WO2004024905A1 - Virus de l'herpes simplex humain recombine pour la production de vecteurs lentivirus - Google Patents

Virus de l'herpes simplex humain recombine pour la production de vecteurs lentivirus Download PDF

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WO2004024905A1
WO2004024905A1 PCT/CN2003/000765 CN0300765W WO2004024905A1 WO 2004024905 A1 WO2004024905 A1 WO 2004024905A1 CN 0300765 W CN0300765 W CN 0300765W WO 2004024905 A1 WO2004024905 A1 WO 2004024905A1
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herpes simplex
vector
simplex virus
recombinant human
virus
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PCT/CN2003/000765
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French (fr)
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Xiaobing Wu
Xiaoyan Dong
Hui Cao
Deyun Hou
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Agtc Gene Technology Company Ltd.
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Priority to AU2003261642A priority Critical patent/AU2003261642A1/en
Publication of WO2004024905A1 publication Critical patent/WO2004024905A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16644—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15051—Methods of production or purification of viral material
    • C12N2740/15052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the invention belongs to the field of biotechnology invention. Specifically, it relates to the construction and use of a set of recombinant human Herpes Simplex Virus type 1 (HSV1) "recombinant HSV1-Lent i-helper virus” mainly used for preparing viral vectors.
  • HSV1 Herpes Simplex Virus type 1
  • Retroviral vectors have a very broad application in the field of gene therapy and gene transfer. It can introduce foreign aid genes into somatic cells including murine and primate 5L ⁇ and integrate them into In the cell genome, the purpose of achieving long-term stable expression makes it a very promising application in many fields including genetic disease treatment, transplantation, and stem cell transduction.
  • Retroviruses are RM viruses with DNA intermediates in their life history.
  • the retroviral particles contain two identical RNA molecules.
  • the core protein of the virus contains replication-related enzymes.
  • the virus envelope is enveloped by the core, which contains glycoproteins encoded by the host cell membrane and the virus.
  • the process of retrovirus infecting cells is as follows: Viral particles interact with receptors on the cell membrane through the glycoproteins on their envelopes, and membrane fusion occurs between the two.
  • the viral core particles enter the cytoplasm through endocytosis, and viral RNA is reverse transcribed into double-strand DNA, the latter is transported into the nucleus, in the nuclear virus DNA is permanently integrated into the chromosome (called provirus), becomes part of the host DNA, and replicates and divides with the host cell.
  • provirus chromosome
  • Proviral DNA is transcribed into RNA, transported to the cytoplasm, and translated here into viral protein precursors, which are subsequently cleaved into viral structural proteins and replicase proteins, assembled with viral RNA to form a viral nucleocapsid, and at the cell membrane Attach an envelope. Further processing of the precursor nucleocapsid protein resulted in mature, infectious progeny virus particles.
  • tumorigenic retroviral vectors There are two main types of retroviral vectors: tumorigenic retroviral vectors and lentiviral vectors.
  • Tumorigenic retroviral vectors Tumorigenic retroviral vectors
  • Murine leukemia virus (oncogenic retroviruses). These viruses have similar genomic structures and can cause transformation in their natural hosts.
  • Murine leukemia virus (oncogenic retroviruses). These viruses have similar genomic structures and can cause transformation in their natural hosts.
  • Murine leukemia virus (oncogenic retroviruses). These viruses have similar genomic structures and can cause transformation in their natural hosts.
  • Murine leukemia virus (oncogenic retroviruses). These viruses have similar genomic structures and can cause transformation in their natural hosts.
  • LTR long terminal repeat
  • pol encodes viral replication enzymes.
  • the above two genes were originally expressed as a Gag-Pol fusion protein. The viral protease cleaves the Gag-Pol precursor and further processes it.
  • the Pol protein becomes a variety of proteases (for cutting viral precursor proteins into mature forms), reverse transcriptase (for replicating viral nucleic acids), and integrase (integrating the viral genome into chromosomal DNA).
  • the Gag protein is further cleaved into the core components of the virus: matrix, caps id and nucleocaps id proteins (which are the same in all retroviruses) and core proteins specific to various viruses.
  • Env encodes a viral envelope glycoprotein, which is cleaved by proteases in the cell into outer and transmembrane proteins. The specificity of the glycoprotein and the cell surface receptors on the envelope determines the cellular affinity of the virus.
