CN113699146A - Promoter element, retroviral genome transcription cassette comprising the same, and vector - Google Patents

Abstract

The application provides a response element TRE based On a Tet-On system or a TRE and a promoter element of an R-U5 functional domain in a Long Terminal Repeat (LTR), and also provides a retrovirus/lentivirus genome transcription cassette and a vector comprising the promoter element.

Description

Promoter element, retroviral genome transcription cassette comprising the same, and vector

Technical Field

The present invention relates to the field of viral vectors, in particular retroviruses, in particular lentiviral vectors; more particularly, it relates to an element for initiating transcription in a viral genome transcription cassette carrying a nucleic acid fragment of interest for use in the preparation of retroviral, especially lentiviral, vectors, and viral genome transcription cassettes and vectors comprising such an element.

Background

Retroviruses, also known as retroviruses, belong to the class of RNA viruses, which are double-stranded RNA viruses with an envelope, which are characterized primarily by the ability to "reverse transcribe" their genome from RNA to DNA. Virions are typically around 100nm in diameter and comprise a dimeric genome (two identical single-stranded positive-stranded RNAs) that form a complex with Nucleocapsid (NC) proteins. Its genome is enclosed IN a protein Capsid (CA) which also contains proteins with enzymatic activity, i.e. Reverse Transcriptase (RT), Integrase (IN) and Protease (PR), which are essential for viral infection. Matrix protein (MA) forms a layer outside the capsid core that interacts with the envelope, which is a lipid bilayer derived from the host cell membrane and surrounding the viral core particle. Anchored to this envelope is the viral envelope glycoprotein (Env), which is responsible for recognizing specific receptors on host cells and initiating the infection process. The envelope protein is formed of two subunits, a Transmembrane (TM) subunit anchoring the protein in the lipid membrane and a Surface (SU) subunit binding to a cell receptor, respectively.

Gamma-retroviral vectors, the most commonly used retroviral vector, account for 17.3% of all transfection procedures used in clinical trials reporting gene therapy in 2017. Currently, there is increasing interest in lentiviruses (lentivirus vectors) derived from complex retroviruses such as human immunodeficiency virus (HIV-1), with 2.9% of clinical trials of gene therapy rising from 2012 to 7.3% of 2017. This is because lentiviruses are capable of transducing non-dividing cells, a property that distinguishes them from other viral vectors (including gamma-retroviral vectors). In addition, lentiviruses have a more favorable lineage of gene insertion sites relative to retroviruses. The most attractive properties of retroviral and lentiviral vectors are: as a gene delivery tool, the gene delivery capacity of the gene delivery tool can reach 9 kb; less immune response in patients and better clinically proven safety; high transduction efficiency in vivo and in vitro; and the ability to permanently integrate foreign genes into the target cell genome, allowing long-lasting expression of the delivered gene.

The prototype lentiviral vector system was developed based on the HIV-1 virus, which is a well studied human pathogenic virus. In addition to HIV-1, other lentiviruses have been developed for use as gene delivery vectors (TVs), but have largely fallen short of clinical research, such as HIV-2, Simian Immunodeficiency Virus (SIV), or non-primate lentiviruses, including Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), or Caprine arthritis-encephalitis virus (CAEV). Vectors based solely on Equine Infectious Anemia Virus (EIAV) have been developed to the stage of clinical use for the treatment of parkinson (ProSavin).

Mainly due to the safety problem caused by the pathogenicity of HIV-1 in human body, three generations of lentivirus vector systems are developed. The first generation of lentiviral vector system is represented by a three-plasmid system consisting of 3 plasmids, a packaging plasmid, an envelope plasmid, and a transfer vector plasmid carrying a viral genome transcription cassette (transfer vector) carrying a nucleic acid fragment of interest. The packaging plasmid is derived from HIV-1 proviral genome, its 5'LTR is replaced by cytomegalovirus early promoter, 3' LTR is replaced by simian virus-40 poly A (SV40polyA) sequence, and HIV-1 envelope gene env is deleted. The packaging plasmid simultaneously expresses rev, vif, vpr, vpu and nef auxiliary genes. The deleted HIV-1 envelope gene was replaced with VSV-G, an envelope protein gene of Vesicular stomatitis virus (Vesicular stomatis virus), and expressed from an envelope plasmid. The transfer vector plasmid carrying the transcription cassette of the viral genome carrying the nucleic acid fragment of interest carries the 5' LTR of HIV-1, and the entire 5' untranslated region, the 5' gag gene of around 300bp, the central polypurine tract (cppt) fragment, and additionally the Rev Response Element (RRE) fragment. This transfer vector plasmid is used to clone the nucleic acid fragment of interest and provide viral genomic RNA upon viral assembly. The second generation lentiviral vector system was modified from the first generation by deleting all the accessory genes (vif, vpr, vpu, and nef genes) of HIV-1 in the packaging plasmid. The removal of these auxiliary genes does not affect the titer and infectivity of the virus, and simultaneously increases the safety of the vector. The third generation lentiviral vector system consisted of four plasmids, which removed the rev gene from the packaging plasmid and placed separately on another packaging plasmid. Furthermore, the third generation lentiviral vector system adds two safety features simultaneously: the first safety feature is the construction of a self-inactivating lentiviral transfer vector plasmid, i.e., deletion of the U3 region of the 3 ' LTR in the transcription cassette of the viral genome, so that the lentiviral vector permanently loses the enhancer and promoter fragments of the U3 regions of the 5' LTR and the 3 ' LTR after completion of the reverse transcription reaction, so that even if all viral proteins are present at this time, the viral cannot be successfully packaged because they can no longer transcribe the viral genomic RNA, and therefore the third generation of transfer vector plasmids is also called self-inactivating (SIN) transfer vector plasmids. The deletion of the U3 region also greatly reduces the carcinogenicity of the vector gene inserted into the host cell. The second safety feature is that the tat gene with the function of transcriptional transactivation is removed, the viral genomic RNA is transcribed by replacing the enhancer and promoter sequence of the U3 region of the 5' LTR with a heterologous promoter sequence, and the heterologous promoter cannot transcribe itself when transcribing the lentiviral genomic RNA, thus further ensuring that the lentiviral vector can only be transfected once by packaging. The third generation lentiviral vector system only retains the gag, pol and rev gene sequences in the original HIV-1 genome, and is therefore safer.

The retrovirus/Lentivirus vector may have different pseudotypes (pseudotypes) by replacing different heterologous envelope glycoproteins, such as Lentivirus (Lentivirus, e.g., human, simian, Feline, bovine immunodeficiency virus (immunodeficiency virus), caprine arthritis-encephalitis virus (hepatitis-encephalititis virus), Equine infectious anemia virus (Equine infectious anemia virus), etc.) envelope proteins, retrovirus (retrovirus, e.g., Murine leukemia virus (Murine leukovirus, 10A1,4070A), Gibbon ape leukemia virus (Gibbape e leukemia virus), Feline leukemia virus (Feline leukemia virus, RD114), Amphotropic retrovirus (Amphivirus), retroviral (Ecotropic leukemia virus), Baboon virus (Baboon leukemia virus), simian leukemia virus (Baboon virus), etc. (paramyxovirus), such as Nippon virus envelope proteins (melanoma virus), etc. (maize virus envelope proteins), and the like, Rhabdoviruses (Rhabdoviruses, such as Rabies Virus (Rabies Virus), Mokola Virus (Mokola Virus), etc.) envelope proteins, Filoviruses (Filoviruses, such as Ebola Virus Zaire Virus (Ebola Zaire Virus), etc.) envelope proteins, trachoma Virus (Arenaviruses, such as Lymphocytic choriomeningitis Virus (Lymphatic choromitis Virus), etc.) envelope proteins, Baculovirus (Baculovir) envelope proteins, Alphaviruses (Alphavirus, such as Chikungunya Virus, Ross River Virus (RoRiriver Virus), Semliki Forest Virus (Semliki Virus), Newcastle disease Virus (Sindbis Virus), Nerviella encephalitis Virus (Veilluevirus), Veillus Virus (Veillonevirus), etc. (Fowlett-packard Virus, etc.), such as Vesicular stomatitis virus (Vesicular stomatis virus), Chandipura virus and Piry virus (Chandipura virus and Piry virus) envelope proteins, most of the lentiviral vectors currently use Vesicular stomatitis virus envelope glycoprotein (VSV-G) because such glycoprotein allows the lentiviral vectors to have a wide transduction spectrum (transduction spectrum) and better stability during downstream processing.

Currently most retroviral/lentiviral vectors are preferably prepared using mammalian cell lines as host cells, the most widely used being the 293T or HEK293 cell lines and derived cell lines screened or engineered based thereon. The HEK293 cell is a human kidney epithelial cell line transfected with adenovirus E1A gene, the 293T cell is derived from the HEK293 cell and expresses SV40 large T antigen, and a plasmid containing an SV40 replication starting point and a promoter region can be replicated in the 293T cell and maintain high plasmid copy number in a period of time, so that the protein expression amount of the plasmid-carried gene is improved. In addition, 293T cells have faster cell growth rate and higher transfection efficiency compared to HEK293 cells. The characteristics of the 293T cell enable the preparation efficiency of the virus vector to be higher. In addition, according to literature reports, production/packaging cells useful for viral vectors also include mammalian cells HepG2 cells, HeLa cells, CHO cells, BHK cells, COS cells, NIH/3T3 cells, Vero cells, HT1080 cells, Te671 cells, CEM cells, NSO cells, and PerC6 cells.

The conventional retroviral/lentiviral vectors are generally prepared by transferring gag gene, pol gene, rev gene (for lentiviral vectors), envelope glycoprotein gene, and a viral genome transcription cassette carrying a nucleic acid fragment of interest (comprising a promoter for transcription-packaging into the RNA genome of retrovirus/lentivirus; various cis-acting sequences required for packaging and transfection of retrovirus/lentivirus genome, such as 5' LTR, PBS, psi packaging signal, cppt (for lentiviral vectors), RRE (for lentiviral vectors), ppt and 3 ' LTR sequences, etc.; a nucleic acid fragment of interest to be transduced, a polyadenylation signal at the 3 ' end, etc.) into mammalian host cells, usually murine or human cells, respectively. This can be achieved by transient transfection of a construct, such as a plasmid, containing the nucleic acid fragment into the host cell and subsequent production and harvesting of the viral vector for 24-72 hours (transient production), or by stable integration of the nucleic acid fragment into the host cell genome to form a stable production cell line (producer cell line) for continued production (stable production).

Most retroviral vectors currently in use are derived from Moloney murine leukemia virus (Mo-MLV), and many design improvements have been made to this vector system over the years. A rapid and efficient method for preparing retroviral vectors is by transient transfection of 293-based packaging cell lines such as Phoenix-ECO and Phoenix-AMPHO (Dr. Gary P. Nolan, Stanford University) or other commercially available packaging cell lines such as Plat-A, Plat-E, and Plat-GP (cell Biolabs); AmphoPak-293; EcoPak 2-293; and RetroPackPT67 (Clontock). To pseudotype retroviral vectors with other envelope proteins, such as vesicular stomatitis virus glycoprotein (VSV-G), the transfer vector plasmid and the plasmid expressing the envelope protein of interest can be transfected simultaneously in a packaging Cell line (e.g., Plat-GP, Cell Biolabs; GP2-293, Clontech) expressing only the Mo-MLV gag and pol genes; or by transient transfection of 293 cells simultaneously with a plasmid expressing the Mo-MLV gag and pol genes (e.g., pUMVC-plasmid, Addgene, #8449), a plasmid expressing the envelope protein of interest, and a transfer vector plasmid.

Method for transient production and utilization of transfection plasmidTo introduce viral gene constructs, using cationic agents that can form complexes with negatively charged DNA, allowing their uptake by cells via endocytosis. Polyethyleneimine (PEI) is one of the most widely used and most efficient cationic reagents, and the PEI transfection method is currently used clinically and industrially to introduce the above-mentioned constructs, but the major problems of unstable process and low titer of toxin are existed after the process is scaled up. Other methods, such as calcium phosphate precipitation, cationic liposome complexation, and non-liposomal transfection reagents, such as Lipofectamine and

however, these methods can only be used for small-scale production or for research purposes, since they are either difficult to scale up or too expensive. Alternatively, viral infection has also been developed and validated for retroviral/lentiviral vector production using baculovirus or adenovirus to introduce lentiviral gene constructs. However, this method requires additional isolation of the retrovirus/lentivirus vector and baculovirus or adenovirus downstream to meet the clinical grade virus production standard, and the process of preparing the retrovirus/lentivirus vector using baculovirus or adenovirus is complicated and the final transfection titer is not high compared to the plasmid DNA transfection method. Alternatively, continuous electroporation has also been developed for large volume cell transfection, and in principle can also be used for transient transfection production of retroviruses/lentiviruses, but this technique is still limited by process scale-up capabilities and expensive equipment and consumables.

