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Expression vector

A technology for expression vectors and variants, applied in the field of expression vectors, can solve the problems of death, inability to achieve long-term expression, etc.

Active Publication Date: 2016-01-13
CANTONBIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very obvious defects in this method, that is, usually after the late (late) or very late (verylate) promoter starts to express, the host cell will die quickly within two or three days, and the purpose of long-term expression cannot be achieved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Host cells

[0076] Tn-5 cell line: BTI-TN-5B1-4 cell line (also known as HighFive or Tn-5) from Trichoplusiani, adapted to suspension culture and protein-free medium.

[0077] Proliferation of Tn-5 cells:

[0078] Tn-5 cells were obtained from commercial sources (LifeTechnologies, Basel, Switzerland) and cultured in suspension in SF900IISFM medium (LifeTechnologies). Cells were cultured in TubeSpin Bioreactor 50 (TS50) or in TubeSpin Bioreactor 600 (TS600) (TPP, Trasadingen, Switzerland) in volumes of 10 mL or 300 mL of the respective medium. Cells were passaged 3 times a week at a density of 1-5x105 cells / mL. All cultures were maintained at a temperature of 28° C. in an ISF1-X shaking incubator (Kühner AG, Birsfelden, Switzerland) with a shaking speed of 180 rpm and a shaking diameter of 5 cm. Cell density and viability were determined by trypan blue exclusion using a Neubauer hemocytometer.

Embodiment 2

[0079] Example 2. Plasmid preparation

[0080]PIEx-XEGFP and PIEx-TNFR-Fc (Table 1) carry the genes of green fluorescent protein and human TNFR-Fc respectively, and are regulated by the Autographacalifornica multicapsidnucleopolyhedrovirus (AcMNPV) homology region 5 (hr5) enhancer and Lizao 1 (ie1) promoter controls, which have been described in a previous article (Shen et al., 2014). Using PIB / V5-His (Life Technologies) as a template, the promoters of Orgyiapseudotsugatamulticapsidnuclearpolyhedrosisvirus (OpMNPV) Opie1 (Opie1) and 2 (Opie2) were amplified by PCR using specific oligonucleotide primers (Table 2). Individual PCR products were digested with XmaI and NotI and subcloned into PIEx–XEGFP digested with the same two restriction enzymes, yielding pOPIE1-EGFP and pOPIE2-EGFP, respectively (Table 2). For these two plasmids, the AcMNPVhr5 enhancer and the iel promoter of PIEx-XEGFP could be replaced by Opie1 or Opie2 promoters, respectively. The metallothionein (MT) pro...

Embodiment 3

[0081] Example 3. Vector Construction

[0082] In the initial transfection of Tn-5 cells, we used an expression vector carrying the AcMNPVie1 promoter and hr5 enhancer. In order to study other promoters, we constructed expression vectors with Opie1, Opie2, and MT promoters, respectively. In addition, vectors carrying Opie1 promoter and AcMNPVhr5 enhancer, and vectors carrying Opie2 promoter and AcMNPVhr5 enhancer were also constructed. The vector carries either the EGFP gene or the TNFR-Fc gene and is constructed from the same starting vector PIEx-X (Shen et al., 2014). We also tested two other vectors: pUC-actEGFP, in which the EGFP gene is controlled by the Drosophila actin 5C promoter, and pMYKEF1-EGFP-puro, which is a vector carrying the EGFP gene (regulated by the hCMV- / enhancer control) mammalian expression vector. The pMYKEF1-EGFP-puro vector was reported to be efficiently expressed in Drosophila cells (Qin et al., 2010).

[0083] At the beginning, the cells were a...

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Abstract

The invention belongs to the field of a biology technology, and relates to an expression vector. The expression vector is an expression vector for insect, and contains an AcIE1 promoter of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and a hr5 enhancer of a homologous region 5 and an Opie2 promoter of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). An insect host cell containing the expression vector and a method for preparing a recombinant protein by using the expression vector are provided.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to an expression vector. Background technique [0002] Several cultured insect cell lines are important hosts for heterologous expression of recombinant proteins in virus-like particles (VLPs) for various research applications and as vaccines (Cherezov et al., 2007; Imasaki et al., 2011; Rasmussen et al., 2007; White et al. et al., 2012), (Metz and Pijlman, 2011; Shrestha et al., 2007; Treanor et al., 2011; Treanor et al., 2011). These processes mainly utilize the baculovirus expression vector system (BEVS) to infect cell lines derived from Lepidoptera. (Berger et al., 2004; Jarvis, 2009; Kost et al., 2005; Summers, 2006). The most commonly used hosts are Sf21 and Sf9 from Spodopterafrugiperda and the BTI-TN-5B1-4 cell line (also known as HighFive or Tn-5) from Trichoplusiani. These cells can grow to very high cell densities in suspension culture and are capable of performing post-transla...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12P21/00
Inventor 沈潇林兴华
Owner CANTONBIO CO LTD
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