  • retroviruses can permanently integrate their genomes into the chromosomes of infected cells, the genes transduced by them can be stably present and expressed in infected cells and their offspring. Therefore, tumorigenic retroviruses have been widely studied and used as gene transfer vectors and gene therapy research. Among them, MLV vectors are the most widely used. MLV is a mouse retrovirus, and some MLV strains, such as the amphiphilic group
  • (amphotropic group) envelope glycoproteins can mediate MLV somatic cells.
  • Retroviruses commonly used as vectors are replication-defective. They can infect and integrate foreign genes into target cells, but the virus cannot replicate and spread to other cells. This is because there are no viral genes in the vector genome, leaving only the cis-elements necessary for a single round of replication.
  • the cis elements retained in the plant genome are: Reverse transcription with RNA Related cis-elements (such as LTR), cis-elements (such as at tsi te) associated with viral DNA integration, cis-elements (LTR) associated with transcription of provirus, and viral RNA packaging-related sites ((, ps i) iso-cis elements.
  • Shield granules containing the above cis-elements can be amplified in cells.
  • this plasmid can be used as a gene transfer vector.
  • the above-mentioned trans element can be provided by wild-type virus DNA. As a result, a wild-type virus capable of infecting and replicating can be obtained at the same time as obtaining a virus vector, which is clinically unacceptable.
  • a third improved method of constructing retroviral vectors is the use of packaging cell lines.
  • the latter expression vector is required for amplification of viral proteins.
  • This method greatly improves the packaging efficiency of the carrier.
  • the usual method is to transduce the structural protein gene and replicase gene of the virus into cells and make them stably expressed or inducible. By transfecting the vector genome into the cells, vector viruses can be produced.
  • This method also does not produce helper viruses, and the vector does not replicate and expand on non-packaging cell lines.
  • this method has two advantages: First, the process of generating a viral vector is simpler and more effective, and the titer of the resulting plant is higher than the co-transfection method. Second, it reduces the chance of recombination between the vector plasmid and the trans protein gene, so it is safer.
  • the lentivirus (lent ivirus) genome is more complex than the tumorigenic retrovirus, resulting in a more complex replication process.
  • the HIV-1 vector is currently the most well-studied lentiviral vector. Its genome is basically the same as that of tumorigenic retroviruses, including gag, pol and env. But HIV-1 also contains other helper genes, including vif, vpr, tat, rev: vpu, nef, and vpx. Some of them play important roles in virus replication. Ratio For example, Tat and Rev proteins are required for efficient expression of viral genes. Tat activates the promoter function of HIV-1 LTR and improves the transcription efficiency of viral RNA. Rev interacts with the Rev-responsive element (RRE) region on the viral genomic RNA to promote the transfer of viral RM from the nucleus to the cytoplasm.
  • RRE Rev-responsive element
  • lentiviral vectors Compared with tumorigenic retroviral vectors, lentiviral vectors have achieved the greatest progress in that they can infect non-dividing and terminally differentiated cells. Although tumorigenic retroviral vectors can also infect non-dividing cells, the viral genome is blocked from the nucleus by the nuclear membrane. Only when the cell divides, the integrity of the nuclear membrane can be broken, and the genome of the plant can be in the nucleus. Therefore, the reverse transcription and integration of RNA from tumorigenic retroviral vectors can only be completed in dividing cells. Although the exact mechanism by which lentivirus infects non-dividing cells is unknown, it appears that HIV can infect non-dividing cells by virtue of the following viral proteins: integrase protein, matrix protein, and helper protein Vpr. Integrase and matrix proteins contain nuclear localization signals, and Vpr may bind directly to the nuclear pore complex.
  • Lentiviral vectors have advantages in many gene therapy programs that require non-dividing cells as target cells (including rarely dividing hematopoietic stem cells, terminally differentiated neural cells, and even tumor cells, etc.), and may become the first choice. Because lentiviruses can infect non-dividing cells (in some cell types, the cells need to be in the GO to Gib phase), the clinical research prospects of lentiviral vectors are very attractive. However, due to the complexity of the lentiviral genome and replication, research on vectors and packaging cell lines is more difficult than oncogenic retroviruses.
  • Packaging cells for tumorigenic retroviral vectors need only provide Gag, Pol, and Env proteins, while HIV vectors also need to provide Tat and Rev proteins, and the functions of other helper genes are unclear, and stable packaging cell lines are also required. It is very difficult, and the reason may be related to the toxicity of certain proteins of HIV.
  • the original packaging system for HIV vectors simply used plasmids containing the core protein of HIV-1 virus, enzymes, helper genes, and VSV-G gene.