Construction of a stable retroviral/lentiviral producer cell line relies on the integration of viral gene constructs into the cell genome to allow constitutive or regulated expression, respectively. Generally, gag, pol, rev (for lentiviral vectors) and env genes are first transferred into cells simultaneously or sequentially and cells are selected for stable insertion of all the genes into the genome and for co-high expression by corresponding resistance selection and clonal selection, thereby establishing a packaging cell line (packaging cell). Thereafter, a viral genome transcription cassette carrying the nucleic acid fragment of interest is introduced to construct a viral vector-producing cell line carrying the nucleic acid fragment of interest. If non-SIN (self-inactivating) vectors are used which are replicable after reverse transcription, this can be achieved directly by viral infection; otherwise, it is necessary to obtain a producer cell line stably integrated in the genome of the packaging cell described above of a transcription cassette of the viral genome carrying the nucleic acid fragment of interest by chemical transfection of the plasmid followed by resistance selection and clonal selection. A very efficient method is to insert a lentivirus genome transcription cassette containing a (selectable) marker gene into primary cells, and to select a production cell line which stably integrates and expresses a virus vector at a high level for a long time by the marker gene, and then to replace the marker gene for constructing the production cell line with a target nucleic acid fragment by a site-specific recombinase (site-specific recombinase), such as FLP-FRT or Cre-lox recombinase system, to rapidly construct a production cell line which stably produces a virus vector carrying the target nucleic acid fragment. This method has been shown to allow the establishment of high titer human retroviral vector producing cell lines (Schucht, R., et al (2006) ' A new generation of retroviral vector. ' Mol. Ther 14(2): 285. cells 292.; Loew, R., et al. (2010) ' A new PG13-base packaging cell line for a stable production of clinical-genetic vector targeted "Gene 17. 272). In addition, viral vector packaging cell lines or production cell line platforms for rapid replacement of nucleic acid fragments of interest and/or envelope proteins can be developed using similar principles. Development of stable retroviral/lentiviral vector producer cell lines is a time-consuming and labor-intensive complex system engineering that takes a year or more to fully develop and characterize the cell line platform, and because of the complexity of the work, many of the published work has ultimately failed to meet industry needs due to issues of titer of produced virus, cell line stability, culture adaptation, etc. However, once a stable virus-producing cell line is successfully obtained through research and development, the stable virus-producing cell line has irreplaceable advantages in the aspects of process reliability, process amplification capacity, production cost, virus product safety and the like in the fields of clinical and industrial applications compared with an instant transfection production process. Firstly, the production process of the stable production cell line is more stable, a completely characterized production platform can be provided, and safer virus vectors can be produced with low batch-to-batch difference; secondly, the process is easier to scale up, and cannot cause the rapid reduction of the production titer along with the increase of the culture volume like an instant production system; in addition, as the DNA plasmid, transfection reagent and other raw and auxiliary materials are not needed, a GMP production line for producing the plasmid is not additionally established; finally, the method has higher unit yield and simpler production process quality control. In the process of expanding the production scale, the production process based on the stable production cell line has further remarkable advantages in the aspects of research and development, production, management, operation and maintenance, cost and the like. These advantages are useful for promoting the technical and pharmaceutical industrialization in the fields of gene therapy and cell therapy.

The retroviral/lentiviral vector titer in the purified pre-cell stock currently available in production was 10⁶To 10⁷Infected particles/mL culture medium. However, the average amount of carrier required to treat a patient in a clinical trial was 10¹⁰Infection particles are at this level per patient. In addition, virus production is generally characterized by a low infectious particle/physical particle ratio (less than 1: 100); at the same time, these viral vectors are very sensitive and lose their infectivity rapidly in cell culture supernatants at 37 ℃ with half-lives of about 8-12 hours, which further increases the demand for viral productivity. It is estimated that based on the current production technology platform, each patient requires approximately 10-100L culture volume of viral vectors produced, and therefore, current retroviral and lentiviral vector production systems behave far below therapeutic requirements, and any increase in the production capacity of current retroviral and lentiviral vector production systems is of great value.

Therefore, there is a strong need in the art to provide a technique for increasing the production capacity of retroviral and lentiviral vectors.

Disclosure of Invention

The invention remarkably improves the production titer of the retrovirus/lentivirus vector by constructing and using a Tet-On inducible expression system Tet Response Element (TRE) to regulate and control the transcription of a retrovirus/lentivirus genome transcription box carrying a target nucleic acid fragment.

In the case of transduction using retroviral/lentiviral vectors, the nucleic acid fragment of interest is loaded into the retroviral/lentiviral RNA genome. The term "nucleic acid fragment of interest" may generally refer to a gene, such as a nucleic acid sequence encoding a protein, depending on the purpose of use; may be a functional ribonucleic acid (RNA), such as small interfering RNA (siRNA), long non-coding RNA (LncRNA), guide RNA (gRNA), transfer RNA (tRNA), Ribosomal RNA (rRNA) or other functional RNA coding sequences of CRISPR gene editing systems; other functional nucleic acid sequences may be present, such as homologous recombination sequences, DNA or RNA sequences capable of binding to proteins, DNA or RNA sequences capable of binding to other nucleic acid fragments (e.g., primers or probes); can be any nucleic acid sequence in nature or an artificial nucleic acid sequence; or a combination of one or more of the above; the nucleic acid fragment of interest may further comprise a nucleic acid sequence regulating gene expression such as a promoter, enhancer, insulator, poly A signal, etc. The RNA genome of a retrovirus and/or lentivirus refers to a ribonucleic acid fragment that can be packaged into a virus when constructing a retrovirus/lentivirus vector, and generally contains sequences necessary for viral packaging and transduction, such as a ψ packaging signal, a Long Terminal Repeat (LTR). In the lentiviral RNA genome, it typically contains all 5 'untranslated regions, about 300bp of the 5' gag gene, a central polypurine tract (cppt) fragment, and additionally a Rev Response Element (RRE) fragment; the absence of one or more fragments may severely affect the packaging or transduction efficiency of the lentivirus. In preparing a retrovirus/lentivirus vector, a viral genomic RNA fragment carrying a nucleic acid fragment of interest is typically obtained by constructing a viral genomic transcription cassette (transcriptional cassette) carrying a nucleic acid fragment of interest, which contains a nucleic acid sequence with promoter function (which may be a retroviral/lentivirus LTR sequence itself or a chimeric promoter constructed from other heterologous promoters and LTRs), a DNA sequence corresponding to the viral RNA genome, and typically a poly a signal sequence that regulates the termination of transcription. In the preparation of retroviral/lentiviral vectors, a construct carrying a viral genomic transcription cassette carrying a nucleic acid segment of interest (e.g., the transcription cassette described above is constructed into a transfer vector plasmid or into a viral vector) is delivered to a host cell, and then transcribed by the transcriptional machinery of the host cell into a corresponding viral genomic RNA segment carrying the nucleic acid segment of interest that can be packaged into a viral vector.

The transcription cassette of the retrovirus genome carrying the nucleic acid fragment of interest is required to provide cis-acting elements necessary for transcription, packaging and transduction, and mainly comprises: (1) long Terminal Repeat (LTR) fragment: the primary functions of the LTRs are to regulate the reverse transcription of the viral genome from RNA to DNA, the integration of DNA proviruses (provirus) in the host cell genome, and the transcription of viral mRNA fragments following integration. The LTR consists of three functional domains, U3(unique-3 '), R (repeat), and U5 (unique-5'), according to the structure of U3-R-U5, wherein the U3 domain provides mainly transcriptional promoter and enhancer functions; the R domain is involved in the reverse transcription process and encodes the 5 'capping sequence (5' cap) and polyadenylation (polyA) signals of the viral RNA genome; the U5 domain is the start site for reverse transcription. The currently used SIN vector eliminates the U3 domain in the 3 'LTR and regulates transcription of the entire viral RNA genome transcription cassette carrying the nucleic acid fragment of interest by a chimeric promoter consisting of a foreign promoter and the R-U5 domain in the 5' LTR. (2) A Primer Binding Site (PBS) located immediately downstream of the 5' LTR and in close proximity to the U5 domain, which binds to a specific transfer RNA (tRNA) in the host cell and initiates reverse transcription using this tRNA as a primer. (3) ψ packing signal (ψ packing signal): located between the primer binding site and the gag open reading frame is an essential element of the packaging of the viral RNA genome into a viral particle. (4) The polypurine tract (PPT) located upstream of the 3' LTR, immediately adjacent to the U3 functional domain, is the primer for plus strand DNA synthesis during reverse transcription. For a lentivirus genome transcription cassette, the following essential cis-acting elements are also provided: (1) central polypurine tract/central termination sequence (cPPT/CTS): the second polypurine tract located in the lentivirus genome is important for the transfer of provirus into host cell nucleus, and can obviously improve the efficiency of integrating provirus complex into host cell genome. (2) Rev Response Element (RRE) binds to lentivirus encoded regulatory protein Rev, which is essential for the transport of post-transcribed unspliced and incompletely spliced viral mRNA from the nucleus to the cytoplasm. The RRE sequence contained in the lentivirus genome transcription box can obviously improve the packaging efficiency of the lentivirus vector. In addition, the expression level of a transgene in a nucleic acid fragment of interest can be significantly increased by linking Posttranscriptional Regulatory elements (Posttranscriptional Regulatory elements) to the 3' end of the nucleic acid fragment of interest in a genomic transcription cassette of a retrovirus/lentivirus vector, such as a Posttranscriptional Regulatory Element of Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE) or a Constitutive Transport Element (CTE) of Mason-Pfizer monkey Virus (Mason-Pfizer monkey Virus).

The term "vector" is a vector comprising a nucleic acid molecule, which is typically used as a vehicle for the artificial entrainment of foreign genetic material, such as the above-described nucleic acid fragments of interest, into another cell, where it replicates and/or is expressed. Functionally, all vectors can generally be used to clone and carry foreign nucleic acid fragments of interest, as well as expression vectors specifically designed for transcription of nucleic acid fragments and expression of proteins. Plasmids, viral vectors, cosmids (cosmids) and artificial chromosomes are four major types of vectors. Among these, the most commonly used vectors are plasmids. All engineered plasmid vectors contain an origin of replication for replication in bacteria, a multiple cloning site for insertion of a nucleic acid fragment of interest and a marker gene for selection of positive strains. Viral vectors are another commonly used vector, and are commonly used to deliver genetic material, such as nucleic acid fragments of interest, into cells. This process can be carried out in vivo (in vivo) or in cell culture (in vitro). The nucleic acid fragment of interest can be efficiently transferred into infected target cells based on various molecular mechanisms of virus self-evolution, such as protection of genetic material, host cell selection based on recipient, delivery of genetic material into host cells, replication and/or expression in host cells, modulation of host cell growth, metabolism, reproductive replication and defense mechanisms, and suppression and/or escape of the immune system in higher animals. In addition to being used in molecular biology research, viral vectors are also commonly used in gene therapy, cell therapy, immunotherapy, and vaccine development. In the present application, the gene segments for packaging retrovirus/lentivirus and the viral genome transcription cassette carrying the nucleic acid segment of interest can be produced by constructing the above-mentioned various vectors and transferring into host cells for viral vector production.

In the present invention, transcription of a viral genome transcription cassette carrying a nucleic acid fragment of interest transferred into a host cell is controlled by an Inducible expression system (Inducible expression system). Examples of Inducible expression systems that may be used in the present invention include, but are not limited to, the Tet-off system (see, e.g., Gossen, M. and H.Bujard (1992). "light control of gene expression in mammarian cells by tetracyclic-responsive promoters." Proc Natl Acad Sci U S89 (12): 5547-. In the Tet-On inducible expression system, a tetracycline dependent transactivator (rtTA) can bind to a Tet-On inducible expression system TRE responsive Element (a nucleic acid sequence containing multiple copies of a contiguous TetO operator sequence and a minimal promoter sequence, hereinafter referred to as a TRE) to initiate transcription of a downstream linked regulated nucleic acid fragment only in the presence of a tetracycline derivative such as tetracycline or doxycycline (Dox). Currently common Tet-On inducible expression systems include the second generation Tet-On inducible expression system (Tet-On Advanced, Clontech, where the transactivator rtTA and the response element TRE are hereinafter referred to as rtTA, respectively_adv(encoding nucleic acid sequence: 13-756bp in SEQ ID NO: 11) and TRE_adv) And the third generation Tet-On inducible expression System (Tet-On 3G, Clontech, where the trans-activator rtTA and the response element TRE are hereinafter referred to as rtTA respectively_3G(encoding nucleic acid sequence: 13-756bp in SEQ ID NO: 10) and TRE_3G). Trans-activators rtTA and TRE in second and third generation Tet-On inducible expression systems can be used in combination, e.g., rtTA_advCan combine TREs_3GUse, rtTA_3GTRE can also be combined_advThe preparation is used.

