Pseudovirion particle and preparation method and application thereof

A pseudo virus particle and retrovirus technology, applied in the field of pseudo virus particles and their preparation, can solve the problems of unreported non-infectivity, safety and reliability, etc.

Pending Publication Date: 2019-03-01
海南出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is also not reported to use a non-infectious, safe, reliable, high stability, and specific external standard positive quality control product containing monkey D-type retrovirus

Method used

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  • Pseudovirion particle and preparation method and application thereof
  • Pseudovirion particle and preparation method and application thereof
  • Pseudovirion particle and preparation method and application thereof

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preparation example Construction

[0044] In another embodiment of the present invention, the present invention provides a kind of preparation method of pseudovirion, comprising:

[0045]S1. Using the first primer pair directed at the MS2 phage genome, and using the MS2 phage gene as a template, perform RT-PCR amplification to obtain the first amplified product;

[0046] S2. Cloning the first amplification product described in step S1 to obtain the first cloning plasmid, and using restriction endonucleases Nco I and BamH I to double digest the first cloning plasmid and plasmid pET-28b(+ ), connect after purification to obtain the first connection product; wherein the cloning is to connect the first amplified product to the clone T vector pMD18-T to construct the pMD18-T-MS2 cloning plasmid; the purification is to combine the enzyme Gel recovery and purification of the cleaved product; the connection can be carried out using the Ligation kit ligase kit;

[0047] S3. Transform the first ligation product describe...

Embodiment 1

[0069] Embodiment 1: the design and construction pMD18-T-MS2 cloning plasmid of the primer pair of the MS2 gene of amplifying MS2 bacteriophage

[0070] Utilize OLIGO7.0 software, refer to the MS2 phage gene sequence (NC_001417) in the GenBank database to design the effective gene sequence 11-1699nt (total 1689bp) that comprises the coding assembly protein of MS2 and envelope protein, simultaneously according to this gene design primer pair MS2F, MS2R, and artificially synthesize it. The designed primer pairs are shown in Table 1 below:

[0071] Table 1. Nucleotide sequences of MS2 gene amplification primers

[0072]

[0073] According to routine operations, using the above primer pair MS2F and MS2R, PCR amplification was performed using the MS2 gene as a template to obtain an amplified product containing the gene sequence encoding the assembly protein and envelope protein of MS2, and the amplified product was connected to the clone T Vector pMD18-T, construct pMD18-T-MS2...

Embodiment 2

[0074] Example 2: Construction of recombinant plasmid pET-28b-MS2

[0075] The cloning plasmid pMD18-T-MS2 constructed above and the empty plasmid pET28b(+) expression vector (both kept in our laboratory) were double-digested with NEB fast enzymes NcoI and BamHI, respectively. The enzyme digestion system used is shown in Table 2 below:

[0076] Table 2. Enzyme digestion system

[0077]

[0078] After enzyme digestion at 37°C for 15 minutes, the target products of enzyme digestion were gel recovered and purified, and the products were named MS2-T(N / B) and pET28b(N / B) respectively.

[0079] Ligate the digested products MS2-T(N / B) and pET28b(N / B) recovered above with the Ligation kit. The connection system is shown in Table 3 below:

[0080] Table 3. Connection system

[0081]

[0082] Immediately after connecting at 16°C for 30 minutes, ice bath. The ligation product was transformed into Escherichia coli BL21(DE3)pLysS competent cells, plated, single clone colonies wer...

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Abstract

The invention discloses a pseudovirion particle and a preparation method and application thereof. The pseudovirion particle comprises RNA fragments of a monkey type D retrovirus encapsulated by MS2 phage capsid protein. The preparation method of the pseudovirion particle is simple, operation is easy, fineness of the obtained pseudovirion particle is high, and the pseudovirion particlecan be used as an external standard quality control product of a nuclein detecting method of monkey type D retrovirus detection. The method has the characteristics of high stability and nuclease resistance, a target template sequence exists only in the form of RNA, infectivity is avoided, and security is high.

Description

technical field [0001] The invention relates to a pseudovirion particle and a preparation method thereof, in particular to a pseudovirus particle containing a monkey D-type retrovirus RNA fragment, a preparation method thereof and an application in detecting monkey D-type retrovirus nucleic acid. Background technique [0002] Simian AIDS Type D Retrovirus (SRV) belongs to Retroviridae, Oncovirinae, Oncovirus D, and has multiple serotypes SRV-D1, 2, 3, 4, 5. Rhesus macaques in Asia are the main host of SRV, with characteristics of endemic infection. The disease manifests as a series of subclinical symptoms, opportunistic infection and tumor formation, which eventually lead to immunosuppression, similar to AIDS. SRV can cause disease in its natural host Asian macaques, and its prevalence is very high in some monkey groups. Once an outbreak occurs, it may cause a large number of deaths in the entire monkey group. Therefore, regular census of SRV infection must be carried out on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02C12N15/70C12Q1/70C12Q1/686
CPCC12N7/00C12N15/70C12Q1/686C12Q1/701C12N2740/10051C12N2740/10023C12Q2600/166
Inventor 李丹丹高慎阳张体银凌宗帅邱索平刘忠梅王绥家
Owner 海南出入境检验检疫局检验检疫技术中心
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