  • VSV G protein can expand the type of infectious cells of lentiviral plants. Moreover, VSV-G can also be used to obtain lentivirus plants with higher titers and better stability.
  • the second-generation lentiviral vector packaging plasmid components were reduced to only HIV-1 gag, pol, tat, rev, and VSV-G.
  • the vector plasmid contained only HIV-1 transcription, packaging, reverse transcription, and integration-related cis-elements.
  • the sequence of the genetic elements included in the vector plasmid is from 5, to 3, in that order: HIV-1 5'LTR, leader sequence, 5, SD site (spl ice donor site), 360 bp gag gene part, 700 bp env (Including RRE and SA sites spl ice acceptor site), internal promoter (ie IE enhancer / promoter of CMV or gk (phosphoglycerate kinase) phosphoglycerate kinase promoter), foreign genes and HIV-1 3. LTR.
  • the recombinant lentiviral vector is produced by co-transfecting three plasmids carrying various necessary elements into 293T cells. The lentiviral vector can activate the transcriptional activity of LTR and the accumulation of uncleaved genomic RNA in the cytoplasm only in the presence of Tat and Rev proteins.
  • the third generation lentiviral vector is based on the second generation, and further deleted some virus sequences, which further reduced the packaging plasmid components to only gag, poK rev, and VSV-G.
  • U3 of LTR was replaced by the RSV (Rous sarcoma virus) enhancer / promoter, so that the transcription of viral mRNA is not restricted by the Tat protein, and the Tat gene and its action site are respectively extracted from the plasmid and the vector plasmid.
  • the U3 region in 3 and LTR was also completely deleted, and the latter is the template for LTR transcription. The deletion of U3 region caused the vector genome to infect cells without generating new progeny viruses, which became "self-inactivating plants". Body
  • Lentiviral vectors are currently only used for HIV replication mechanisms and research on lentiviral vector systems. They still lag behind tumorigenic retroviral vectors in clinical applications of gene therapy. There are two main reasons for this: First, people are more concerned and concerned about the safety of HI V plants than on tumorigenic retroviruses. The former contains certain cis prerequisites, such as the RRE sequence, in the vector plasmid and the helper plasmid. Both exist, so there is the possibility of recombination of wild-type viruses. In addition, the packaging signal of HI V extends to the gag gene. Therefore, the HIV vector contains part of the gag gene sequence, which may also cause homologous recombination of the vector and the helper plasmid.
  • VSV-G is toxic to cells, so Difficult to construct HIV vector package Load cell lines.
  • a patent (US PATENT 6218181) uses a regulated promoter such as a tetracycline promoter to start the expression of VSV-G, thereby constructing an HIV vector packaging cell line, and then transfecting the vector plasmid into the cell line to produce Out of the HIV vector. But the current general method is still co-transfection of multiple (2-4) plasmids, so the vector titer is low, and it can produce recombinant, replicable vectors.
  • a regulated promoter such as a tetracycline promoter
  • Human herpes simplex virus type 1 The human herpes simplex virus HSV1 genome is 152 kb in length, with a total of 72 genes, of which about 1/3 are non-essential genes, that is, deletion or inactivation of these genes does not affect the infection and HSV1 vector cells. Breed. The insertion of foreign genes below 15 kb in the HSV1 genome does not affect its genome packaging and virus infectivity. In addition, HSV1 has a high level of infection in most animal or human cells, and the foreign genes it carries can be expressed at higher levels in these cells. Therefore, HSV1 is an ideal vector for carrying foreign genes into cells. Summary of the invention
  • the invention describes a recombinant human herpes simplex virus type 1 (HSV1) "recombinant HSVl-Lent i-helper virus", which inserts into the genome various trans-forms required for lentiviral vector packaging Protein expression cassettes (gag, pol, VSV-G, Rev, etc.).
  • HSV1 virus is used to infect plant cell lines, which can express proteins such as gag, poK VSV-G, and Rev at a high level, thereby saving the lentiviral vector genome in the form of provirus in the chromosome of the vector cell line and packaging the lentivirus Carrier.
  • Affinity chromatography, ion adsorption chromatography, or ultra-ionization can be used to separate the lentiviral vector from HSV1 for purification purposes.
  • the method is characterized in that a viral infection method is used instead of a plasmid transfection method to produce a lentiviral vector.
  • the vector titer is high, the cost is low, and it is conducive to large-scale production.