The present invention relates to the regulation of transcription of a viral genome transcription cassette carrying a nucleic acid fragment of interest during the production of a retroviral/lentiviral vector based on a tetracycline dependent nucleic acid sequence (Tet response element, TRE). Specifically, the Tet-responsive element TRE comprises at least 2 copies of a TetO-operator (TetO) sequence that binds to tetracycline dependent transactivator (rtTA), 1 copy of a minimal promoter sequence comprising a TATA box sequence; preferably, the TRE sequence is shown as SEQ ID NO 17 and SEQ ID NO 18. In one aspect of the invention, the chimeric promoter regulating transcription of the transcription cassette of the viral genome carrying the nucleic acid fragment of interest comprises the above-described Tet response element TRE and the R-U5 functional domain in the LTR sequence of a retrovirus/lentivirus; preferably, the R-U5 domain is linked downstream of and spaced 20-24 base pairs from the TATA box sequence in the Tet-responsive element, more preferably spaced 24 base pairs apart. In one aspect of the invention, the R-U5 domain is the R-U5 domain of HIV-1 lentivirus, preferably the chimeric response element consisting of the Tet response element and the R-U5 domain of HIV-1 lentivirus is TRE1-RU5 (as shown in SEQ ID NO: 19) and TRE2-RU5 (as shown in SEQ ID NO: 20).

In the present invention, the gamma-retroviral genomic transcription cassette carrying the nucleic acid fragment of interest may be derived from a transfer vector plasmid of an MSCV (mouse Stem Cell Virus) retroviral expression system using a 5' LTR as a promoter, such as pMSCV-IRES-mCherry FP (Addge, #52114), pMSCV-IRES-GFP (Addge, # 20672); transfer vector plasmids such as pBABE-hygro-hTERT (Addgene, #1773), pBABE-puro (Addgene, #1764) in the MoMLV (Moloney Murine Leukavirus) reverse transcription expression system using 5' LTrz as a promoter; and SIN transfer vector plasmids based on CMV-R-U5 chimeric promoter and deleted U3 functional domain of the above two reverse transcription expression systems, such as pmko.1gfp (Addgene, #10676), TtRMPVIR (Addgene, #27995), pRetroX GFP T2A Cre (Addgene, #63704), pRXTN (Addgene, # 47916). The viral genome transcription cassettes in the retroviral transfer vector plasmids described above are in principle suitable for use in the methods of the invention described herein for regulating transcription of viral genome transcription cassettes using Tet-responsive elements.

In the present invention, the lentiviral genome transcription cassette carrying the nucleic acid fragment of interest may be derived from a transfer vector plasmid of a second generation lentiviral vector such as pLVPRT-tTR-KRAB (Addgene, #11648), pLenti CMVlight eGFP Puro (w771-1) (Addgene, #26431) or a transfer vector plasmid of a third generation lentiviral vector such as pSLIK-Hygro (Addgene, #25737), pHIV-EGFP (Addgene, #21373), pSico (Addgene, #11578), pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene, #12252), Tet-pLKO-Puro (Addgene, #21915), pLenti-Puro (Addgene, #39481), pLVUT-tTR-KRAB (Addgene, #11651) or the like. Most of the viral genome transcription cassettes of the third generation lentiviral vector and the second generation lentiviral vector share the nucleic acid sequences of LTR, 5' non-coding fragment, HIV-1 psi packaging signal, RRE, cPPT and gag partial sequences and the like which play key roles in the virus packaging and transduction process, and the main difference of the third generation lentiviral genome transcription cassette compared with the second generation lentiviral vector is that a constitutive active promoter such as CMV or RSV is used for replacing the U3 sequence which plays the promoter function in the 5' LTR sequence, and the U3 sequence of the 3 ' -LTR sequence is deleted, so that the lentiviral transfer vector becomes an SIN (self-activating) vector. In the third generation of lentiviral genome transcription cassettes, pSLIK-Hygro, pHIV-EGFP, pSico vector' transcribed lentiviral genomic RNA using the CMV promoter, while pRRLSIN. cPPT. PGK-GFP. WPRE, Tet-pLKO-puro, pLenti-puro transcribed lentiviral genomic RNA using the RSV promoter. Tet-pLKO-puro and pLenti-puro do not contain WPRE sequence in the lentiviral genome transcription cassette, as compared to other third generation lentiviral transfer vector plasmids. The lentivirus genome transcription cassette in the lentivirus transfer vector plasmid is basically applicable to the method for preparing the virus vector by the virus genome transcription cassette which is controlled by the Tet response element and carries the target nucleic acid segment. In one aspect of the invention, the sequence of the lentiviral genome transcription cassette used in the invention was designed based on the nucleic acid sequence in the prrlsin. cppt. pgk-gfp. wpre (Addgene, #12252) transfer vector plasmid and a plasmid construct containing this sequence was constructed.

In the present invention, retroviruses include, but are not limited to, lentiviruses such as Murine Leukemia Virus (MLV), Human Immunodeficiency Virus (HIV), Equine Infectious Anemia Virus (EIAV), Murine Mammary Tumor Virus (MMTV), Rous Sarcoma Virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine sarcoma virus (FBRMSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelomatosis virus-29 (MC29), and Avian Encephalomyelitis Virus (AEV), Simian immunodeficiency virus (Simian immunodeficiency virus, SIV), Feline immunodeficiency virus (Feline immunodeficiency virus, FIV), Bovine immunodeficiency virus (Bovine immunodeficiency virus, BIV), Caprine immunodeficiency virus (Caprine-encephalitis virus), Simian leukemia virus (Caprine-virus), Simian immunodeficiency virus (Caprine-virus, Caprine-associated with arms), and Simian leukemia virus (Caprine-associated with arms) Feline leukemia virus (Feline leukemia virus), Amphotropic retrovirus (Amprotropic retrovirus), tropic retrovirus (Ecotropic retrovirus), Baboon ape leukemia virus (Baboon ape leukemia virus), and all other retroviruses of the family Retroviridae (Retroviridae).

The retrovirus/lentivirus vector can be prepared by transferring a gag gene, a pol gene, a rev gene (for a lentivirus vector), an envelope glycoprotein gene and a viral genome transcription cassette carrying a target nucleic acid fragment, the transcription of which is controlled by a Tet response element (at the same time, a trans activator rtTA for controlling the Tet response element is required), into a host cell. The above-mentioned retrovirus/lentivirus vector production method can be realized by "transient production" which is represented by transferring a vector, such as a plasmid or a virus vector, containing a viral genome transcription cassette carrying a nucleic acid fragment of interest (hereinafter referred to as viral genome transcription cassette) whose transcription is regulated by a Tet-responsive element according to the present invention into a host cell, and preparing the virus vector without integration into the host cell genome. In one aspect of the invention, a retroviral/lentiviral vector is prepared by transferring into a host cell a vector construct (e.g., a plasmid or viral vector) comprising the gag gene, pol gene, rev gene (for lentiviral vectors), envelope glycoprotein gene, transactivator rtTA coding sequence, and viral genome transcription cassette, all by transient transfection. In one aspect of the invention, the transactivator rtTA coding sequence is first stably integrated into the genome of the host cell, such that the host cell stably expresses the rtTA protein. Then based on the genetically modified host cell, a retrovirus/lentivirus vector is prepared by transferring into the host cell a vector construct (e.g., a plasmid or a viral vector) comprising the gag gene, pol gene, rev gene (for a lentiviral vector), envelope glycoprotein gene, and viral genome transcription cassette, all by transient transfection. In one aspect of the invention, a trans-activator rtTA coding sequence is stably integrated into the genome of a host cell, and one, more or all of a gag gene, a pol gene, a rev gene (for lentiviral vectors), and an envelope glycoprotein gene are stably integrated into the genome of the host cell to construct a packaging cell line, and then a retrovirus/lentiviral vector is prepared by transferring the gag gene, the pol gene, the rev gene (for lentiviral vectors), and the envelope glycoprotein gene, which are not stably integrated into the packaging cell, into the packaging cell in the form of a vector construct (e.g., a plasmid or a viral vector) and a vector construct (e.g., a plasmid or a viral vector) of a viral genome transcription cassette by transient transfection. Preferably, the trans-activator rtTA coding sequence, gag gene, pol gene, rev gene (for lentiviral vectors), envelope glycoprotein gene are all stably integrated into the genome of the host cell to construct the packaging cell. The viral genome transcription cassette is then transferred into the packaging cells described above by transient transfection to produce retroviral/lentiviral vectors. In the above transient production method, tetracycline or a derivative thereof is added to induce the preparation of a retrovirus/lentivirus vector. The above-mentioned retrovirus/lentivirus vector production method can also be achieved by "stable production" which is characterized by stably integrating a viral genome transcription cassette carrying a nucleic acid fragment of interest under the control of transcription by a Tet-responsive element (hereinafter referred to as a viral genome transcription cassette) of the present invention together with a packaging gene such as a gag gene, pol gene, rev gene (for a lentivirus vector), envelope glycoprotein gene and transactivator rtTA coding sequence into the genome of a host cell to construct a production cell line, and inducing the production of a retrovirus/lentivirus vector by adding tetracycline or a derivative thereof. In one aspect of the invention, the packaging cells and producer cells described above for construction of retroviral/lentiviral vectors may be realized by a transposon system such as the Sleeping Beauty (SB) transposon system and/or the piggybac (pb) transposon system.

In the present invention, the host cell useful for preparing the retrovirus/lentivirus vector is a mammalian cell. Examples of host cells suitable for use in the present disclosure are 293T cells, HepG2 cells, Hela cells, CHO cells, BHK cells, HEK293 cells, COS cells, NIH/3T3 cells, Vero cells, HT1080 cells, Te671 cells, CEM cells, NSO cells or PerC6 cells, and derivatives thereof. In one aspect, the host cell is a HEK293 cell or a cell derived from a HEK293 cell. In one aspect, the host cell is a 293T cell. In one aspect, the host cell can be cultured adherent or suspension. In one aspect, the host cell can be cultured in the presence or absence of serum.

In the present invention, tetracycline and its derivatives useful in the Tet-On inducible expression system include compounds similar in structure to tetracycline, which are capable of binding to the tetracycline-dependent transactivator rtTA of the present invention with a binding constant Ka of at least 10-6M; preferably, the binding constant Ka is at or above 10-9M. The tetracycline derivative may for example be selected from: doxycycline (Dox), anhydrotetracycline (Atc), chlorotetracycline, oxytetracycline, and doxycycline.

In one aspect, the invention provides the following:

a nucleic acid sequence comprising the response element TRE of the Tet-On system and the R-U5 domain in the retroviral Long Terminal Repeat (LTR).

The nucleic acid sequence of item 2 item 1, wherein the R-U5 functional domain is downstream of and spaced from the TATA box of a TRE by 15-30bp, preferably by 24 bp.

The nucleic acid sequence of item 3 or 1, wherein the sequence of TRE is as shown in SEQ ID NO 17 or SEQ ID NO 18.

The nucleic acid sequence of item 4 or 1, which has the sequence shown in SEQ ID NO. 19 or SEQ ID NO. 20.

A retroviral genome transcription cassette comprising a nucleic acid sequence of any one of the response element TRE or items 1 to 4 of the Tet-On system for controlling transcription of said transcription cassette, a cis-acting element for retroviral packaging located downstream of the response element TRE or the nucleic acid sequence of any one of items 1 to 4 of the Tet-On system and a multiple cloning site for insertion of a nucleic acid fragment of interest.

The retroviral genome transcription cassette of item 6.5, wherein the cis-acting element comprises a Long Terminal Repeat (LTR), a Primer Binding Site (PBS), and a viral packaging signal (phi signal).

The retroviral genome transcription cassette of item 7. 5, wherein the retrovirus is a lentivirus.

The retroviral genome transcription cassette of item 8. 7, wherein the cis-acting element comprises a Long Terminal Repeat (LTR), a Primer Binding Site (PBS), a viral packaging signal (phi signal), a central polypurine tract (cPPT), and a rev protein response element (RRE), and preferably further comprises a woodchuck hepatitis virus post-transcriptional regulatory sequence (WPRE).

The retroviral genome transcription cassette of any one of items 9, 6 or 8, wherein the long terminal repeat is a self-replicating wild-type U3-R-U5 sequence or a self-suppressing SIN sequence with the U3 sequence deleted.

