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Lentiviral expression vector, as well as preparation method and application of lentiviral expression vector, and preparation method of recombinant lentivirus

A technology of recombinant lentivirus and expression vector, which is applied in the direction of virus/bacteriophage, botanical equipment and methods, biochemical equipment and methods, etc., and can solve problems such as inconsistent expression levels

Inactive Publication Date: 2016-04-13
SHENZHEN GENTARGET BIOTHERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two methods can realize the expression of double genes, but there are two main disadvantages: 1. The expression levels of genes located upstream and downstream of the vector are inconsistent
There is no lentiviral vector that can solve the above problems in the prior art

Method used

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  • Lentiviral expression vector, as well as preparation method and application of lentiviral expression vector, and preparation method of recombinant lentivirus
  • Lentiviral expression vector, as well as preparation method and application of lentiviral expression vector, and preparation method of recombinant lentivirus
  • Lentiviral expression vector, as well as preparation method and application of lentiviral expression vector, and preparation method of recombinant lentivirus

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0064] Such as figure 2 The method for preparing the aforementioned lentiviral expression vector includes the following steps:

[0065] S10. Design the dual gene expression box Dual, and commission the synthesis of the dual gene expression box Dual to obtain the E. coli bacteria liquid containing the pUC-Dual plasmid.

[0066] The synthesis of the dual-gene expression box Dual was commissioned by a commercial company to directly obtain the E. coli bacteria liquid containing the pUC-Dual plasmid.

[0067] The pUC-Dual plasmid contains the dual gene expression box Dual. The dual gene expression box Dual includes the following structures arranged in sequence from 5'to 3': the first multiple cloning site-furin cleavage site-V5 tag-Spacer- 2A peptide-second multiple cloning site, "-" stands for connection.

[0068] S20. Mix the Escherichia coli broth containing pUC-Dual plasmid obtained from S10 with selective LB liquid medium, and incubate in a constant temperature shaker at 37°C at 300r...

Embodiment 1

[0098] Example 1 Design of double gene expression cassette

[0099] The core of the dual gene expression cassette is to express 2A peptide (SEQIDNo.7), which has the first multiple cloning site (SEQIDNo.2), 2A peptide (SEQIDNo.7) and the second multiple cloning site (SEQIDNo.3). It can be used to achieve equal expression of double foreign genes. However, the expression of the foreign gene obtained by this method is not high, and 13 amino acid residues derived from the 2A peptide will be added to the end of the polypeptide sequence upstream of the 2A peptide, which may have a certain impact on the characteristics of the polypeptide .

[0100] Further, inserting a Spacer (SEQ ID No. 6) between the first multiple cloning site of the double gene expression cassette in the previous step and the 2A peptide can effectively increase the expression level of the foreign gene.

[0101] Further, insert a furin cleavage site (SEQ ID No. 4) between the first multiple cloning site of the double g...

Embodiment 2

[0104] Example 2 Construction of pRRLSIN.cPPT.MSCV-Dual.WPRE vector

[0105] Shanghai Shenggong Bioengineering Technology Service Co., Ltd. was entrusted to synthesize the sequence of the double gene expression cassette, and the obtained sequence was contained in the pUC57-DGEC vector in E. coli. The Escherichia coli was expanded and cultured, and the pUC57-DGEC vector was extracted, and then the purity and concentration were determined. The results are shown in the following table.

[0106] Purity and concentration of pUC57-DGEC vector

[0107] Recombinant vector

A260 / A280

Concentration (ng / μL)

pUC57-DGEC

1.93

626.3

[0108] The recombinant pUC57 vector and pRRLSIN.cPPT.MSCV / GFP.WPRE vector were double digested with AscI and SalI, and the small fragments in the digested product of the recombinant pUC57 vector and pRRLSIN.cPPT.MSCV / GFP.WPRE vector were recovered after electrophoresis. Large fragments of digested products. According to the fragment of pUC51 vector digestion...

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Abstract

The invention discloses a lentiviral expression vector, as well as a preparation method and an application of the lentiviral expression vector, and a preparation method of a recombinant lentivirus. The lentiviral expression vector is a pRRLSIN.cPPT.MSCV-Dual.WPRE vector, which is obtained by replacing the GFP sequence of a pRRLSIN.cPPT.MSCV / GFP.WPRE vector with a double gene expression box Dual, wherein the double gene expression box Dual comprises the following structure sequentially arranged from the 5' terminal to the 3' terminal: a first polyclonal site-a furin cleavage site-a V5 label-Spacer-2A peptide-a second polyclonal site, the mark '-' represents the connection relation. According to the lentiviral expression vector, 2A peptide is utilized for realizing the expression of double exogenous genes in cells of mammals, 2A peptide has the 'self-shearing' property, in the translation process of protein, 2A peptide changes the activity of ribosome, promotes the hydrolysis of ester chains between peptide residue Gly and tRNAGly of 2A, releases upstream polypeptides and starts the translation of upstream polypeptides from a transcribed compound, and the realizes double gene expression in the level of translation.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a lentivirus expression vector, a preparation method and application thereof, and a preparation method of a recombinant lentivirus. Background technique [0002] Gene expression is the process by which cells transcribes and translates genetic information stored in DNA sequences into protein molecules with biological activity during their life. Through genetic engineering, the expression of exogenous genes in biological cells and even organisms can be achieved, so as to achieve the purpose of artificially regulating the properties of cells or solid objects. [0003] The introduction of exogenous genes in eukaryotic cells, especially mammalian cells, is mainly accomplished through physical, chemical or biological methods. Physical methods mainly include DNA microinjection, electroporation, and metal particle bombardment methods; chemical methods mainly include liposome-mediated and r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N7/01
CPCC12N7/00C12N2740/15041C12N2840/10C12N15/66C12N15/867
Inventor 杨世成毛侃琅
Owner SHENZHEN GENTARGET BIOTHERAPEUTICS CO LTD
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