  • the foreign DNA fragment inserted into the HSV1 genome includes:
  • gag / pol retrovirus, especially human immunodeficiency virus type 1 (HIV-1) Viral core protein / replication and integrase encoding genes.
  • the promoter upstream of the gag / pol gene is a strong eukaryotic promoter, such as the human cytomegalovirus CMV promoter containing a human bet ag lob in intron enhancer.
  • the downstream of the gag / pol gene is the Rev-1 response element (RRE) of HIV-1. Under the action of Rev, RRE expresses Gag and Pol proteins. Downstream of the RRE are eukaryotic transcription termination and tailing signals, such as human beta-globin polyA.
  • RRE Rev-1 response element
  • Rev a gene encoding a retrovirus, especially a human immunodeficiency virus type 1 (HIV-1) regulatory protein.
  • Upstream is a strong eukaryotic promoter, such as the enhancer / promoter of Rous sarcoma virus RSV.
  • Downstream of the Rev gene are eukaryotic transcription termination and tailing signals, such as the polyA of HIV-1 LTR.
  • a schematic representation of Rev's DNA fragment composition is shown in Figure 2 of the accompanying drawings.
  • Non-HIV-1 envelope proteins Genes that include, but are not limited to, envelope proteins of the following viruses: Moloney murine leukemia virus (MoMuLV), human immunodeficiency virus (HIV), human vesicular stomatitis virus G protein (VSV-G) and so on.
  • the promoter upstream of the gene is a strong eukaryotic promoter, such as a human cytomegalovirus CMV promoter containing a human beta-globin intron enhancer.
  • Downstream are eukaryotic transcription termination and tailing signals, such as human beta-globin polyA.
  • the three DNA fragments in (1), (2), and (3) above are inserted or substituted into non-essential gene regions of HSV1 by methods such as enzymatic digestion or homology arm recombination.
  • the three DNA fragments in (1), (2), and (3) above can be inserted into the genomes of three herpes simplex viruses, respectively, or (1), (2), and (3) can be inserted into the same strain of herpes simplex virus. Or (1), (3) inserted into the genome of the same strain of herpes simplex virus, (2) inserted into the genome of another strain of herpes simplex virus.
  • the so-called "enzymatic digestion” method refers to: using a set of cosmid system SetC, to recombinate the three DNA fragments (1), (2), and (3) by enzyme digestion, ligation and co-transfection.
  • Set C cosmid is composed of five cosmids that sequentially load the entire genome of HSVl disease 17 strains) according to the HSVl virus gene sequence: cos6, cos28, cosl4, cos56, cos48.
  • cos6, cos28, cosl4, cos56, cos48 Presented to Davis ion AJ (Cormingham C, Davis ion AJ. Virology, 1993, 197: 116-124).
  • the HSV1 viral genome fragment loaded in cos28 The 3 'terminus of the HSV1 virus genome loaded in cosl4 overlaps with the 5, terminal sequence, and so on, and so on, and the 3, terminus of the HSV1 virus genome loaded in cosl4 and the HSV1 virus genome loaded in cos56 5, the terminal sequence repeats, the 3 'end of the HSV1 virus genome fragment loaded in cos56 and the 5' end sequence of the HSV1 virus genome fragment loaded in cos48, and the HSV1 virus genome fragment loaded in cos48 3, the ends are repeated with the 5, end sequences of the HSV1 virus genome fragment loaded in cos6.
  • This is the 5 ftij of 5 HSV1 genomic fragments that undergo homologous recombination in cells to produce recombinant HSV1.
  • a schematic of the SetC cosmid map is shown in Figure 4 of the accompanying drawings.
  • HSVl-Lent i-he lper virus ".
  • the three types of DNA fragments in (1), (2), and (3) above can be inserted into the Xbal single cut point of UL2 of cos6, the Xbal single cut point of UL44 of cos56, or the Ns il single cut point of TK of cos28, Unlimited locations.
  • the so-called “homologous arm recombination method" refers to: ⁇ ?
  • the three types of DNA fragments in (1), (2), and (3) above can be inserted into any non-essential gene region of HSV1 by homologous recombination, and their positions are not limited.
  • the retroviruses mentioned in (1) and (2) above may be other retroviruses other than HIV-1.
  • Recombinant HSV1 virus obtained by the above method can be prepared from eukaryotic cells sensitive to HSV1 (such as BHK-21) and stably passaged for a long time, or stored at -70 ° C.
  • Application of recombinant herpes simplex virus proposed by the present invention can be prepared from eukaryotic cells sensitive to HSV1 (such as BHK-21) and stably passaged for a long time, or stored at -70 ° C.
