JPH06277074A - Expression vector - Google Patents

Abstract

PURPOSE:To provide a new expression vector useful for getting a desired protein in sufficiently high production efficiency. CONSTITUTION:The expression vector (e.g. pACELltat) is composed of a promoter, a 1st cistron coding the objective protein, a 2nd cistron coding a protein having autocatalytic promotion action of the promoter and an internal translation initiation signal positioned between both cistrons. The exemplified expression vector can be produced by constructing an IRES of an HCV group at the downstream side of an HIV-1LTR promoter originated from HIV virus, a luciferase gene as an upstream side cistron interposing the IRES and a tat gene as a downstream side cistron of the IRES by conventional construction process.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel expression vector capable of efficiently producing a target protein.

[0002]

2. Description of the Related Art The development of expression vectors in eukaryotic cells is an important subject that underlies many molecular biological studies. Depending on the purpose, efficient large-scale expression or inducible transient expression is required. To meet such demands,
In these expression systems, the conventional concept of constructing an expression vector depends on the innate functions of the promoter and enhancer. That is, as a means for increasing the production efficiency of a target protein, a strong promoter or enhancer is searched for in a natural gene, and the DN of the target protein is searched for.
It has been exclusively performed to increase the transcription efficiency of mRNA by linking to A. For efficient large-scale expression, SV40 (Simian Virus 40) or SR
Promoters such as α are known as strong promoters, and expression vectors incorporating these promoters are mainly used for mammalian cells. In addition, it has also been attempted to increase the transcription efficiency of a gene and the protein production efficiency by incorporating an enhancer sequence into a vector. On the other hand, in the case of performing transient expression, a method using an expression inducer is known, for example, mouse papillary cancer virus (MMTV = Mouse).
Dexamethason (dexamethason), an expression inducer, in an expression system using the Mammalian Tumour Virus promoter
Methods such as addition when e) is desired to be expressed are used.

[0003]

However, the expression vectors obtained by these methods do not always provide sufficient production efficiency, and there has been a long-felt demand for the appearance of more efficient expression vectors. Therefore, it is an object of the present invention to provide a novel expression vector which is not bound by the conventional concept of constructing an expression vector in order to increase the production efficiency of the target protein.

[0004]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have conducted extensive studies and as a result, have found a promoter, a first cistron encoding a target protein, and a protein having an autocatalytic enhancing effect on the promoter. The above problems have been solved by an expression vector having a second cistron that encodes and an internal translation initiation signal provided between the first cistron and the second cistron.

The positional relationship between the first cistron and the second cistron is that the first cistron is on the upstream side,
It is preferable that the second cistron is on the downstream side. In addition, in the combination of the above promoter and the second cistron, the above promoter is the HIV-1 HIV-1 LTR promoter derived from the HIV virus, and the second cistron is t.
It is preferably atDNA. Furthermore, the internal translation initiation signal is preferably derived from hepatitis C virus. The above problem can also be solved by a transformant transformed with the above expression vector.

The expression vector of the present invention will be described in detail below. The present invention is directed to a promoter and its autocatalytic expression effect.
ction) and the properties of the internal translation initiation signal. In general, "autocatalysis" means that a product promotes the reaction itself in an enzymatic reaction, but "autocatalytic enhancing action" in the present invention means that a gene product whose expression is controlled by a promoter is a promoter. It means to enhance the activity of and to promote gene expression.

The promoter and the protein having its autocatalytic enhancing action include, for example, the promoter of the AIDS (hereinafter referred to as HIV) virus, the tat protein, and HT.
A combination of LV promoter and tax protein is known. These promoters explosively enhance the promoter activity when the tat protein or the tax protein is present in a trace amount. The mechanism of the promoter and the protein having the autocatalytic enhancing action will be described by taking the combination of the HIV virus promoter and the tat protein as an example. The tat protein is a transcriptional activator that acts on trans-activation of HIV, and is a mR transcribed from the HIV gene.
It is known that tat protein attaches to the stem-loop structure called TAR-RNA from the 1st to 57th positions on the 5'side of NA to promote transcription (Haseltine.WA et a
l .: in Harvard AIDS Institute Series on Gene Regul
ation of Human Retroviruses, Vol.1, Raven Press, New
York 1991). The DNA sequence of this tat protein is A
See rya et al. Science 229 69-73, 1985.

Further, an internal translation initiation signal (IRES =
Internal Ribosome Entry Site) is a signal found in picornaviruses, etc., and is 1 in hepatitis C virus.
Was discovered by Kyoko Ohara et al. In 992 years (J.Viol. 66 .1
476-1483 1992). The initiation of protein synthesis in mRNA includes the initiation of cap-dependent translation that is usually performed by eukaryotic cells, and the initiation of independent translation that is observed in the above picornavirus and the like. The cap-dependent initiation of translation is a prerequisite that the ribosome bound to the mRNA recognizes the cap structure at the initiation of translation.
Initiate RNA translation. On the other hand, cap-independent translation initiation is thought to occur when the ribosome enters a certain binding point upstream of the translation initiation point, moves to reach the translation initiation point, and initiates translation. Signals are said to be one of the ones that initiate cap-independent translation. Next, the structure of the internal translation initiation signal is shown as hepatitis C virus (hereinafter referred to as HCV).
The internal translation initiation signal of will be described as an example. HCV
The genome of is a single-stranded positive RNA, about 10,000
It consists of nucleotides. This RNA is about 301
It is the only open reading frame (ORF = Open reading frame) for the synthesis of large single-chain proteins consisting of 0 amino acids.
HCV carries out independent translation initiation, and the ribosome is located upstream of the translation initiation site, that is, 5'UTR (untranslated region).
It was found that the ribosome binds to the Initial-Sequence of E.coli and the translation starts when the ribosome moves to reach the translation start point, and that sequence was identified as IRES (Kyoko Ohara et al. J. Viol. .66 .1
476-1483, 1992 and Kyoko Ohara et al. Virus Gene.Viol.
5 .243-254, 1991).

