermore, existing production methods often lack sufficient flexibility and operability, making it difficult to meet the needs of nucleic acid detection for different pathogens.

[0005] Therefore, it is necessary to develop a new method that can produce MS2-VLP-based nucleic acid standards simply and at low cost. These standards should have excellent resistance to nucleases, high temperatures, and freeze-thaw cycles, and should be able to effectively mimic the form of nucleic acids present in clinical samples to meet the quality control requirements of clinical nucleic acid testing. Summary of the Invention

[0006] This invention aims to address the technical bottlenecks of existing nucleic acid standards (such as naked DNA, plasmids, or in vitro transcribed RNA) in clinical sample matrices, which are easily degraded by nucleases, cannot simulate the actual sample extraction process, and are difficult to meet the requirements of detection validation and proficiency testing. To this end, a simple, low-cost, and highly stable nucleic acid standard preparation method and system based on MS2 virus-like particles (MS2-VLP) is provided. Through a two-step method of "one-step PCR-self-assembly encapsulation," exogenous DNA is efficiently loaded and protected, ultimately yielding nuclease-resistant, heat-resistant, freeze-thaw resistant, and quantitatively detectable nucleic acid standards suitable for quality control and standardization in clinical nucleic acid testing.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a plasmid vector containing a CP-CP and CP-CP-His chimeric CP dimer sequence capable of assembling into MS2 virus-like particles. The His tag facilitates subsequent purification steps. The specific technical solution is described below.

[0008] (1) MS2-VLP expression and purification An MS2 capsid protein (CP) expression plasmid carrying a His tag was constructed and transformed into TOP10 competent cells. After expression induced by L-arabinose, high-purity MS2-VLP was obtained by nickel affinity chromatography (AKTA system). The purity and integrity of VLP were verified by SDS-PAGE and agarose gel electrophoresis, confirming that it exhibited a characteristic band of approximately 30 kDa capsid protein dimer.

[0009] (2) Preparation of exogenous DNA template and exposure of PAC sites Design PCR primers containing a pac site (19 nt stem-loop) to amplify the target fragment in one step using the target as a template. During the denaturation and annealing process in PCR, the pac site will spontaneously expose the MS2 CP binding site.

[0010] (3) Self-assembly encapsulation and purification MS2-VLP was lysed with glacial acetic acid and neutralized to pH 7.4 with 5% Tris solution (pH 10–11) to restore capsid protein activity. The capsid protein was then mixed with DNA at a molar ratio of ≤10:1 and incubated at 37 °C for 12–16 h, allowing for self-assembly and encapsulation driven by CP-pac site-specific binding. DNase I was added to digest the free DNA, and the MS2-VLP-DNA complex was purified using His magnetic beads. Agarose gel electrophoresis and PCR confirmed successful encapsulation of the exogenous DNA and its resistance to nuclease degradation. Attached Figure Description Figure 1 This is a map of the MS2-VLP plasmid vector. Figure 2 The image shows the verification results after MS2-VLP purification. Figure 3 This is a diagram showing the verification results of the PCR amplification of exogenous DNA products. Figure 4 A schematic diagram illustrating the principle of EB virus nucleic acid encapsulated by the dimer CP protein. Figure 5 To verify the agarose gel electrophoresis results of CP dimer encapsulating exogenous DNA and the standard curve for q-PCR detection of internal DNA. Figure 6 The figure shows the validation results of multiple stability features of MS2-VLP-DNA.

Claims

1. A nucleic acid standard preparation system based on MS2 virus-like particles, characterized in that: This system only requires mixing purified MS2-VLP dimer CP protein and a mixture of dimer CP protein with His Tag at the carboxyl terminus with a DNA sequence containing a pac site to achieve self-assembly and encapsulate the DNA sequence, effectively preventing DNA from being degraded by nucleases, and can be used as a nucleic acid detection standard.

2. The MS2-VLP dimer CP protein for preparing nucleic acid standards according to claim 1, comprising two conserved sequences as shown in SEQ 1, wherein the carboxyl terminus of the first SEQ 1 and the amino terminus of the second SEQ 1 are linked by three or more amino acid sequences. A His Tag-carrying dimer CP protein can be prepared by fusing a His Tag (SEQ 2) to the carboxyl terminus of the second SEQ 1.

3. According to claim 1, the DNA sequence containing the pac site can be single-stranded DNA, double-stranded DNA, or a PCR product, and the pac site sequence is shown in SEQ 3.

4. According to claim 1, the dimer CP protein mixture as described in claim 2 is mixed with the DNA sequence as described in claim 3, the pH is adjusted to between 7.0 and 9.0, the mixture is allowed to stand for more than 5 minutes, DNase I is added to digest the unencapsulated DNA, and finally MS2-VLP encapsulated with exogenous DNA is obtained by nickel ion affinity chromatography purification.

5. The dimer CP protein according to claim 2 and the mixture of dimer CP proteins with His Tag at the carboxyl terminus can be obtained by lysing virus-like particles with His Tag on their surface.

6. The virus-like particles with His Tag on their surface as described in claim 5 can be expressed by transfecting a single plasmid vector into a phage display strain and obtained by ion affinity chromatography.

7. According to claim 5, the virus-like particle lysis step is as follows: mix with pre-cooled glacial acetic acid at a volume ratio of 1:2, incubate on ice for 30 minutes, centrifuge to collect the supernatant, add RNase to degrade residual nucleic acid impurities, and then adjust the pH to 7.1-7.4 with 5% Tris solution.

8. The vector of a single plasmid according to claim 6, the dimeric CP protein expressed by the key sequence as shown in SEQ 4, and the His Tag fused to its carboxyl terminus as shown in SEQ 5.

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