A retroviral genome transcription cassette obtained by inserting a nucleic acid fragment of interest into the multiple cloning site of the retroviral genome transcription cassette of any one of items 5 to 9.

The vector according to claim 11, wherein the vector comprises the nucleic acid sequence according to any one of items 1 to 4 or the retroviral genome transcription cassette according to any one of items 5 to 10.

The vector of items 12 and 11, which is a plasmid vector or a viral vector.

A host cell comprising the nucleic acid sequence of any one of items 1 to 4, the retroviral genome transcription cassette of any one of items 5 to 10, or the vector of item 11 or 12.

Use of a nucleic acid sequence according to any of items 14, 1 to 4, a retroviral genomic transcription cassette according to any of items 5 to 10, a vector according to items 11 or 12, or a host cell according to item 13 for the production of a retroviral vector carrying a nucleic acid fragment of interest.

The use according to item 15, item 14, wherein the nucleic acid sequence according to any one of items 1 to 4, the retroviral genomic transcription cassette according to any one of items 5 to 10, the vector according to item 11 or 12, or the host cell according to item 13 is used for the transient or stable production of the retroviral vector carrying the nucleic acid segment of interest.

Brief Description of Drawings

FIG. 1 is a schematic diagram of the real part of the plasmid of the present invention.

FIG. 2 shows comparison of the production titer of each transfer vector plasmid by transient transfection in 293T cells according to one example. The abscissa is the combination of each transfer vector plasmid and rtTA expression plasmid, and the ordinate is the RLU value of the Luciferase experiment for detecting the virus transfection titer.

FIG. 3 shows comparison of the virulence titres of each transfer vector plasmid by transient transfection in 293T cells stably expressing rtTA according to one example. The abscissa is the chimeric promoter or chimeric response element used for each transfer vector plasmid and the ordinate is the RLU value of the Luciferase assay to detect the viral transfection titer.

FIG. 4 shows comparison of the production titer of each transfer vector plasmid by transient transfection in EuLV293T packaging cells according to one example. The abscissa is the chimeric promoter or chimeric response element used for each transfer vector plasmid and the ordinate is the RLU value of the Luciferase assay to detect the viral transfection titer.

FIG. 5 shows the effect of various promoters or response elements that regulate transcription of viral genome transcription cassettes on productive titer in a lentivirus stable producer cell, according to one embodiment. The abscissa is the chimeric promoter or chimeric response element used to transcribe the viral genome transcription cassette in each lentivirus stable producer cell line, and the ordinate is the RLU value in the Luciferase assay to detect viral transfection titres.

Detailed Description

The following examples are provided to illustrate the technical solutions of the present invention and should not be construed as limiting the scope and spirit of the present invention.

Example 1: plasmid construction method

The molecular cloning techniques used in the following examples, such as PCR amplification of DNA fragments, restriction endonuclease cleavage of DNA fragments, gel recovery of DNA fragments, T4DNA ligase ligation of two or more DNA fragments, transformation of ligation competent cells, plasmid minipreparation and identification, are all well known in the art. The following reagents are referred to in the examples below: PCR enzymes (Thermo, F-530S); restriction enzymes (NEB); t4DNA ligase (Invitrogen, 15224041); DNA fragment gel recovery kit (Omega, D2500-02); plasmid miniprep kit (TIANGEN, DP 105-03); competent cells (XL-10Gold, Hu nan Fenghui Biotech Co., Ltd., JZ 011); the nucleic acid sequences shown in SEQ ID NO 1 to SEQ ID NO 16 were synthesized by Kisry and used for the plasmid construction according to the present invention, and the plasmid sequencing identification was performed by Invitrogen corporation. A map of a part of the plasmid used in the following examples is schematically shown in FIG. 1; table 1 shows the primer information for plasmid construction; table 2 shows the element composition of the sequences SEQ ID NO 1 to SEQ ID NO 20; table 3 is a description of the functional elements in the plasmid; table 4 shows the plasmid numbers and corresponding names constructed according to the present invention. Information on functional element sequences used in the following examples to illustrate the present invention, those skilled in the art can expect that the effect of the present invention can be achieved by replacing the functional element sequences on the plasmids used in the following examples with other sequences having similar biological functions, including but not limited to backbone sequences (such as replication origin (replication origin), resistance genes, etc.), restriction enzyme site sequences, transposon repeat sequences, inducible system response element sequences, Insulator (Insulator) sequences, promoter sequences, intron sequences, poly a signal (PolyA) sequences, different codon-optimized gene sequences, mutants of the above functional element sequences and gene sequences, and cloning positions, cloning sequences and cloning directions of the functional element sequences and gene sequences. The specific plasmid construction method is as follows:

1. plasmids 18BF007 and 18BF004 were constructed: the synthetic sequences SEQ ID NO 2(2900bp) and SEQ ID NO 3(1386bp) were digested with NotI and AsiSI, and ligated to NotI and AsiSI cleavage sites of plasmid 18BF003(SEQ ID NO 1, 1893bp), respectively, to construct plasmids 18BF007 and 18BF004, respectively.

2. Construction of plasmids 18BF011 and 18BF 063: the 18BF007 plasmid is digested by MluI and SphI, the 1730bp fragment is recovered from gel and is connected with the MluI and SphI digestion sites of the 18BF003 plasmid, so that the plasmid 18BF011 is constructed. The synthetic sequence SEQ ID NO. 4(915bp) is cut by MluI and ClaI and is connected to the MluI and ClaI cutting sites of 18BF007 to replace the CMV-BGI fragment to construct the plasmid 18BF 063.

3. Construction of plasmid 18BF 072: PCR-amplified Rev gene fragment (380bp) using pRSV-Rev (Addgene, #12253) as template and C-Rev-F (SEQ ID NO:23) and C-Rev-R (SEQ ID NO:24) as primers, and then digested with ClaI and XhoI and ligated to ClaI and XhoI cleavage sites of 18BF063 plasmid to construct 18BF072 plasmid.

4. Construction of plasmid 18BF068: the VSV-G gene fragment (1565bp) was PCR-amplified using pMD2.G (Addgene, #12259) as a template and C-VSVG-F (SEQ ID NO:21) and C-VSVG-R (SEQ ID NO:22) as primers, and then cleaved with ClaI and XhoI and ligated to ClaI and XhoI cleavage sites of the 18BF063 plasmid to construct a plasmid 18BF 068.

5. Construction of plasmids 18BF074 and 19BF 126: PCR amplification of RRE gene fragment (400bp) with pMDLg/pRRE (Addgene, #12251) as template and C-RRE-F (SEQ ID NO:25) and C-RRE-R (SEQ ID NO:26) as primers; C-GagPol-F (SEQ ID NO:27) and C-GagPol-R (SEQ ID NO:28) were used as primers to PCR amplify the gag/pol gene fragment (4336bp), and then the two DNA fragments were digested with XbaI and XhoI and EcoRI and XbaI, respectively, and ligated to EcoRI and XhoI cleavage sites of the 18BF007 plasmid to construct the 18BF074 plasmid. The 18BF074 plasmid was digested with BstBI, and the 8758bp (18BF074) fragment was gel recovered and ligated with T4 ligase to construct plasmid 19BF 126.

6. Plasmids 19BF257, 19BF256 and 19BF075 were constructed: the synthetic sequences SEQ ID NO 7(633bp) and SEQ ID NO 8(1496bp) are respectively cut by ClaI, XhoI and SpeI and AgeI and are sequentially connected with ClaI, XhoI cutting sites and AvrII and AgeI cutting sites of the 18BF007 plasmid, so as to construct the 19BF073 plasmid. The synthetic sequence SEQ ID NO 9(1979bp) is cut by MluI and AgeI and is connected to MluI and AgeI cutting sites of the 18BF007 plasmid to replace a CMV-BGI-MCS-pA fragment, so that the 18BF008 plasmid is constructed. The synthetic sequences SEQ ID NO 10(768bp) and SEQ ID NO 11(765bp) were digested with ClaI and XhoI respectively and ligated to ClaI and XhoI restriction sites of the 18BF008 plasmid, respectively, to construct 18BF085 and 18BF084 plasmids, respectively. The synthetic sequence SEQ ID NO 8(1496bp) is cut by SpeI and AgeI and is respectively connected with the AvrII and AgeI cutting sites of the 18BF085 and 18BF084 plasmids, so as to respectively construct 19BF257 and 19BF256 plasmids. Plasmid 19BF073 was digested with SpeI and AgeI, and the 3821bp fragment was gel recovered and ligated to the AvrII and AgeI cleavage sites of 18BF085 plasmid to construct 19BF075 plasmid.

7. Plasmids 18BF019 and 18BF031 were constructed: the synthetic sequences SEQ ID NO 13(1044bp) and SEQ ID NO 12(1320bp) were digested with BamHI and XhoI and BglII, respectively, and ligated to BamHI and BglII sites of the 18BF011 plasmid to construct an 18BF019 plasmid. The synthetic sequences SEQ ID NO:14(1806bp) and SEQ ID NO:12(1320bp) were digested with BamHI and XhoI and BglII, respectively, and ligated to BamHI and BglII cleavage sites of the 18BF011 plasmid to construct an 18BF031 plasmid.

8. Construction of plasmid 19BF 081: PCR amplification of PGK gene fragment (706bp) with pRRLSIN. cPPT. PGK-GFP. WPRE (Addgene, #12252) as template and hPGK-F (SEQ ID NO:29) and hPGK-R (SEQ ID NO:30) as primers; PCR amplification of luciferase gene fragment (1728bp) was performed using pGL3-Basic (Promega, E1751) as template and Luc-F (SEQ ID NO:31) and Luc-R (SEQ ID NO:32) as primers, after which MluI and BamHI (gel recovery of 538bp fragment) and BamHI and XhoI were used to cleave both DNA fragments separately and ligate them at MluI and XhoI cleavage sites of 19BF126 plasmid to replace the original plasmid DNA sequence and construct 18YYH26 plasmid. The synthetic sequence SEQ ID NO 15(3610bp) is cut by SpeI and AgeI and is connected with SpeI and AgeI cutting sites of the 18BF004 plasmid so as to construct a 19BF080 plasmid. The synthetic sequence SEQ ID NO 16(1320bp) was digested with XhoI and BglII and ligated to the XhoI and BamHI cleavage sites of the 19BF080 plasmid to construct a 19BF214 plasmid. The plasmid 18YYH26 was digested with PacI and XhoI, and the DNA fragment 2272bp was gel-recovered, respectively, and the recovered fragment was ligated to the PacI and XhoI cleavage sites of the 19BF214 plasmid to construct a 19BF081 plasmid.

9. Construction of plasmids 19BF123, 19BF124, 19BF 125: the plasmid 19BF120 was constructed by using 18BF007 as a template, PCR-amplifying a gene fragment (585bp) using CMV (SpeI) -F (SEQ ID NO:33) and Fu-CMV-RU5-R (SEQ ID NO:35) as primers, PCR-amplifying a gene fragment (725bp) using the synthetic sequence SEQ ID NO:15(3610bp) as a template, Fu-CMV-RU5-F (SEQ ID NO:34) and GAG (ClaI) -R (SEQ ID NO:40) as primers, ligating these two DNA fragments by fusion PCR and PCR-amplifying a gene fragment (1282bp) using CMV SpeI) -F (SEQ ID NO:33) and GAG (ClaI) -R (SEQ ID NO:40) as primers, and then enzymatically cleaving with SpeI and ClaI and ligating to the SpeI and CalI sites on the 19BF080 plasmid. The gene fragment (318bp) was amplified by PCR using the synthetic sequence SEQ ID NO 6(315bp), the TRE (SpeI) -F (SEQ ID NO:36) and the Fu-CMV-RU5-R (SEQ ID NO:35) as primers, the gene fragment (725bp) was amplified using the synthetic sequence SEQ ID NO:15(3610bp) as a template and the Fu-CMV-RU5-F (SEQ ID NO:34) and GAG (ClaI) -R (SEQ ID NO:40) as primers, the two DNA fragments were ligated by the fusion PCR method and PCR-amplified using the TRE SpeI-F (SEQ ID NO:36) and GAG (ClaI) -R (SEQ ID NO:40) as primers to obtain a gene fragment (1015bp), and then the SpeI and CalI sites ligated to the 19BF080 plasmid were digested with SpeI and ClaI to construct a plasmid 19BF 121. The gene fragment (308bp) was PCR-amplified using the synthetic sequence SEQ ID NO:5(302bp) as a template, TRE2(SpeI) -F (SEQ ID NO:39) and Fu _ TRE2-RU5-R (SEQ ID NO:38) as primers, the gene fragment (725bp) was PCR-amplified using the synthetic sequence SEQ ID NO:15(3610bp) as a template, and Fu-TRE2-RU5-F (SEQ ID NO:37) and GAG (ClaI) -R (SEQ ID NO:40) as primers, the two pieces of DNA were ligated by fusion PCR and PCR-amplified using TRE2(SpeI) -F (SEQ ID NO:39) and GAG (ClaI) -R (SEQ ID NO:40) as primers to obtain a gene fragment (1004bp), after which was cleaved with SpeI and ClaI and ligated to the SpeI and CalI site on 19BF080 plasmid to construct 19BF 122. The plasmid 19BF081 is cut by PacI and PvuII, the gene fragment 4224bp is recovered by gel, and is respectively connected with the PacI and PvuII cutting sites of the plasmids 19BF120, 19BF121 and 19BF122 to respectively construct plasmids 19BF123, 19BF124 and 19BF 125.