The expression mechanism of the expression plasmid of the present invention will be described below with reference to FIG. The foreign gene sequence of FIG. 1 incorporated in a plasmid such as pUC19 is HIV-
The 1LTR promoter directs transcription of a structural gene under the control of the promoter into mRNA. This transcription gives the mRNA encoding the luciferase-IRES-tat protein. And this m
RNA is translated to synthesize proteins, luciferase and tat protein, respectively. As described above, the tat protein synthesized by the instruction of the HIV-1 LTR promoter acts to enhance the function of the promoter, and causes large-scale expression of the target protein luciferase.

[0010]

INDUSTRIAL APPLICABILITY Since the expression vector of the present invention is an expression vector having an autocatalytic enhancing action using the characteristics of IRES, it is a novel construction concept that does not depend on the innate functions of the promoter and enhancer. And a transformant into which the expression vector is introduced can exhibit high expression efficiency.

[0011]

EXAMPLES The following is a method for preparing the expression vector of the present invention,
The production of the target protein by the transformed cells containing the vector, which was carried out to confirm the expression of the vector, will be described in more detail with reference to luciferase as the target protein. "Example"

HIV virus-derived H as a promoter
Expression having an IV-1LTR promoter, an IRES of HCV group 1 on the downstream side thereof, a luciferase gene encoding a luciferase as a target protein as a cistron on the upstream side of the IRES, and a tat gene as a cistron on the downstream side of the IRES The plasmid, pACELItat, was constructed.

That is, each DNA fragment was prepared as follows. The vector DNA portion is Sal derived from pUC19.
(1065) -Sal (3721) to HIV-1LT
The R promoter is a splice junction (Splice Junct) that was isolated from HIV cDNA by PCR.
ion) is a 10s Splice Junction derived from pSV2,
The RES part is upstream of 342 bp from p22 ATG of HCV group 1, and luciferase ORF is plasmid p.
What was separated from GEM-lucDNA (trade name: manufactured by Promega) was tatORF from HIV cDNA to PC.
Those separated by the R method and poly A derived from a pCD vector were prepared according to a conventional method. These were constructed according to a conventional method to prepare an expression vector. The restriction enzyme map of this expression plasmid pACE LItat is shown in FIG. The HIV-1 LTR promoter and tat gene are described in Arya et al. Science.
The base sequence is described in ce 229 69-73,1985.

<Test Example> In order to confirm the protein-producing ability of the expression plasmid prepared as described above, HeLa cells were transformed with the expression plasmid according to a conventional method to prepare a transformant. And the productivity was measured. In addition, each of the constructed expression plasmids,
HeLa by calcium phosphate method or lipofectin method
The cells were transformed.

(1) Preparation of cell extract The culture medium is removed 24-48 hours after the transformation. The cells were washed twice with PBS (-). 0.25 ml (60 m
cell lysate (using mφ plate) -25 mM Tris
Buffer solution (pH 7.8) / 2mM DTT / 2mM 1,2
-Diaminocyclohexane-N, N, N ', N'-tetraaceti
c acid / 10% glycerol / 1% TritonX
-100- was added and the mixture was left at room temperature for 10 to 15 minutes. The cell suspension was scraped from the petri dish, transferred to an Eppendorf tube, and centrifuged at 10,000 rpm for 5 seconds.
The supernatant after centrifugation was used as a sample.

(2) Measurement of protein productivity A luciferase produced as a target protein was labeled with Co
It was measured by the luciferase assay method using enzyme A. That is, luminescent substrate solution- [PBS buffer (137 mM NaCl / 2.7 mM KCl / 4.3 m
M Na ₂ HPO ₄ /1.4 mM KH ₂ PO ₄ ) with luminescent substrate (20 mM Tricine / 1.07 mM (MgCO ₃ ))
_{4 Mg (OH) 2 · 5H} 2 O / 2.67mM MgSO 4/0.
1 mM EDTA / 33.3 mM DTT / 270 μM co
enzyme A / 470μM luciferin / 530μM AT
Dissolve P). Alternatively, P is used as a prepared luminescent substrate.
available from Romega] -100 μl (1)
An appropriate amount of the cell extract sample obtained in step 1 was added and mixed. The measurement was started 30 seconds after mixing, and the amount of luminescence for 20 seconds was measured as a relative light amount unit with a general-purpose luminometer or a scintillation counter. The relative light intensity unit is pSV2
/ L-AΔ5 ′ was used as a control.

The results are shown in FIG. PSV2 / L- using a typical SV40 viral promoter as a control
It was compared with AΔ5 ′. Expression of the IRES-deficient tat gene is not expected, and pACE / LΔItat, which shows the function of the HIV promoter alone, shows a relatively weak promoter activity of 0.33, but pACE / LItat
, The promoter activity was autocatalytically enhanced by the tat gene product, and a very high activity of 11.69 was obtained. From these, it is clear that the mechanism of autocatalytic expression enhancement by the tat gene product is working.

[Brief description of drawings]

FIG. 1 is a conceptual diagram of the expression vector of the present invention.

FIG. 2 Restriction enzyme map of the expression vector of the present invention

FIG. 3 is a diagram showing the ability of the expression vector of the present invention to produce a target protein.

Claims (1)

[Claims] 1. A promoter, a first cistron encoding a protein of interest, a second cistron encoding a protein having an autocatalytic enhancing action of the promoter, and provided between the first cistron and the second cistron. And an internal translation initiation signal obtained.