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Pseudovirion vector and preparation method and application thereof

A pseudovirus and vector technology, applied in the field of genetic engineering, can solve the problems of research affecting the quantification of pseudovirus particles, large loss of RNA, false positives in PCR, etc. Effect

Active Publication Date: 2013-05-15
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is cumbersome to operate and requires high equipment (requires a high-speed low-temperature centrifuge). More importantly, there is still a certain amount of nucleic acid in the solution containing pseudovirus particles recovered by gradient centrifugation, and the residual nucleic acid is identified by PCR as containing pseudovirus. During the purity of the particle solution, it is easy to cause false positives in PCR, which further affects the quantitative study of pseudoviral particles.
Although some researchers extract RNA from the purified solution containing pseudovirion particles, and then perform DNase I enzyme digestion to remove residual nucleic acid interference, this will cause a large loss of prepared RNA and also cause inaccurate quantification in the next step

Method used

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  • Pseudovirion vector and preparation method and application thereof
  • Pseudovirion vector and preparation method and application thereof
  • Pseudovirion vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Propseudoviral vector pTrcHis-MS2 construction

[0048] 1) Extract MS2 phage (ATCC 15597-B1, purchased from ATCC, USA) RNA, refer to the one step RT-PCR kit for RT-PCR amplification, recover and purify the PCR product and connect it into pGEM-T Easy Vector to construct MS2-T plasmid. The MS2-T plasmid (preserved in our laboratory) and the expression vector pTrcHis2A (purchased from Invitrogen) were respectively digested with NcoI and BglII, and the enzyme digestion system was as follows: NcoI / BglII 10U, 10×K buffer 2ul, 0.1%BSA 2ul, Template 1μg, add water to 20ul. 37°C 2h. The digested products were gel-recovered and purified, and the products were named MS2-T(N / B) and pTrcHis2A(N / B), respectively.

[0049] 2) Ligate the digested product with T4 DNA ligase, the ligation system is as follows: 10×buffer 1ul, T4DNase 0.5ul, pTrcHis2A(N / B) 50ng, MS2-T(N / B) 120ng, 25°C, 10min, Then immediately put on ice.

[0050] 3) Transform the ligation product, pick a sin...

Embodiment 2

[0051] Example 2 Construction of Pseudovirus Vector pTrcMS

[0052] Using the method of gene insertion, the 6His sequence is inserted between 15-16 amino acids of the coat protein gene coding sequence in MS2 on the pTrcHis-MS2 vector to obtain the pseudoviral vector pTrcMS, and its specific construction method is as follows:

[0053] ① Introduce mutated bases into the PCR primers. The former pseudovirus vector pTrcHis-MS2 was used as a template, and Primer1 and Primer2 were used as primer pairs respectively. STAR HS DNA Polymerase was used for PCR amplification respectively, and the amplified products were named as PCR products I and II. Wherein, the PCR amplification primers are:

[0054] Primer1-F: 5'-AGGTAACATGCTCGAGGGCCTT-3',

[0055]Primer1-R: 5'-GACATCACCATCACCATCACACTGGCGACGTGGCTGTCGCCCCA-3';

[0056] Primer2-F: 5'-GTGTGATGGTGATGGTGATGTCCGCCATTGTCGACGAGAACG-3',

[0057] Primer2-R: 5'-GTTCGGGCCCAAGCTTCGAATTCCC-3'.

[0058] The reaction system for 50ul PCR amplifica...

Embodiment 3

[0066] The development of embodiment 3 Nipah (NP) pseudovirus particles

[0067] 1. Construction of pTrcMS-NP plasmid

[0068] The pGEM-T-NP plasmid and the pTrcMS plasmid prepared in Example 2 were digested with pstI and HindIII respectively, gel recovered and purified, ligated and sequenced to construct pTrcMS-NP. For the specific method, refer to the construction method of the propseudovirus vector pTrcHis-MS2 in Example 1.

[0069] 2. Purification and preparation of pTrcMS-NP pseudovirus particles

[0070] The pTrcMS-NP recombinant bacteria were induced to express, the expression product was treated as in Example 2, and the recovered pseudovirus particles were identified by PCR and RT-PCR respectively. The electrophoresis of PCR identification products showed no target bands, and the electrophoresis of RT-PCR identification products showed obvious target bands, indicating that the solution contained Nipah pseudovirus particles and no DNA contamination.

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Abstract

The invention discloses an animal pathogen microbial pseudovirion vector and a preparation technology thereof. The gene coding sequences of a maturase protein and a coat protein of an MS2 phage and a cDNA (complementary Deoxyribose Nucleic Acid) sequence which corresponds to a 5' non-coded sequence of a gene regulating element sequence included by a genome part are connected to the downstream of a pTrcHis2A vector promoter, so that a pseudovirion vector is constructed. A 6His purification tag is added between the fifteenth and sixteenth amine acids of the coat protein in the MS2 phage througha gene insertion method, a pseudovirion particle comprising an exogenous gene is expressed into an RNA (Ribonucleic Acid)-protein complex, and the pseudovirion particle is captured with a protein purifying method, so that the complex operating process for preparing the pseudovirion particle can be simplified, and the purification quality of the pseudovirion particle is enhanced simultaneously. The pseudovirion vector is pTrcMS of which the nucleotide sequence is shown as SEQ ID No.1.

Description

technical field [0001] The invention relates to the field of genetic engineering, and relates to a preparation method and application of an animal pathogenic microorganism pseudovirus vector. Background technique [0002] At the end of the 20th century, Pasloske et al. proposed a new RNA quality control product preparation technology, that is, armored RNA (Armored RNA) technology (Pasloske B L, et al.Armored RNAtechnology for production of ribonuclease-resistant viral RNA controls and standards[J ].J Clin Microbiol, 1998, 36(12): 3590-3594; Walker Peach CR, et al.Ribonuclease resistant RNA controls (Armored RNA) for reverse transcription-PCR, branched DNA, and genotyping assays for hepatitis C virus[ J]. Clin Chem, 1999, 45(12): 2079-2085). This technology solves the problems of traditional RNA quality control products (or poor stability, or hidden dangers of biological infection, or failure to achieve the purpose of monitoring nucleic acid extraction and reverse transcript...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12R1/92
Inventor 吴绍强邓俊花林祥梅张永宁王彩霞
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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