RU2364627C2 - Expression vector for protein synthesis in mammalian cells - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to field of biotechnology. Claimed is eucariotic vector which consists of the following elements: promoter of early genes of human cytomegalovirus, sequence of translation initiation, site of cloning, modified signal of polyadenilation of mRNA of bull growth hormone gene, signal of polyadenilation of mRNA of virus SV40 early genes, gene of resistance to ampicillin and site of Col El replication initiation, intron-sequence IVS2 from beta-globin of rabbit located between promoter and sequence of translation initiation, and post-transcription regulatory element WPRE from woodchuck hepatitis virus, located between site of cloning and signal of polyadenilation.

EFFECT: invention can be used for transfection of wide spectrum of eucariotic cells, in particular, for gene therapy.

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Description

The invention relates to the field of biotechnology, and in particular to vectors for the expression of transgenes in mammalian cells for the purpose of their use in cell engineering and in gene therapy. The vectors in accordance with this invention can be used to transfect a wide range of eukaryotic cells. They can also be used for transfection of mammalian cells in vivo, in particular for gene therapy.

Currently, plasmid vectors are one of the possible means of transgene delivery and expression of proteins. A traditional plasmid vector for transgene expression in mammalian cells (for example, the Invitrogen pcDNA3.1 vector) contains functional elements such as the early promoter of human cytomegalovirus (CMV) genes, the cloning site, the bovine growth hormone gene mRNA polyadenylation signal, and the antibiotic resistance gene G418 , β-lactamase gene providing ampicillin resistance, Col E1 replication initiation site [Invitrogen, Catalog 2002, p. 159].

In order to enhance transgene expression, it was proposed to introduce into its composition such regulatory elements as intron sequences of various genes, translation initiation sequences, post-transcriptional and transport elements, etc. [Romano G. "Current development of nonviral-mediated gene transfer." Drug News Pespect. (2007), v.20, pp. 227-231]. Each of these elements individually allows one to increase transgene expression, and the magnitude of the effect, as a rule, depends on the chosen model expression system.

As an intron sequence, in particular, the rabbit beta-globin gene IVS2 sequence is used, which is inserted into the plasmid vector immediately after the promoter [van Ooyen A., van den Berg J., Mantei N., Weissmann C. "Comparison of total sequence of a cloned rabbit beta-globin gene and its flanking regions with a homologous mouse sequence. " Science (1979), v.206, pp.337-344]. This element is used, for example, in the Clontech plasmid vector pVSV-G, for expression of the vesicular stomatitis virus coat protein [BD Biosciences Clontech, 2002 Catalog, p.128]. According to published data, a similar element allows to increase the transgene expression by 1.6 times [Yew N.S., Wysokenski D.M., Wang K.X., Ziegler R.J., Marshall J, McNeilly D., Cherry M., Osburn W., Cheng S.H. "Optimization of plasmid vectors for high-level expression in lung epithelial cells." Hum Gene Ther. (1997), v.8, pp.575-584].

It is known to use, as a post-transcriptional regulatory element, which apparently facilitates the transport of mRNA from the nucleus to the cytoplasm, the North American woodchuck hepatitis WPRE sequence, which is placed in the plasmid vector between the transgene insertion site and the polyadenylation signal. This element allows you to increase the level of gene expression by 5-8 times [Zufferey R., Donello J.E., Trono D., Hope T.J. "Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors." Journal of Virology (1999), v.73, pp. 2886-2892]. It is used, in particular, in SBI's pCDH-CMV-MCS lentiviral vector [http://www.systembio.com/cDNA_Cloning_Vectors.htm].

Closest to the claimed is the vector pGL3 -Promoter Vector by Promega [Promega, Catalog 2000, p.10.7], which is intended for expression of the luciferase gene and consists of the following elements: promoter of the early SV40 virus genes, translation initiation sequence, Photinus luciferase gene Modified codon Pyralis, SV40 late gene polyadenase signal, E. coli origin replication initiation sequence, β-lactamase gene providing ampicillin resistance, origin replication initiation sequence aha F1 and an additional synthetic polyadenylation signal. The elements of the vector are listed in the order of their arrangement. An additional functional element in this vector is the sequence of translation initiation.