  • the recombinant HSV1 virus "recombinant HSVl-Lenti-helper virus” containing (1), (2), and (3) three DNA fragments obtained by the above method is mainly used for the production of 'it virus vectors carrying foreign genes, especially HIV -1 carrier.
  • the specific method is to first construct a vector plasmid, which contains the following components: 1.
  • the promoter of the gene is a strong eukaryotic promoter, such as the IE promoter of the human cytomegalovirus CMV, and the eukaryotic transcription termination and tailing signals are downstream of the foreign gene, such as the polyA of the SV40 virus; Contains lentivirus, especially HIV-1 Two cis-elements psi (packaging signal) and RRE (carrying the complete viral RNA genome out of the nucleus) required for genome packaging and localization; 3.
  • LTR long-term repeat
  • Resistant gene expression cassettes expressed in eukaryotic cells such as neo r Blas t icidin r , kan r, etc .; 4.
  • the replication origin sequence such as pUC ori, and resistance screening genes, such as amp r , to make the plasmid replicate in prokaryotic cells, such as E. coli.
  • the vector plasmid is transduced into HSV1-sensitive eukaryotic cells (such as BHK-21) by transfection reagents such as liposomes, and antibiotics of appropriate concentration are added to the culture medium. After a certain period of culture, the vector is selected.
  • the plasmid is a cell line that is stably carried and / or integrated into the chromosome of the cell.
  • the cell line was infected with recombinant HSV1 virus "recombinant HSVl-Lent i-helper virus" (three DNA fragments on the same recombinant HSV1 virus genome) containing three DNA fragments (1), (2), and (3), and expressed
  • the gag / pol, VSV-G and Rev proteins reverse-transcribe the vector DNA integrated into the chromosome of the cell into the lentiviral RM genome, and package the lentiviral vector.
  • This cell line was infected with two recombinant HSV1 viruses containing (1), (3) two kinds of DNA fragments and (2) one kind of DNA fragments at a certain ratio, and then expressed gag / pol, VSV-G and Rev proteins, which will integrate
  • the vector DNA into the cell chromosome is reverse transcribed into the lentiviral RNA genome, and the lentiviral vector is packaged.
  • Recombinant herpes simplex virus (“recombinant HSV1-Lent i” -helper virus "), which is cytotoxic to animal or human cells and needs to be removed. It can be removed by affinity chromatography for VSV-G protein, ion adsorption chromatography, or CsCl density ladder centrifugation.
  • FIG. 1 of the accompanying drawings of the specification is a schematic diagram of the structure of the DNA fragment composed of gag / pol. Its upstream (ie, 5 'end) is the human cytomegalovirus CMV promoter (CMV promoter), followed by: beta-globin intron enhancer, gag / po K RRE, beta-g lobin po lyA.
  • CMV promoter human cytomegalovirus CMV promoter
  • FIG. 2 of the accompanying drawings of the specification is a schematic diagram of the structure of the Rev DNA fragment.
  • the 5, end of the Rev is the RSV promoter (RSV enhancer / promoter), Rev, and LTR polyA of HIV.
  • FIG. 3 of the accompanying drawings of the specification is a schematic diagram of the structure of the D fragment of a non-HIV-1 envelope protein.
  • the 5 'end from upstream to the next is a human cytomegalovirus CMV promoter (CMV promoter) and beta-globin intron enhancement.
  • CMV promoter human cytomegalovirus CMV promoter
  • beta-globin intron enhancement Sub, VSV_G, beta-g lobin polyA.
  • FIG. 4 of the description of the specification is a schematic diagram of the structure of SetC.
  • Set C cosmids sequentially load the entire genome of HSV1 virus (17 strains) according to the entire genetic sequence of HSV1 virus, and the first DNA sequence of adjacent cosmids are partially identical. Consisting of 5 cosmids cos6, cos28, cos l4, cos56, cos48. detailed description
  • VSV-G expression cassette (CMV promoter / beta-g lobin enhancer + VSV-G + beta-globin polyA) in the plasmid pLP / VSVG (produced by Invitrogen) was used for PCR.
  • the method is called up (about 3770 bp in length), and the primers are: upstream 5, TCTAGACTTGGCCCATTGCATA 3, (SEQ ID NO: 1); downstream 5, TCTAGAACTGCCATGTCGAGGG 3, (SEQ ID NO: 2).
  • the response is: 94'C, 30 seconds; 56 ° C, 30 seconds; 72 ° C, 4 minutes.