TABLE 1 primer information List

TABLE 2 sequence element composition description

TABLE 3 plasmid functional element description

TABLE 4 plasmid numbering and nomenclature

Example 2: lentiviral vector preparation by transient transfection of Tet-responsive element regulated viral genome transcription cassette constructs in 293T cells

This example induced the transactivator rtTA of the expression System by packaging the Lentiviral genes Rev, VSV-G, gag/pol and Tet-On_advOr rtTA_3GAnd a construct (transfer vector plasmid) of the viral genome transcription cassette carrying the target nucleic acid fragment regulated by the Tet response element of the present invention transiently transfected 293T cells to produce lentiviruses, and the viral genome transcription cassette regulated by the Tet response element of the present invention was compared to the production titer of a viral genome transcription cassette regulated using a constitutively active promoter. This example demonstrates in total 4 transfer vector plasmids 19BF081 (the constitutively active promoter RSV-RU5), 19BF123 (the constitutively active promoter CMV-RU5), 19BF124(TRE1-RU5) and 19BF125(TRE2-RU5) to regulate transcription of a lentiviral genome expression cassette carrying a transgene of interest using RSV or CMV constitutively active promoters, or TRE1 or TRE2Tet response elements as described herein, respectively. In the embodiment, an hPGK-Luciferase-IRES-EGFP sequence is taken as a target nucleic acid fragment, and the virulence production titer of each transfer vector plasmid under the transient production condition is compared based on the Luciferase activity. The specific experimental method is as follows.

293T cells were seeded at 1.5E +05 cells per well in 24-well plates and cultured at 37 ℃ with 5% CO₂In the environment, the culture volume was 500. mu.L of DMEM (Sigam, D6429) medium containing 10% FBS (ExCell, 11H 116). After 24 hours of inoculation, cells were transfected according to the PEI method, as follows: transfection reagents were added at 50. mu.L per well at the time of transfection, containing 0.8. mu.g total plasmid and 3.2. mu.g PEI MAX (Polysciences, 24765-1), where the molar ratio of plasmids pMD2.G (Addge 12259): pMDLg/PRRE (Addge 12251): pRSV-Rev (Addge 12253): rtTA (19BF257 or 19BF 256): the transfer vector plasmid was 1:1:1:1: 1. Wherein the transfer vectors are 19BF081, 19BF123, 19BF124 and 19BF 125; the Tet-On transactivator encoding plasmid was 19BF257 (rtTA)_3G) Or 19BF256 (rtTA)_adv) Multiple wells were set for each sample. 3 hours after transfection, the medium was changed and the inducer (2mmol/L sodium butyrate (Sigma, 303410), 1. mu.g/ml DOX (doxycycline hydrochloride (DOX), Biotechnology (Shanghai) GmbH, A600889)) was added to one replicate well of each sample, and only 2mmol/L sodium butyrate was added to the other replicate well. After further culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500rpm for 15 minutes.

The method for detecting the titer of the Luciferase virus of HT1080 cells is used for detecting the titer of the produced virus of each sample, and the specific operation is as follows: after 24 hours induction of toxigenicity, HT1080 cells were seeded in a 96-well plate (Corning 3916) at 1E +04 cells per well in DMEM complete medium. 1 hour before the titer was detected, the medium of HT1080 cells was changed to DMEM complete medium containing 8. mu.g/ml polybrene (Sigam, H9268). The virus supernatants from the above centrifugation were then added to 96-well plates in 50. mu.l/well of three wells per sample, and 50. mu.L of DMEM complete medium was added to negative control wells. After 48 hours of culture, the mixture is used

The Luciferase Assay System (Promega, E2610) kit was used to detect relative light units RLU (relative light unit) in wells according to the protocol (Promega, FB037) using a fluorescent microplate reader (Perkin Elmer Victor V). The results of the detection are shown in FIG. 1.

The results in FIG. 2 show that when packaging lentiviruses using the method of transient transfection of 293T cells, the production of viral titers using TRE1-RU5(19BF124) and TRE2-RU5(19BF125) Tet chimeric response elements for the control of transcription of the lentivirus genome expression cassette is significantly higher than that of conventional transfer vector plasmids using constitutively active promoters. Use of the 19BF124 plasmid with TRE1-RU5 chimeric response element in combination with rtTA_advIn trans-activator, the toxin-producing titer is 2.93 times or 2.33 times that of RSV-RU5 or CMV-RU5 respectively; in collocation rtTA_3GThe toxin-producing titer was 2.55-fold or 2.02-fold greater than that of RSV-RU5 or CMV-RU5, respectively. Use of the 19BF125 plasmid with TRE2-RU5 chimeric response element in combination with rtTA_advIn the case of transactivator, the titer of toxin production is respectively1.75 times or 1.39 times that of RSV-RU5 or CMV-RU 5; in collocation rtTA_3GThe toxin-producing titer was 1.83-fold or 1.45-fold that of RSV-RU5 or CMV-RU5, respectively.

Example 3: lentiviral vector preparation by transient transfection of Tet response element regulated viral genome transcription cassette constructs in 293T cells stably expressing rtTA

This example produced lentiviruses by transient transfection of 293T cells stably expressing transactivator rtTA with constructs (transfer vector plasmids) comprising the genes rev, VSV-G, gag/pol required for packaging lentiviruses and the viral genome transcription cassettes carrying the nucleic acid fragments of interest under the control of the Tet response element according to the invention, and compared the titer of viral production of the viral genome transcription cassettes under the control of the Tet response element according to the invention versus the use of constitutively active promoters. The transfer vector plasmids used in this example were in accordance with those described in example 2 and were 19BF081, 19BF123, 19BF124 and 19BF125, respectively. The specific experimental method is as follows.

1. 293T-rtTA is constructed by a 'Sleeping Beauty, SB' transposon system_advAnd 293T-rtTA_3GCell line:

293T cells were seeded at 1.5E +06 cells per 60mm dish at 37 ℃ with 5% CO₂In DMEM (Sigam, D6429) complete medium supplemented with 10% FBS (ExCell, 11H 116). After 24 hours of culture, transfection is carried out according to a PEI method, the total plasmid quantity is 5.5ug, and the mass ratio of PEI to plasmid is 4:1, wherein transfection was performed in accordance with a molar ratio of plasmid 19BF256:18BF019 (expressing SB transposase) of 10:1 to obtain 293T-rtTA_advA cell; transfection was carried out according to the molar ratio 10:1 of plasmid 19BF257:18BF019 to obtain 293T-rtTA_3GA cell. After at least three generations of drug selection with Hygromycin (Hygromycin, raw a600230-0001) after transfection, the following experiment was performed after cells were grown under drug selection pressure consistent with the original 293T cells.

2. Comparison of the production titer of the respective transfer vector plasmids by transient transfection in 293T cells stably expressing rtTA

Mix 293T-rtTA_advAnd 293T-rtTA_3GTwo kinds of thinCell lines were seeded at 1.5E +05 cells per well in 24-well plates in a 500. mu.L DMEM complete medium. After 24 hours of inoculation, cells were transfected according to the PEI method, as follows: at the time of transfection, 50. mu.L of transfection reagent containing 0.8. mu.g of total plasmid and 3.2. mu.g of PEIMAX (Polysciences, 24765-1) was added per well at the time of transfection, wherein the molar ratio pMD2.G (Addgene 12259) pMDLg/PRRE (Addgene 12251) pRSV-Rev (Addgene 12253) and transfer vector plasmid were 1:1:1: 1. Wherein the transfer vector was 19BF081(RSV-RU5), 19BF123(CMV-RU5), 19BF124(TRE1-RU5) or 19BF125(TRE2-RU5), and each sample was provided with a plurality of wells. 3 hours after transfection, the medium was changed and the inducer (2mmol/L sodium butyrate, 1. mu.g/ml DOX) was added to one replicate well of each sample and only 2mmol/L sodium butyrate was added to the other replicate well. After further culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500rpm for 15 minutes. The assay for the titer of Luciferase virus in HT1080 cells was performed in the same manner as in example 2, and the results are shown in FIG. 3.

The results in FIG. 3 show that the use of the TRE1-RU5(19BF124) and TRE2-RU5(19BF125) chimeric response elements to regulate transcription of the lentiviral genome expression cassette produces significantly higher viral titers under DOX induction than the traditional transfer vector plasmids using the constitutively active promoters RSV and CMV. At 293T-rtTA_advIn the cells, the induced toxigenic titers of 19BF124(TRE1-RU5) and 19BF125(TRE2-RU5) were 1.57E +06RLU and 1.38E +06RLU, respectively; 6.7 times and 5.9 times of the 19BF081(RSV-RU5) transfer vector plasmid, and 2.48 times and 2.18 times of the 19BF123(CMV-RU5) transfer vector plasmid, respectively. At 293T-rtTA_3GIn the cells, the induced toxigenic titers of 19BF124(TRE1-RU5) and 19BF125(TRE2-RU5) were 2.36E +06RLU and 1.97E +6RLU, respectively; 7.7 times and 6.5 times of the 19BF081(RSV-RU5) transfer vector plasmid, and 2.4 times and 2.0 times of the 19BF123(CMV-RU5) transfer vector plasmid, respectively. Transfection of 19BF124(TRE1-RU5) and 19BF125(TRE2-RU5) transfer vector plasmids in 293T-rtTA under non-inducible conditions_advAnd 293T-rtTA_3GNo significant toxic titer was observed in the cells, demonstrating that the TRE1-RU5 and TRE2-RU5 chimeric Tet response elements are able to transcribe lentiviral genomic RNA and package the virus only in the presence of an inducer.

Example 4: lentiviral vector preparation by transient transfection of viral genome transcription cassette constructs regulated by Tet response elements in EuLV293T packaging cells stably expressing lentiviral packaging genes

In this example, a transposon system of Sleeping Beauty (SB) was first constructed to stably integrate rtTA in genes rev, VSV-G and gag/pol required for lentiviral packaging in 293T genome and in a Tet-On inducible expression system_3GTrans-activator and Cumate induced a EuLV293T stable packaging cell line expressing the CymR repressor of the system. Wherein the expression of rev gene and VSV-G gene is regulated by Tet-On and Cumate composite induction expression system, and plasmids for constructing cell line are 18BF072 and 18BF 068; the expression of the gag/pol gene is regulated by a CMV promoter, and a plasmid for constructing a cell line is 18BF 074; trans activator rtTA_3GAnd the expression of the repressor CymR protein are regulated by the CAGGS promoter and the CMV promoter, respectively, and expression cassettes (expression cassettes) for the two proteins are constructed together on a 19BF075 plasmid. The viral titer of the viral genomic transcription cassette regulated by the Tet response element of the invention versus a constitutively active promoter was then compared by transient transfection of the construct (transfer vector plasmid) of the viral genomic transcription cassette carrying the nucleic acid fragment of interest of the invention into the EuLV293T packaging cells described above. The transfer vector plasmids used in this example were in accordance with those described in example 2 and were 19BF081, 19BF123, 19BF124 and 19BF125, respectively. The specific experimental method is as follows:

1. construction of EuLV293T Lentiviral packaging cell line by the SB transposon System

The experimental procedures of cell culture, PEI transfection and hygromycin screening were as described in examples 2 and 3. 293T cells (ATCC, CRL3216) were seeded as 1.5E +06 cells in a60 mm dish in 3ml DMEM complete medium (DMEM (Sigma, D6429) supplemented with 10% FBS (ExCell, 11H116)) at 37 ℃ with 5% CO₂Incubated under conditions for 24 hours. And (3) carrying out plasmid transfection by a PEI transfection method, wherein the total plasmid amount is 5 mu g, and the mass ratio of PEI to the total plasmid is 4:1, wherein the transfection is carried out according to the molar ratio of 19BF075:19BF72:18BF068:19BF74:18BF019 being 3:3:2:12: 2. Post-transfection 2After 4 hours, the cells were inoculated into a 100mm dish and cultured, and 200. mu.g/ml hygromycin (Bio-worker A600230-0001) was added for selection. The screening pressure is maintained for subculturing until the cells stably grow, and the judgment basis is as follows: (1) the growth rate of the cells is the same as that of original 293T cells, (2) the SB expression plasmid 18BF019 basically disappears, and the proportion of ECFP positive cells drops to below 1%, and (3) the proportion of dead cells is less than 5%. Finally obtaining rtTA stably integrated in genome_3GCymR, rev, VSV-G and gag/pol protein coding sequences and designated EuLV 293T.