The technical problem solved by the authors of the present invention was the development of a more effective eukaryotic vector, which allows to achieve a higher level of transgene expression in cells.

The technical result was achieved by creating a vector, the general scheme of which is presented in figure 1.

The vector consists of the following elements: promoter of the early human cytomegalovirus (CMV) genes, intron, translation initiation sequence, cloning site, post-transcriptional regulatory element, bovine growth hormone gene mRNA polyadenylation signal, SV40 early gene mRNA polyadenylation signal, β-lactamase gene providing ampicillin resistance, Col E1 replication initiation site. The elements of the vector are listed in the order of their arrangement.

As a translation initiation sequence, a translation initiation sequence from the Promega pGL3-Promoter Vector can be used. The rabbit beta-globin gene IVS2 sequence is used as an intron, and the modified posttranscriptional regulatory element of the woodchuck hepatitis virus (WPRE) is used as the post-transcriptional regulatory element.

The complete nucleotide sequence of the proposed vector in the case of using these elements is shown in Fig.2.

Significant differences of the claimed invention is that instead of expression vectors with traditional functional elements in the form of a promoter and a polyadenylation signal, as well as vectors of the next generation containing one additional regulatory element, which enhances transgene expression, a vector is constructed that contains three such additional elements at once whose effects can be mutually reinforced. In particular, in the closest vector pGL3-Promoter Vector two of these elements are missing, namely the intron and the post-transcriptional regulatory element.

Functional diagram of the proposed vector is shown in figure 1.

The essence and industrial applicability of the invention (in biotechnology and gene therapy) are illustrated by the following examples.

Example 1. The creation of the plasmid vector pC4W for highly efficient transgene expression.

The vector was obtained using standard genetic engineering methods, commercially available plasmids, and chemically synthesized oligonucleotides [Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. 2nd ed. New York, NY: Cold Spring Harbor Laboratory Press; 1989].

The vector consists of the functional elements listed below in the order of their arrangement:

1) the promoter of the early genes of human cytomegalovirus (CMV) is intended for transcription of mRNA encoding a transgenic protein;

2) the intron IVS2 of the rabbit beta-globin gene is intended for splicing mRNA and enhancing transgene expression;

3) the translation initiation sequence is intended for efficient binding of ribosomes;

4) the cloning site is designed to embed a DNA sequence encoding a transgenic protein;

5) a modified posttranscriptional regulatory element of the woodchuck hepatitis virus (WPRE) is designed to efficiently transport mRNA from the nucleus to the cytoplasm and enhance transgene expression;

6) the mRNA polyadenylation signal of the bovine growth hormone gene is designed to correctly process mRNA and add the poly (A) sequence to the 3'-end of the mRNA;

7) the mRNA polyadenylation signal of the early SV40 virus genes is intended to improve mRNA processing;

8) the gene for resistance to ampicillin (β-lactamase) is intended for the selection of bacteria containing the pC4W vector during its production;

9) Col E1 replication initiation site is designed to replicate the pC4W vector in E. coli bacteria.

Functional diagram of the vector pC4W indicating the coordinates of the functional elements is presented in figure 1, and the complete nucleotide sequence of this vector is shown in figure 2. The arrangement of functional elements is essential for the effective operation of the vector.

Example 2. Comparison of two vectors that differ in the presence of one additional functional element (sequence of translation initiation from the vector pGL3-Promoter Vector by Promega).

HEK293 cells seeded in the wells of a 24-well plate (50,000 cells per well) in DMEM medium containing 2% fetal calf serum were transfected with pcDNA3-Luc or pcDNA3-K-Luc vectors carrying the luciferase cDNA sequence. The pcDNA3-K-Luc vector also contains a translation initiation sequence from the pGL3-Promoter Vector of Promega (K). Transfection was performed using 1 μg DNA and 1 μl Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's protocol. 24, 48 and 72 hours after transfection, the cells were lysed and luciferase activity was measured using a Beckton-Dickinson kit and a LKB 1250 luminometer. The measurements were carried out in 3 parallels and the mean value and standard deviation were determined. The data obtained are presented in figure 3.

Example 3. Comparison of vectors with a different set of additional functional elements.