  • gag / pol expression cassette (CMV promoter / beta-globin enhancer + gag / pol + RRE + beta-globin polyA) in plasmid pLP1 (produced by Invitrogen) was called up by PCR (about 6837 bp in length), The primers are: upstream 5,
  • TCTAGAACTGCCATGTCGAGGG 3 (SEQ ID NO: 4).
  • Xbal sites are at both ends.
  • the reaction conditions were: 94 C, 30 seconds; 56 °, 30 seconds; 72, 7 minutes.
  • the PCR product was digested with Xbal and loaded into the Xbal site of cos56 to obtain a cosmid.
  • the Rev expression cassette (RSV promoter + Rev + HIV polyA) in plasmid pLP2 was called out by PCR (about 971 bp in length), and the primers were: upstream 5, TCTAGACAATGTAGTCTTATGC 3, (SEQ ID NO: 5); downstream 5, TCTAGACCAGGGTTTTCCTGAT 3, (SEQ ID NO: 6). Both ends are Xbal sites.
  • the reaction conditions were: 94. C, 30 seconds; 56, 30 seconds; 72, 1 minute.
  • the PCR product was digested with Xbal and loaded into the Xbal site of cos6 to obtain cosmid cos6-rev.
  • rHSVl-VSVG-gag / pol recombinant virus 24 hours after transfection, 2% FBS (fetal calf Serum) of 1640 culture medium 37. C culture, change the fluid once a day. After 5 days, the cells began to show lesions. After the cells were completely damaged, the culture supernatant was collected, centrifuged at 2000 r / min for 5 minutes, and the supernatant was stored in -20 aliquots. Plaque purification was performed on the obtained recombinant virus twice to obtain pure rHSVl-VSVG-gag / pol recombinant virus.
  • Cos6-rev is equimolarly mixed with 5 cosmids such as cos56, cosl4, cos28, cos48, etc., and the cos skeleton is cut with Pacl (without separation and removal), and extracted with phenol, phenol / chloroform (1: 1) and chloroform. Once, the supernatant was pipetted and the DNA was precipitated with 2.5 times absolute ethanol. Using lipofactamine (produced by GIBCO BRL), 20ul and 10 ug DNA were transfected into 80% confluent BHK-21 cells (approximately 2 ⁇ 10 ⁇ ) cells according to the product instructions. Five HSV1 fragments will undergo homologous recombination in the cells and separate.
  • rHSV-rev recombinant virus Production of rHSV-rev recombinant virus. 24 hours after transfection, the cells were cultured with 1640 medium containing 2% FBS, and the medium was changed once a day. After 5 days, the cells began to show lesions. After the cells were completely damaged, the culture supernatant was collected, centrifuged at 2000 r / min for 5 minutes, and the supernatant was aliquoted and stored at -20X. Plaque purification was performed on the obtained recombinant virus twice to obtain a pure rHSVl-rev recombinant virus.
  • Example 6 Construction of the vector plasmid pLenti-EGFP carrying the reporter gene EGFP
  • the EGFP gene in pEGFP was retrieved by PCR (about 730 bp in length).
  • the primers were: upstream 5, cacc atg gtg age aag ggc gag 3, (SEQ ID NO: 7); downstream 5, gaattc cct cta gag tcg egg ccg ctt t 3, (SEQ ID NO: 8).
  • PCR reaction H 94V, 30 seconds; 56 ° C, 30 seconds; 72 ° C, 1 minute.
  • pLenti6 / D-T0P0 a recombinant vector plasmid pLenti-EGFP was obtained.
  • Example 7 Construction of a Stable Integration Vector Plasmid pLenti-EGFP Vector Cell Line
  • the plant plasmid pLenti-EGFP was transfected with 1 ipofactamine (manufactured by GIBCO BRL) to 80% confluent BHK-21 cells (about 2 x 10 ⁇ ) cells. After 24 hours, the antibiotic "Blasticdin" (blasticidin) at a final concentration of 50 g / ml was added to the 1640 culture medium.
  • the supernatant of the lentiviral vector Lenti-EGFP mixed with the recombinant type 1 herpes simplex virus was added to an affinity chromatography column containing a VSV-G monoclonal antibody.
  • the vector Lenti-GFP was eluted to obtain a purified 'virus vector Lent i-EGFP.