2. Comparison of the production titer of each transfer vector plasmid by transient transfection in EuLV293T packaging cells

EuLV293T cells were cultured at 37 ℃ with 5% CO₂The medium was DMEM (Sigam, D6429) medium containing 10% FBS (ExCell, 11H116) in the environment. Cell lines were seeded at 1.5E +05 cells per well in 24-well plates at a culture volume of 500 μ L. At the time of transfection, 50. mu.L of a transfection reagent containing 0.26. mu.g of the transfer vector plasmid, 0.54. mu.g of the 18BF003 plasmid and 3.2. mu.g of PEI MAX (Polysciences, 24765-1) was added to each well, and the wells were each set up. 3h after transfection, the medium was changed to complete medium containing 5mmol/L sodium butyrate, and 1. mu.g/ml DOX and 200. mu.g/ml Cumate (Aladdin, I107765) were added to one of the wells of each sample replicate to set as an induction group; the other well was filled with an equal amount of medium and set as a non-induced group. After further culturing for 48 hours, the virus supernatant was collected by centrifugation at 4500rpm for 15 minutes. The assay for the titer of Luciferase virus in HT1080 cells was performed in the same manner as in example 2, and the results are shown in FIG. 4.

FIG. 4 shows that the transfer vector plasmids using TRE1-RU5(19BF124) and TRE2-RU5(19BF125) Tet chimeric response elements to regulate transcription of lentiviral genome expression cassettes are significantly higher in virus titer under induction by addition of DOX and Cumate than the conventional transfer vector plasmids using constitutively active promoters RSV and CMV. TRE1-RU5(19BF124) and TRE2-RU5(19BF125) had induced toxigenic titers of 2.42E +06RLU and 2.14E +06RLU, respectively; 3.4 times and 3.0 times of the 19BF081(RSV-RU5) and 1.6 times and 1.4 times of the 19BF123(CMV-RU5) transfer vector plasmid, respectively. Under non-inducing conditions, all transfer vector plasmids had no apparent challenge titers in EuLV293T cells, demonstrating that EuLV293T cells transcribe the lentiviral genome and package lentiviruses only in the presence of an inducer.

Example 5: production of lentiviruses in lentivirus-stable producer cell lines by viral genome transcription cassettes regulated by Tet-responsive elements

This example first stably integrates viral genome transcription cassettes carrying hPGK-Luciferase-EGFP target nucleic acid fragments, whose transcription is regulated by different promoters or Tet-responsive elements, into the genome of EuLV293T packaging cells prepared in example 4 via a "piggybac (pb) transposon system derived from Trichoplusia ni (Trichoplusia ni), wherein the transfer vector plasmids for constructing lentivirus-producing cell lines are: 19BF081, 19BF123, 19BF124 and 19BF 125. And then preparing lentiviruses in an induction mode, and comparing the virus genome transcription cassette regulated by the Tet response element with the virus genome transcription cassette regulated by a constitutive active promoter to generate the titer of the virus. The specific experimental method is as follows:

1. construction of lentivirus-stable producer cell lines by the PB transposon system

EuLV293T cells were seeded at 1.5E +06 cells per well in 60mm dishes in 3ml DMEM complete medium at 37 ℃ with 5% CO₂After 24 hours of culture under the conditions, transfection was performed according to the PEI method, the total plasmid amount was 5.5ug, wherein transfection was performed according to the molar ratio of transfer vector plasmid: 18BF031 (expressing PB transposase) of 10:1, and the total PEI amount was 22 ug. Wherein the transfer vector plasmid is 19BF081, 19BF123, 19BF124 or 19BF 125. At least 3 passages were screened for cell line stability by the addition of 2.5. mu.g/ml Puromycin (Puromycin, Alantin P113126) 24 hours after transfection to give 4 lentivirus stable production cell lines, EuLV293T-19BF081, EuLV293T-19BF123, EuLV293T-19BF124 and EuLV293T-19BF125, respectively.

2. Comparison of the production Capacity of lentivirus Stable production cell lines constructed Using different transfer vector plasmids

The four lentivirus-stable producer cells described above were seeded at 1.5E +05 cells per well in 24-well plates at a culture volume of 500. mu.L, with duplicate wells set for each cell. After 24 hours of incubation, the medium was replaced and the induction group was set by adding the inducer (5mmol/L sodium butyrate, 1. mu.g/ml DOX and 200. mu.g/ml Cumate) to one of the duplicate wells and the non-induction group was set by adding only the same amount of sodium butyrate to the duplicate wells. After further incubation for 48h, the virus supernatant was collected by centrifugation at 4500rpm for 15 minutes. The assay for the titer of Luciferase virus in HT1080 cells was performed in the same manner as in example 2, and the results are shown in FIG. 5.

FIG. 5 shows the results of stable producer cell lines using TRE1-RU5(19BF124) and TRE2-RU5(19BF125) Tet chimeric response elements to regulate transcription of lentiviral genome expression cassettes at significantly higher titer of virus production induced by addition of DOX and Cumate than stable producer cell lines using conventional RSV-RU5 and CMV-RU5 chimeric promoters to regulate transcription of lentiviral genome expression cassettes. Induced titer of 3.64E +07RLU and 2.37E +07RLU in EuLV293T-19BF124(TRE1-RU5) and EuLV293T-19BF125(TRE2-RU5) producer cell lines; 9.1-fold and 5.9-fold induction of virulence titres in EuLV293T-19BF081(RSV-RU5) producer cells, respectively; 2.46-fold and 1.61-fold induction of toxin titer in EuLV293T-19BF123(CMV-RU5) producer cells, respectively. Under non-inducing conditions, no virus titers were detected in all lentivirus stable producer cell lines.

Sequence listing

<110> Shenzhen Shenyan Biotech Co., Ltd

<120> promoter element, retroviral genome transcription cassette comprising the same, and vector

<130> P20200002B

<160> 40

<170> PatentIn version 3.5

<210> 1

<211> 1893

<212> DNA

<213> Artificial sequence

<400> 1

actgcggccg ccctgcaggt caactagtga cgtcttaatt aattgccggc tggaacgcgt 60

ttcgaacatc gattgaattc tggccaagtg gatccgctag ctctagagtc gacggtacca 120

agcttgcctc gagccatgga gatctgcatg ccctaggtcc ggaaccggtt ggcgcgccat 180

ctggcagcga tcgccgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 240

atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 300

tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 360

tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 420

gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 480

aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 540

gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 600

ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 660

gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 720

gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 780

atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 840

ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 900

cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 960

cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 1020

gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 1080

cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 1140

cactgattaa gcattggtaa cgtacggaag ttagagaaaa ggcataagta gaaaagatca 1200

aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 1260

caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 1320

taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 1380

gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 1440

cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 1500

taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 1560

agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 1620

ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 1680

gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 1740

acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 1800

acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcatctca 1860

tgaaaattat gcaaattgag ccagtcaggc agt 1893

<210> 2

<211> 2900

<212> DNA

<213> Artificial sequence

<400> 2

gcggccgcat ctatacagtt gaagtcggaa gtttacatac acttaagttg gagtcattaa 60

aactcgtttt tcaactactc cacaaatttc ttgttaacaa acaatagttt tggcaagtca 120

gttaggacat ctactttgtg catgacacaa gtcatttttc caacaattgt ttacagacag 180

attatttcac ttataattca ctgtatcaca attccagtgg gtcagaagtt tacatacact 240

aagttgactg tgcctttaaa cagcttggaa aattccagaa aatgatgtca tggctttagc 300

ctgcagggag ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 360

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 420

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 480

ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tggggggata cggggaaaag 540

gacctgcagg tcaactagtg acgtcttaat taattgccgg ctggacgtac gcgtctagtt 600

attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta 660

cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 720

caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 780

tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta 840

cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 900

ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg 960

tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 1020

caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 1080

ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 1140

gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc 1200

cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgc ggattcgaat 1260

cccggccggg aacggtgcat tggaacgcgg attccccgtg ccaagagtga cgtaagtacc 1320

gcctatagag tctataggcc cacaaaaaat gctttcttct tttaatatac ttttttgttt 1380

atcttatttc taatactttc cctaatctct ttctttcagg gcaataatga tacaatgtat 1440

catgcctctt tgcaccattc taaagaataa cagtgataat ttctgggtta aggcaatagc 1500

aatatttctg catataaata tttctgcata taaattgtaa ctgatgtaag aggtttcata 1560

ttgctaatag cagctacaat ccagctacca ttctgctttt attttatggt tgggataagg 1620

ctggattatt ctgagtccaa gctaggccct tttgctaatc atgttcatac ctcttatctt 1680

cctcccacag ctcctgggca acgtgctggt ctgtgtgctg gcccatcact ttggcaaaga 1740

attgggattc gaacatcgat tgaattctgg ccaggatccg ctagctctag agtcgacggt 1800

accagtacta agcttgcctc gagccatgga gatctacggg tggcatccct gtgacccctc 1860

cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat 1920

aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag 1980

gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta 2040

ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg 2100

ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg caagcatgac 2160

caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct 2220

ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt 2280

acaggcgtga accactgctc ccttccctgt ccttgcatgc cctaggcagc tgtccggaac 2340

cggtgtttaa acaggccttt tccccgtatc cccccaggtg tctgcaggct caaagagcag 2400

cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg 2460

cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct 2520

cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg 2580

ctgtccctca ggccttggcg cgccctaaag ccatgacatc attttctgga attttccaag 2640

ctgtttaaag gcacagtcaa cttagtgtat gtaaacttct gacccactgg aattgtgata 2700

cagtgaatta taagtgaaat aatctgtctg taaacaattg ttggaaaaat gacttgtgtc 2760

atgcacaaag tagatgtcct aactgacttg ccaaaactat tgtttgttaa caagaaattt 2820

gtggagtagt tgaaaaacga gttttaatga ctccaactta agtgtatgta aacttccgac 2880

ttcaactgta tagcgatcgc 2900

<210> 3

<211> 1386

<212> DNA

<213> Artificial sequence

<400> 3

gcggccgcaa cacgcagcta gattaaccct agaaagataa tcatattgtg acgtacgtta 60

aagataatca tgcgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt 120

tattaatttg aatagatatt aagttttatt atatttacac ttacatacta ataataaatt 180

caacaaacaa tttatttatg tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc 240

