Sindbis virus overall length plasmid type carrier and its structuring method

A construction method and plasmid technology, applied in the direction of viruses/phages, vectors, viruses, etc., can solve the problems of poor RNA preservation, cumbersome operation process, limited promotion and application, etc. Effect

Inactive Publication Date: 2017-10-17
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before use, the main carrier and the auxiliary carrier need to be transcribed into RNA in vitro, and then co-transfected into cells. This operation process is cumbersome and the RNA is not easy to preserve. The cost is high, which limits its popularization and application.

Method used

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  • Sindbis virus overall length plasmid type carrier and its structuring method
  • Sindbis virus overall length plasmid type carrier and its structuring method
  • Sindbis virus overall length plasmid type carrier and its structuring method

Examples

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Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction of the full-length plasmid vector pSinRep5-DB-ESP of Sindbis virus

[0050] 1. Construction of Sindbis virus plasmid vector pSinRep5-ESP regulated by ESP strong promoter

[0051] 1) Design ESP primers

[0052] Design a pair of primers based on the ESP promoter sequence on the PEGFP-ESP plasmid to amplify the full-length ESP promoter sequence and introduce a SacI restriction site. The sequence is as follows, and the italicized restriction site is the restriction site.

[0053] ESP-F: 5'-CATCC ATGCATTAGTTATTAATAGTAATCAATTAC-3';

[0054] ESP-R: 5'-GCATC CCGGTCCGTAGGCACGTCT-3';

[0055] Among them, the parts in italics are enzyme cutting sites, and the primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0056] 2) PCR amplification

[0057] Using the PEGFP-ESP plasmid as a template and the primer pair in step 1) as primers, a PCR amplification reaction was performed, and finally 8 μL of the PCR product was taken for electroph...

Embodiment 2

[0076] Example 2 Verification of the function of the Sindbis virus full-length plasmid vector

[0077] After analyzing the structure of each gene of PRRSV, the GP5-M complex gene was selected as the exogenous gene to verify the function of the Sindbis virus full-length plasmid vector, as follows:

[0078] 1. Design and synthesis of PRRSV-GP5 gene primers and extraction of PRRSV total RNA

[0079] Primers were designed according to the GP5-M gene sequence published on Genebank, and XbaI, StuI and Kozak sequences were introduced, marked as GP5-F and GP5-R, and the primer sequences were as follows:

[0080] GP5-F: 5'-CACCTCTAGAGCCACCATGTTGGGGAAGTGCTTGACCG-3';

[0081] GP5-R: 5'-GCATAGGCCTTCGAGGCTGCTTGCCGTTGTTAT-3';

[0082] Primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0083] PRRSV total RNA was extracted according to the instructions of Trizol reagent.

[0084] 2. RT-PCR reaction

[0085] First, cDNA is synthesized from RNA by reverse transcription ...

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Abstract

The invention discloses a Sindbis virus overall length plasmid type carrier and its structuring method. The Sindbis virus overall length plasmid type carrier is an expression carrier recombined and structured by a main carrier PsinRep5 and an assistant carrier DH-BB and ESP promoter; firstly, ESP promoter is lead in a main carrier PsinRep5 of the Sindbis virus, and then inserted to DH-BB structure protein gene to obtain the Sindbis virus overall length plasmid type carrier pSinRep 5-DB-ESP; the carrier can directly transfect DNA to the cell, thus the operation of the virus carrier for expressing an exogenous gene is greatly simplified, thus the carrier can be applied to gene expression and also transformation of other viruses; the structuring method has the advantages of being simple in operation, good in safety, able to avoid appearance of recombined wild virus, low in cost, and convenient to popularize and apply.

Description

technical field [0001] The invention belongs to the field of vector construction, and in particular relates to a Sindbis virus full-length plasmid vector and a construction method thereof. Background technique [0002] Sindbis virus (SINV) belongs to the alphavirus genus of the Togaviridae family and is a representative strain of the alphavirus genus. It is an arbovirus transmitted by blood-sucking mosquitoes and the like. It can cause "Sindbis syndrome syndrome", manifested as fever, Burnout, headaches, joint pains and skin rashes, etc. can be cured by themselves. [0003] Sindbis virus has a wide range of hosts, but gene therapy requires the virus to be targeted. For this, Ohno et al. constructed a SINV vector with Staphylococcus aureus protein A chimerized on the spike protein. The targeting of the vector can be achieved by This is accomplished by altering the IgG targeting various cells. Because SINV has strong apoptosis-inducing effect and high-efficiency foreign gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/66
CPCC12N15/86C12N2770/36143C12N2830/34
Inventor 刘成倩易建中李红
Owner SHANGHAI ACAD OF AGRI SCI
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