  • Example 10 Lenti-EGFP titer assay
  • the lentiviral vector Lenti-EGFP diluted 10-fold with PBS was used to infect 80% of BHK-21 cells. After 24 hours, the culture medium (10% FBS 1640, ie, 1640 medium containing 10% fetal bovine serum) ) Add antibiotic "Blasticidin” (blasticidin) with a final concentration of 5 () g / ml, and after about 10 days, observe under a fluorescence microscope and calculate the number of cell clones, to obtain the lentivirus vector Lenti-EGFP Titer.

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Description

用于生产慢病毒载体的人重组单纯疱疹病毒 发明领域
本发明属于生物技术发明领域。 具体涉及一组主要用于制备 病毒载体的重组人 1型单纯疾奢病毒 ( Herpes Simplex Virus type 1， HSV1 ) "重组 HSV1- Lent i-helper病毒" 的构建及其用途。 背景技术
有关逆转录病毒： 逆转录病毒栽体（retroviral vector)在基因 治疗和基因转移领域有非常广阔应用， 它可以将外援基因导入包括 鼠类、 灵长类5L ^内的体细胞中，并整合到细胞基因组中， 达到长 期稳定表达的目的， 使其在包括遗传病治疗、 移植、 干细胞转导等 许多领域的应用都有非常好的前景。
逆转录病毒是 RM病毒， 其生活史中有 DNA中间体。 逆转录病 毒颗粒包含 2条完全相同的 RNA分子， 病毒核心蛋白中含有与复制 相关的酶类， 核心外包裹病毒的包膜， 其上有来自宿主细胞膜和病 毒编码的糖蛋白。 逆转录病毒感染细胞的过程如下： 病毒颗粒通过 其包膜上的糖蛋白与细胞膜上的受体作用， 两者发生膜融合， 病毒 核心颗粒通过内吞进入细胞浆，病毒 RNA逆转录成双链 DNA,后者被 转运进细胞核， 在核内病毒 DNA永久整合至染色体中 （称为原病毒 provirus ) ， 成为宿主 DNA的一部分， 并随宿主细胞复制和分裂。
Proviral DNA转录成 RNA, 并转运到胞浆， 并在此处翻译成病毒蛋 白前体， 后者随后被分别切割成病毒结构蛋白和复制酶蛋白， 与病 毒 RNA组装成病毒核衣壳， 在细胞膜处装上包膜。 经过对前体核衣 壳蛋白的进一步处理最终得到成熟的、 有感染性的子代病毒颗粒。
逆转录病毒载体主要有两种： 致瘤逆转录病毒载体和慢病毒载 体。 致瘤逆转录病毒栽体
早期的逆转录病毒载体来源于禽类或鼠的致瘤性逆转录病毒
( oncogenic retroviruses ) ， 这些病毒有着相似的基因组结构， 在它们的自然宿主中都能引起转化。 其代表病毒是鼠白血病病毒
( MLV )。 所有的逆转录病毒都有以下三种基因： gag、 pol 和 env。 在受感染的细胞内， provirus基因组在 5，LTR (长末端重复序列， long terminal repeat)启动子和 3， LTR的终止子和 polyA的作用 下，转录出单链前体 mRNA。 gag编码病毒核心蛋白（ core protein ) , pol编码病毒复制酶类。 以上两个基因最初是以 Gag-Pol融合蛋白 形式表达的， 病毒的蛋白酶将 Gag-Pol前体切开， 并进一步加工。
Pol蛋白成为各种蛋白酶（用于将病毒前体蛋白切割成成熟形式） 、 逆转录酶（用于复制病毒核酸）和整合酶（将病毒基因组整合进染 色体 DNA )。 Gag蛋白被进一步切割成病毒核心组分：基质 ( matrix ) , 衣壳 ( caps id )和核衣壳 ( nucleocaps id )蛋白 (在所有逆转录病 毒中都相同）以及各种病毒特有的核心蛋白。 env编码病毒包膜糖蛋 白， 后者被细胞中蛋白酶切割成外膜蛋白和穿膜蛋白。 包膜上糖蛋 白与细胞表面受体的特异性， 决定了病毒的细胞亲嗜性。