ttctataaag taacaaaact tttatcctgc agggagggac agcccccccc caaagccccc 300

agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc 360

tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac 420

ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca 480

gacacctggg gggatacggg gaaaaggacc tgcaggtcaa ctagtgacgt cttaattaat 540

tgccggctgg acgtacgcgt tttggcaaaa tcgattgaat tctggccaag tggatccgct 600

agctctagag tcgacggtac cagtactaag cttgcctcga ggatatccca tggagatcta 660

tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt 720

cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagcat 780

gccctaggca gctgtccgga accggtgttt aaacaggcct tttccccgta tccccccagg 840

tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc gtgccacctt 900

ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg agcgccggac 960

cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac gtaattacat 1020

ccctgggggc tttggggggg ggctgtccct caggccttgg cgcgccatat ctataacaag 1080

aaaatatata tataataagt tatcacgtaa gtagaacacg aaataacaat ataattatcg 1140

tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg 1200

ttgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc 1260

caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga 1320

gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaagc 1380

gatcgc 1386

<210> 4

<211> 915

<212> DNA

<213> Artificial sequence

<400> 4

acgcgttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata 60

gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc 120

ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct 180

acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata 240

gagaacgtat taggcgtgta cggtgggcgc ctataaaagc agagctcgtt tagtgaaccg 300

tcagatcgcc tggagcaatt ccacatacaa acagaccaga ttgtctgttt gttacacttt 360

tgtcttatac caactttccg taccacttcc taccctcgta aattcgaatc ccggccggga 420

acggtgcatt ggaacgcgga ttccccgtgc caagagtgac gtaagtaccg cctatagagt 480

ctataggccc acaaaaaatg ctttcttctt ttaatatact tttttgttta tcttatttct 540

aatactttcc ctaatctctt tctttcaggg caataatgat acaatgtatc atgcctcttt 600

gcaccattct aaagaataac agtgataatt tctgggttaa ggcaatagca atatttctgc 660

atataaatat ttctgcatat aaattgtaac tgatgtaaga ggtttcatat tgctaatagc 720

agctacaatc cagctaccat tctgctttta ttttatggtt gggataaggc tggattattc 780

tgagtccaag ctaggccctt ttgctaatca tgttcatacc tcttatcttc ctcccacagc 840

tcctgggcaa cgtgctggtc tgtgtgctgg cccatcactt tggcaaagaa ttgggattcg 900

aaatcgattg aattc 915

<210> 5

<211> 302

<212> DNA

<213> Artificial sequence

<400> 5

actagtgcgt cgagattcgc gttactccct atcagtgata gagaacgtat gaagagttta 60

ctccctatca gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt 120

ataaggagtt tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt 180

gatagagaac gtatctacag tttactccct atcagtgata gagaacgtat atccagttta 240

ctccctatca gtgatagaga acgtattagg cgtgtacggt gggcgcctat aaaagcagag 300

ct 302

<210> 6

<211> 315

<212> DNA

<213> Artificial sequence

<400> 6

actagtcgag gatcgttcga gcgagtttac tccctatcag tgatagagaa cgtatgtcga 60

gtttactccc tatcagtgat agagaacgat gtcgagttta ctccctatca gtgatagaga 120

acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt tactccctat 180

cagtgataga gaacgtatgt cgagtttatc cctatcagtg atagagaacg tatgtcgagt 240

ttactcccta tcagtgatag agaacgtatg tcgaggtagg cgtgtacggt gggaggccta 300

taaaagcaga gctcg 315

<210> 7

<211> 633

<212> DNA

<213> Artificial sequence

<400> 7

atcgatgcca ccatgtctcc aaagaggaga acccaggcag agagggcaat ggagacacag 60

ggcaagctga tcgccgccgc cctgggcgtg ctgagggaga agggatacgc aggcttccgc 120

atcgccgatg tgccaggagc cgccggcgtg tcccggggcg cacagtctca ccacttccct 180

accaagctgg agctgctgct ggccacattt gagtggctgt atgagcagat caccgagagg 240

agccgcgcca ggctggcaaa gctgaagcca gaggacgatg tgatccagca gatgctggac 300

gatgccgccg agttctttct ggacgatgac tttagcatct ccctggatct gatcgtggcc 360

gccgatagag accccgccct gagggagggc atccagagga cagtggagag aaacaggttc 420

gtggtggagg atatgtggct gggcgtgctg gtgtctcgcg gcctgagccg ggatgacgca 480

gaggacatcc tgtggctgat ctttaacagc gtgcggggcc tggccgtgag atccctgtgg 540

cagaaggaca aggagcggtt cgagcgcgtg cggaattcca ccctggagat cgccagagag 600

aggtacgcca agtttaagag atgataactc gag 633

<210> 8

<211> 1496

<212> DNA

<213> Artificial sequence

<400> 8

actagtgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg 60

caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca 120

ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact 180

ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta 240

atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag 300

tgaggaggct tttttggagg ccataggctt ttgcaaaaag ctatgaaaaa gcctgaactc 360

acagcgactt ctgttgagaa gtttctgatc gaaaagttcg acagcgttag cgacctgatg 420

cagctctcgg agggcgagga atctagggct ttcagcttcg atgtaggagg gcgtggatat 480

gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac 540

tttgcatcgg ctgcgctccc gattcccgaa gtgcttgaca ttggggagtt cagcgagagc 600

ctgacctatt gcatctcccg ccgcgcacag ggcgtaactt tgcaagacct ccctgaaacc 660

gaactgcccg ctgttctaca acctgtcgcg gaggctatgg acgctattgc tgctgccgat 720

ctttcccaga cttccgggtt cggcccattt ggaccgcaag gaatcggtca atacactaca 780

tggcgtgatt tcatttgcgc gattgctgat ccccatgtgt atcattggca aactgtgatg 840

gatgataccg tcagcgcgag tgtcgcgcag gctctcgatg agctgatgct ttgggccgag 900

gattgccccg aagttcgcca cttggtccac gcggatttcg gcagcaacaa tgtcctgaca 960

gataatggcc gcataacagc ggtcattgat tggagcgaag ctatgttcgg ggattcccaa 1020

tacgaggtcg ctaacatctt tttctggcgt ccttggttgg cttgtatgga gcagcaaacg 1080

cgctactttg aaagacgaca tccagagctt gcaggatcgc ctcggctccg ggcgtatatg 1140

ctccgcattg gtcttgacca actctatcag agcttggtgg acggcaattt cgatgatgct 1200

gcttgggcgc agggtcgatg tgatgcaatc gtccgaagtg gagccgggac tgtcgggcga 1260

acacaaatcg cccgcagaag cgcagccgtc tggaccgatg gctgtgtaga agttctcgcc 1320

gatagtggaa acagacgccc ctctactcgt ccgagggcaa aggaatagaa cttgtttatt 1380

gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 1440

ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta accggt 1496

<210> 9

<211> 1979

<212> DNA

<213> Artificial sequence

<400> 9

acgcgtgaca ttgattattg acatgttatt aatagtaatc aattacgggg tcattagttc 60

atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120

cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180

tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240

tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300

ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360

acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc 420

ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg 480

cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg 540

ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa 600

agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc 660

gggcgttcga aggagtcgct gcgacgctgc cttcgccccg tgccccgctc cgccgccgcc 720

tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac 780

ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg 840

tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg 900

gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc 960

ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg 1020

agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc 1080

gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa 1140

ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc 1200

cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg 1260

ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc 1320

cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg 1380

tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg 1440

cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa 1500

atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct 1560

cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc 1620

ttctggcgtg tgaccggcgg ctcttgagcc tctgctaacc atgttcatgc cttcttcttt 1680

ttcctacagc ttcgaacctg ggcaacgtgc tggttattgt gctgtctcat cattttggca 1740

aaatcgattg aattctggcc aagtggatcc gctagctcta gagtcgacgg taccagtact 1800

aagcttgcct cgaggatatc ccatggagat ctatggggac atcatgaagc cccttgagca 1860

tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt 1920

gtgtctctca ctcggaagga catatgggag catgccctag gcagctgtcc ggaaccggt 1979

<210> 10

<211> 768

<212> DNA

<213> Artificial sequence

<400> 10

atcgatgcca ccatgagccg cctggataag tccaaagtga tcaactctgc cctggagctg 60

ctgaatggag tgggaatcga gggactgacc acaaggaagc tggcacagaa gctgggagtg 120

gagcagccta ccctgtactg gcacgtgaag aacaagcgcg ccctgctgga cgcactgcca 180

atcgagatgc tggatcggca ccacacacac agctgcccac tggagggaga gtcctggcag 240

gattttctgc ggaacaatgc caagtcttat agatgtgcac tgctgagcca cagggacgga 300

gcaaaggtgc acctgggaac caggcccaca gagaagcagt acgagaccct ggagaaccag 360

ctggccttcc tgtgccagca gggcttttcc ctggagaatg ccctgtatgc cctgtctgcc 420

gtgggccact ttaccctggg atgcgtgctg gaggagcagg agcaccaggt ggccaaggag 480

gagagagaga caccaaccac agatagcatg ccccctctgc tgaagcaggc catcgagctg 540

ttcgacaggc agggagcaga gccagccttc ctgtttggcc tggagctgat catctgcggc 600

ctggagaagc agctgaagtg tgagtccgga ggacctacag acgcactgga cgatttcgac 660

ctggatatgc tgccagccga tgccctggac gattttgacc tggatatgct gcccgccgac 720

gccctggatg actttgacct ggacatgctg cctggctgat aactcgag 768

<210> 11

<211> 765

<212> DNA

<213> Artificial sequence

<400> 11

atcgatgcca ccatgtccag actggacaag agcaaagtca taaacggcgc tctggaatta 60

ctcaatggag tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt 120

gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctgcca 180

atcgagatgc tggacaggca tcatacccac ttctgccccc tggaaggcga gtcatggcaa 240

gactttctgc ggaacaacgc caagtcattc cgctgtgctc tcctctcaca tcgcgacggg 300

gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag 360

ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc 420

gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt agcaaaagag 480

gaaagagaga cacctaccac cgattctatg cccccacttc tgagacaagc aattgagctg 540

ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc 600

ctggagaaac agctaaagtg cgaaagcggc gggccggccg acgcccttga cgattttgac 660

ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac 720

gctcttgacg attttgacct tgacatgctc cccgggtaac tcgag 765

<210> 12

<211> 1320

<212> DNA

<213> Artificial sequence

<400> 12

ctcgaggccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag 60

gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga 120

gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 180

ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 240

gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 300

ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc 360

agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 420

tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 480

ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga 540

accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggtg 600

agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 660

gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 720

ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 780

accaccctga cctggggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 840

gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 900

gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 960

cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 1020

gagtacaact acatcagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc 1080

aaggccaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 1140

taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 1200

agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 1260

gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagatct 1320

<210> 13

<211> 1044

<212> DNA

<213> Artificial sequence

<400> 13

ggatccgcca ccatgggcaa gtccaaggag atctctcagg acctgagaaa gaggatcgtg 60

gatctgcaca agagcggcag ctccctggga gcaatctcca agcgcctggc agtgcctcgg 120

tctagcgtgc agaccatcgt gcgcaagtac aagcaccacg gcaccacaca gccttcttat 180

cggagcggcc ggagaagggt gctgagccca cgcgacgagc ggacactggt gcgcaaggtg 240

cagatcaacc cccggaccac agccaaggat ctggtgaaga tgctggagga gaccggcaca 300

aaggtgtcca tctctaccgt gaagagagtg ctgtacaggc acaacctgaa gggccactcc 360

gccagaaaga agcctctgct gcagaatagg cacaagaagg caaggctgag gttcgcaacc 420

gcacacggcg acaaggatcg cacattttgg cggaacgtgc tgtggtctga cgagaccaag 480

atcgagctgt tcggccacaa tgatcacaga tacgtgtgga ggaagaaggg cgaggcctgc 540

aagcccaaga ataccatccc tacagtgaag cacggaggag gctccatcat gctgtgggga 600

tgttttgcag caggaggaac aggcgccctg cacaagatcg acggcatcat ggatgccgtg 660

cagtatgtgg acatcctgaa gcagcacctg aagacctctg tgagaaagct gaagctgggc 720

aggaagtggg tgttccagca cgacaacgat ccaaagcaca caagcaaggt ggtggccaag 780

tggctgaagg acaataaggt gaaggtgctg gagtggccca gccagtcccc tgatctgaac 840

ccaatcgaga atctgtgggc cgagctgaag aagagagtga gggcccggag acccaccaac 900

ctgacacagc tgcaccagct gtgccaggag gagtgggcca agatccaccc aaattactgt 960

ggcaagctgg tggagggcta tcccaagagg ctgacccagg tgaagcagtt taagggcaac 1020

gccacaaagt attgataact cgag 1044

<210> 14

<211> 1806

<212> DNA

<213> Artificial sequence

<400> 14

ggatccgcca ccatgggcag ctccctggac gatgagcaca tcctgtccgc cctgctgcag 60

tctgacgatg agctggtggg cgaggacagc gattccgagg tgagcgacca cgtgtccgag 120

gacgatgtgc agagcgacac agaggaggcc ttcatcgatg aggtgcacga ggtgcagcca 180

acctctagcg gcagcgagat cctggatgag cagaacgtga tcgagcagcc tggctcctct 240

ctggcctcca ataagatcct gaccctgcca cagaggacaa tccgcggcaa gaacaagcac 300

tgctggtcta ccagcaagcc tacacggaga tcccgggtgt ctgccctgaa ccacgtgcgg 360

tcccagagag gcccaaccag gatgtgccgc aatatctacg accccctgct gtgctttaag 420

ctgttcttta cagatgagat catcagcgag atcgtgaagt ggaccaacgc cgagatctcc 480

ctgaagaggc gcgagagcat gacctccgcc acattcaggg acaccaatga ggatgagatc 540

tacgccttct ttggcatcct ggtcatgaca gccgtgcgga aggacaacca catgagcacc 600

gacgatctgt ttgatagatc cctgtctatg gtgtacgtga gcgtgatgag cagggaccgc 660

ttcgattttc tgatccggtg cctgagaatg gacgataagt ccatccggcc tacactgaga 720

gagaatgacg tgttcacccc agtgaggaag atctgggatc tgtttatcca ccagtgtatc 780

cagaactaca caccaggagc acacctgacc atcgacgagc agctgctggg cttccggggc 840

agatgccctt ttcgcgtgta catcccaaat aagccctcta agtatggcat caagatcctg 900

atgatgtgcg atagcggcac caagtacatg atcaacggca tgccatatct gggcaggggc 960