HEK293 cells seeded in the wells of a 24-well plate (50,000 cells per well) in DMEM containing 2% fetal calf serum were transfected with the following vectors: pCMV-K-GL3 (containing the translation initiation sequence as the only additional functional element, K) , pCMV-INT-K-GL3 (containing, in addition to element K, a second additional element — an IVS2 sequence from the rabbit beta globin gene, INT), pCMV-K-GL3-WPRE (containing, in addition to element K, a second additional element — a modified post-transcript woodchuck hepatitis virus element, WPRE) and pCMV-INT-K-GL3-WPRE (containing all three additional functional elements - K, INT and WPRE). Transfection was performed using 1 μg DNA and 1 μl Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's protocol. 24, 48 and 72 hours after transfection, the cells were lysed and luciferase activity was measured using a Beckton-Dickinson kit and a LKB 1250 luminometer. The measurements were carried out in 3 parallels and the mean value and standard deviation were determined. The data obtained are presented in figure 4.

Example 4. The ability of plasmid vectors pC4W and pcDNA3 (Invitrogen) to express luciferase from Firefly Photinus pyralis in human kidney cell culture HEK293 was compared.

HEK293 cells seeded in wells of a 24-well plate (50,000 cells per well) in DMEM medium containing 2% fetal calf serum were transfected with pC4W-Luc or pcDNA3-Luc vectors carrying the luciferase cDNA sequence. Transfection was performed using 1 μg DNA and 1 μl Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's protocol. 24, 48 and 72 hours after transfection, the cells were lysed and luciferase activity was measured using a Beckton-Dickinson kit and a LKB 1250 luminometer. The measurements were carried out in 4 parallels and the mean value and standard deviation were determined. The data obtained are presented in figure 5.

Example 5. The ability of plasmid vectors pC4W and pcDNA3 (Invitrogen) to express human urokinase in human HEK293 kidney cell culture was compared.

HEK293 cells seeded in the wells of a 24-well plate (50,000 cells per well) in DMEM containing 2% fetal calf serum were transfected with pC4W-UPA or pcDNA3-UPA vectors carrying the human urokinase cDNA sequence as well as the control vector pC4W. Transfection was performed using 1 μg DNA and 1 μl Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's protocol. 24, 48 and 72 hours after transfection of the cell, the culture medium was collected and replaced with fresh. The accumulation of urokinase in the culture medium was analyzed for every 24 hours between medium shifts. The urokinase inhibitor present in fetal calf serum was inactivated by adding hydrochloric acid to the medium to pH 3.0 for 15 min, after which the medium was neutralized by adding a NaOH solution to pH 7.5. Urokinase activity was measured in the medium thus treated using the chromogenic substrate S-2444 (Sigma). The measurements were carried out in 4 parallels and the mean value and standard deviation were determined. The data obtained are presented in Fig.6.

Example 6. The ability of plasmid vectors pC4W and pcDNA3 to express the red fluorescent protein DsRed-Express from Discosoma marine anemone in cells in a HEK293 human embryo kidney cell culture was compared.

HEK293 cells seeded in the wells of a 24-well plate (50,000 cells per well) in DMEM medium containing 2% fetal calf serum were transfected with pC4W-DsRed or pcDNA3-DsRed vectors carrying the modified DsRed cDNA sequence, as well as the pC4 control vector. Transfection was performed using 1 μg DNA and 1 μl Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's protocol. 24, 48, and 72 hours after transfection, cells were harvested, fixed with 1% formaldehyde and the average red fluorescence level was measured at an excitation wavelength of 488 nm using a FACS Calibur flow cytometer (BD). The measurements were carried out in 4 parallels and the mean value and standard deviation were determined. The obtained data are presented in Fig.7.

Claims (1)

A eukaryotic vector for expression of transgenes in eukaryotic cells, comprising a promoter of early human cytomegalovirus genes, a translation initiation sequence, a cloning site, a modified bovine growth hormone gene mRNA polyadenylation signal, an SV40 virus early mRNA polyadenylation signal, an ampicillin resistance gene and an El replication initiation site characterized in that it also further comprises an intron sequence of the IVS2 from the rabbit beta-globin gene located between the promoter ohm and translation initiation sequence and posttranscriptional regulatory element WPRE of HCV North American woodchuck disposed between cloning site and polyadenylation signal.

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