acccagacaa atggcgtgcc cctgggcgag tactatgtga aggagctgtc caagcctgtg 1020

cacggctctt gccgcaacat cacatgtgac aattggttca cctctatccc cctggccaag 1080

aacctgctgc aggagcctta taagctgacc atcgtgggca cagtgaggag caacaagcgc 1140

gagatccccg aggtgctgaa gaatagcagg tcccgccctg tgggcacatc catgttctgc 1200

tttgatggcc cactgaccct ggtgtcttac aagcccaagc ctgccaagat ggtgtatctg 1260

ctgagctcct gtgacgagga tgcctctatc aacgagagca ccggcaagcc ccagatggtc 1320

atgtactata atcagacaaa gggcggcgtg gacaccctgg atcagatgtg cagcgtgatg 1380

acctgttccc ggaagacaaa tagatggcct atggccctgc tgtacggcat gatcaacatc 1440

gcctgcatca attctttcat catctatagc cacaacgtgt ctagcaaggg cgagaaggtg 1500

cagagcagga agaagttcat gcgcaatctg tacatgggcc tgacatcctc ttttatgcgg 1560

aagagactgg aggcccccac cctgaagagg tatctgcgcg acaacatctc caatatcctg 1620

cctaaggagg tgccaggcac ctccgacgat tctacagagg agccagtgac caagaagcgg 1680

acctactgca catattgtcc ctccaagatc cggagaaagg cctctgccag ctgcaagaag 1740

tgtaagaaag tgatctgtag agagcacaac atcgacatgt gccagtcttg tttttgataa 1800

ctcgag 1806

<210> 15

<211> 3610

<212> DNA

<213> Artificial sequence

<400> 15

actagttaag tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta 60

gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg 120

gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac 180

tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc 240

tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 300

taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 360

ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg 420

cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact 480

cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa 540

attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg 600

gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat 660

aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc 720

ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag 780

acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat 840

caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac 900

aaaagaatcg atattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga 960

aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac 1020

tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt 1080

gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac 1140

agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga 1200

tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc 1260

ttggaatgct agttggagaa ttcttcgaac ctgcaggatg gtaccaaggc ctttttaaaa 1320

gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag 1380

acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt gtttaaactt 1440

aattaattta aatacgcgtt gcgctagctc tagacccggg ctcgaggtcg accgggatcc 1500

gatatcatat gaatcaacct ctggattaca aaatttgtga aagattgact ggtattctta 1560

actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta 1620

ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt 1680

atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg 1740

caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt 1800

tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag 1860

gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc 1920

catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc 1980

cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc 2040

ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc 2100

ctggcggccg cttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa 2160

aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg 2220

cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag 2280

ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc 2340

gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa 2400

tctctagcag gcgatcgcaa cttgtttatt gcagcttata atggttacaa ataaagcaat 2460

agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 2520

aaactcatca atgtatctta cctagtgtgt gtcagttagg gtgtggaaag tccccaggct 2580

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 2640

agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 2700

ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 2760

ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 2820

ctgagctatt ccagaagtag tgaggaggct tttttggagg ccataggctt ttgcaaaaag 2880

ctatgactga gtacaagccc acggtgcgcc tcgccacccg cgacgatgtt cctagagccg 2940

tccgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtagaccctg 3000

accgccacat cgagagggtc acagagctgc aagagctgtt tctcacgcgc gtcgggctcg 3060

acatcggcaa ggtgtgggtc gcggacgacg gcgcagcggt ggcggtctgg accacgccgg 3120

agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg 3180

gttcccggct ggccgcgcag caacagatgg agggacttct ggcaccgcac cgacccaagg 3240

agcccgcgtg gttcctggct accgtcggtg tctcgcccga ccaccagggc aagggtctgg 3300

gctccgccgt cgttctcccc ggagtggagg cagccgagag agccggggtg cccgcctttc 3360

tggaaacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg 3420

ccgatgtcga ggtgcccgaa ggaccgcgta cctggtgcat gacccgcaag cccggtgcct 3480

gaaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 3540

caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 3600

cttaaccggt 3610

<210> 16

<211> 1320

<212> DNA

<213> Artificial sequence

<400> 16

ctcgaggccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag 60

gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga 120

gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 180

ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 240

gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 300

ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc 360

agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 420

tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 480

ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga 540

accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggtg 600

agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 660

gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 720

ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 780

accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 840

gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 900

gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 960

cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 1020

gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc 1080

aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 1140

taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 1200

agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 1260

gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagatct 1320

<210> 17

<211> 275

<212> DNA

<213> Artificial sequence

<400> 17

tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgat 60

gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga 120

tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttatc 180

cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg 240

tcgaggtagg cgtgtacggt gggaggccta taaaa 275

<210> 18

<211> 269

<212> DNA

<213> Artificial sequence

<400> 18

tccctatcag tgatagagaa cgtatgaaga gtttactccc tatcagtgat agagaacgta 60

tgcagacttt actccctatc agtgatagag aacgtataag gagtttactc cctatcagtg 120

atagagaacg tatgaccagt ttactcccta tcagtgatag agaacgtatc tacagtttac 180

tccctatcag tgatagagaa cgtatatcca gtttactccc tatcagtgat agagaacgta 240

ttaggcgtgt acggtgggcg cctataaaa 269

<210> 19

<211> 479

<212> DNA

<213> Artificial sequence

<400> 19

tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgat 60

gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga 120

tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttatc 180

cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg 240

tcgaggtagg cgtgtacggt gggaggccta taaaagcaga gctcgtttgc ttgtactggg 300

tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 360

cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 420

gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcag 479

<210> 20

<211> 473

<212> DNA

<213> Artificial sequence

<400> 20

tccctatcag tgatagagaa cgtatgaaga gtttactccc tatcagtgat agagaacgta 60

tgcagacttt actccctatc agtgatagag aacgtataag gagtttactc cctatcagtg 120

atagagaacg tatgaccagt ttactcccta tcagtgatag agaacgtatc tacagtttac 180

tccctatcag tgatagagaa cgtatatcca gtttactccc tatcagtgat agagaacgta 240

ttaggcgtgt acggtgggcg cctataaaag cagagctcgt ttgcttgtac tgggtctctc 300

tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 360

cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 420

ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cag 473

<210> 21

<211> 38

<212> DNA

<213> Artificial sequence

<400> 21

gctcatcgat gccaccatga agtgcctttt gtacttag 38

<210> 22

<211> 35

<212> DNA

<213> Artificial sequence

<400> 22

caggctcgag ctattacttt ccaagtcggt tcatc 35

<210> 23

<211> 34

<212> DNA

<213> Artificial sequence

<400> 23

cgatatcgat gccaccatgg caggaagaag cgga 34

<210> 24

<211> 34

<212> DNA

<213> Artificial sequence

<400> 24

catgctcgag ttactattct ttagctcctg actc 34

<210> 25

<211> 29

<212> DNA

<213> Artificial sequence

<400> 25

catggatcta gaaggagctt tgttccttg 29

<210> 26

<211> 33

<212> DNA

<213> Artificial sequence

<400> 26

caggctcgag aagcttgtgt aattgttaat ttc 33

<210> 27

<211> 33

<212> DNA

<213> Artificial sequence

<400> 27

gcacgaattc gccaccatgg gtgcgagagc gtc 33

<210> 28

<211> 36

<212> DNA

<213> Artificial sequence

<400> 28

gcagtctaga ctattaatcc tcatcctgtc tacttg 36

<210> 29

<211> 29

<212> DNA

<213> Artificial sequence

<400> 29

catacgcgtg cttgatatcg aattccacg 29

<210> 30

<211> 24

<212> DNA

<213> Artificial sequence

<400> 30

cagatgaact tcagggtcag cttg 24

<210> 31

<211> 31

<212> DNA

<213> Artificial sequence

<400> 31

tcaggatcca tctgcgatct aagtaagctt g 31

<210> 32

<211> 29

<212> DNA

<213> Artificial sequence

<400> 32

tcaactcgag ctagaattac acggcgatc 29

<210> 33

<211> 36

<212> DNA

<213> Artificial sequence

<400> 33

gcactcacta gtcgcgtcta gttattaata gtaatc 36

<210> 34

<211> 64

<212> DNA

<213> Artificial sequence

<400> 34

tacggtggga ggcctataaa agcagagctc gtttgcttgt actgggtctc tctggttaga 60

ccag 64

<210> 35

<211> 30

<212> DNA

<213> Artificial sequence

<400> 35

gctctgcttt tataggcctc ccaccgtaca 30

<210> 36

<211> 28

<212> DNA

<213> Artificial sequence

<400> 36

cgatccacta gtcgaggatc gttcgagc 28

<210> 37

<211> 64

<212> DNA

<213> Artificial sequence

<400> 37

gtacggtggg cgcctataaa agcagagctc gtttgcttgt actgggtctc tctggttaga 60

ccag 64

<210> 38

<211> 29

<212> DNA

<213> Artificial sequence

<400> 38

agctctgctt ttataggcgc ccaccgtac 29

<210> 39

<211> 31

<212> DNA

<213> Artificial sequence

<400> 39

cgaccgacta gtgcgtcgag attcgcgtta c 31

<210> 40

<211> 30

<212> DNA

<213> Artificial sequence

<400> 40

gtcatatcga ttcttttgtt ttgctcttcc 30

Claims (15)

1. A nucleic acid sequence comprising the response element TRE of the Tet-On system and the R-U5 domain in the retroviral Long Terminal Repeat (LTR).

2. The nucleic acid sequence of claim 1, wherein the R-U5 functional domain is downstream of and spaced from the TATA box of a TRE by 15-30bp, preferably by 24 bp.

3. The nucleic acid sequence of claim 1, wherein the sequence of the TRE is as set forth in SEQ ID NO 17 or SEQ ID NO 18.

4. The nucleic acid sequence of claim 1, which has the sequence shown in SEQ ID NO 19 or SEQ ID NO 20.

5. A retroviral genomic transcription cassette comprising a response element TRE of the Tet-On system or the nucleic acid sequence of any one of claims 1 to 4 for controlling transcription of said transcription cassette, a cis-acting element for retroviral packaging located downstream of the response element TRE of the Tet-On system or the nucleic acid sequence of any one of claims 1 to 4 and a multiple cloning site for insertion of a nucleic acid fragment of interest.

6. The retroviral genome transcription cassette of claim 5, wherein the cis-acting element comprises a Long Terminal Repeat (LTR), a Primer Binding Site (PBS), and a viral packaging signal (phi signal).

7. The retroviral genome transcription cassette of claim 5, wherein the retrovirus is a lentivirus.

8. The retroviral genomic transcription cassette of claim 7, wherein said cis-acting element comprises a Long Terminal Repeat (LTR), a Primer Binding Site (PBS), a viral packaging signal (phi signal), a central polypurine tract (cPPT) and a rev protein response element (RRE), and preferably further comprises a woodchuck hepatitis virus post-transcriptional regulatory sequence (WPRE).

9. The retroviral genomic transcription cassette of claim 6 or 8, wherein the long terminal repeat is a self-replicating wild-type U3-R-U5 sequence or a self-suppressing SIN sequence with the U3 sequence deleted.

10. A retroviral genome transcription cassette obtained by inserting a nucleic acid fragment of interest into the multiple cloning site of the retroviral genome transcription cassette of any one of claims 5-9.

11. A vector comprising the nucleic acid sequence of any one of claims 1 to 4 or the retroviral genome transcription cassette of any one of claims 5 to 10.

12. The vector of claim 11, which is a plasmid vector or a viral vector.

13. A host cell comprising the nucleic acid sequence of any one of claims 1 to 4, the retroviral genome transcription cassette of any one of claims 5 to 10, or the vector of claim 11 or 12.

14. Use of a nucleic acid sequence according to any of claims 1 to 4, a retroviral genomic transcription cassette according to any of claims 5 to 10, a vector according to claim 11 or 12, or a host cell according to claim 13 for the production of a retroviral vector carrying a nucleic acid fragment of interest.

15. Use according to claim 14, wherein the nucleic acid sequence according to any one of claims 1 to 4, the retroviral genomic transcription cassette according to any one of claims 5 to 10, the vector according to claim 11 or 12, or the host cell according to claim 13 is used for the transient or stable production of the retroviral vector carrying the nucleic acid segment of interest.

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