由于逆转录病毒能永久性地将自身基因组整合到受其感染的细 胞的染色体中， 从而使被其转导的基因在受感染的细胞及其子代中 稳定存在和表达。 因此致瘤逆转录病毒被广泛研究， 用来作为基因 转移载体和基因治疗研究中。 其中 MLV载体是其中应用最多和最广 的。 MLV是鼠逆转录病毒， 某些 MLV病毒株， 比如双嗜性群
( amphotropic group ) 的包膜糖蛋白可以介导 MLV 体细胞。
通常作为载体的逆转录病毒都是复制缺陷型的， 它们可以感染 并将外源基因整合到靶细胞中， 但病毒无法复制和传播到其它细胞 中。 这是因为载体基因组中无病毒基因， 只剩下一些单轮复制所必 需的顺式元件。 栽体基因组中被保留的顺式元件有： 与 RNA逆转录 相关的顺式元件 （如 LTR ) 、 与病毒 DNA整合相关的顺式元件（如 at t s i te ) 、 与 provirus的转录相关的顺式元件（LTR ) 以及病毒 RNA包装相关的位点 ( Ψ , ps i )等顺式元件。
包含以上顺式元件的盾粒在细胞中可以扩增， 在反式提供 gag、 pol和 env基因的 下，该质粒可以作为基因转移载体。上述反式 元件可以由野生型病毒 DNA提供，其结果是在得到病毒栽体的同时， 得到有感染和复制能力的野生型病毒， 这在临床上是无法接受的。
随后出现了不使用野生型病毒的构建方式。即将野生型病毒 DNA 中扩增所需的所有顺式元件去除， 再与载体 DNA共转染细胞， 由于 构建体只表达病毒蛋白， 但不含复制所需的顺式元件， 因此不产生 辅助病毒。 用这种方法得到的载体感染正常细胞， 由于细胞不表达 病毒蛋白， 所以载体不再扩增。 这种方法得到的载体滴度较低。
第三种改进的构建逆转录病毒载体的方法是运用包装细胞株。 后者表达载体扩增所需的病毒蛋白。 这种方法大大提高了载体的包 装效率。 通常的做法是将病毒的结构蛋白基因和复制酶基因转导到 细胞中， 并使其稳定表达或可诱导表达。 将载体基因组转染到该细 胞中， 就能产生载体病毒。 这种方法同样不产生辅助病毒， 载体在 非包装细胞株上也不会复制和扩增。 与上一种方法相比， 该方法的 优点有二： 第一， 产生病毒载体的过程更简单而有效， 产生的栽体 滴度高于共转染法。 第二， 减少了载体质粒与反式蛋白基因间发生 重组的几率， 因此更安全。
慢病毒载体
慢病毒（ lent ivirus )基因组比致瘤逆转录病毒更复杂一些， 从而导致其复制过程也更复杂。 HIV-1载体是目前研究得最充分的慢 病毒载体。其基因组基本组成与致瘤逆转录病毒一样，包括 gag, pol 和 env。但 HIV-1还含有另外一些辅助基因，包括 vif， vpr, tat, rev: vpu, nef 和 vpx等。 其中某些在病毒复制过程中起着重要作用。 比 如， Tat和 Rev蛋白是病毒基因高效表达所必需的。 Tat激活 HIV- 1 的 LTR中的启动子功能，提高病毒 RNA的转录效率。 Rev与病毒基因 组 RNA上的 RRE ( Rev-respons ive element ) 区域相互作用， 促进 病毒 RM从胞核向胞浆的转移。
慢病毒载体与致瘤逆转录病毒栽体相比， 取得的最大进展是它 可以感染非分裂细胞和终末分化的细胞。 致瘤逆转录病毒载体虽然 也能感染非分裂细胞， 但病毒基因组被核膜阻挡在细胞核之外， 只 有细胞分裂时， 核膜的完整性被打破， 栽体基因组才能^核内。 因此致瘤逆转录病毒载体的 RNA的反转录和整合只有在分裂细胞中 才能得以完成。 虽然慢病毒感染非分裂细胞的准确机制还不清楚， 但目前看来 HIV能感染非分裂细胞得益于以下几种病毒蛋白： 整合 酶蛋白、 基质蛋白以及辅助蛋白 Vpr等。 整合酶和基质蛋白含有核 定位信号， Vpr可能与核孔复合物直接结合。
慢病毒栽体在许多需要以非分裂细胞作为靶细胞的基因治疗 (包括极少分裂的造血干细胞、 终末分化的神经细胞甚至肿瘤细胞 等） 方案中具有优势， 将可能成为首选。 由于慢病毒可以感染非分 裂细胞， （某些细胞类型中， 需要细胞处于 GO 期到 Gib期） ， 慢 病毒载体的临床研究前景是非常诱人的。 但是， 由于慢病毒基因组 和复制的复杂性， 导致载体和包装细胞株的研究进展比致瘤逆转录 病毒更困难。致瘤逆转录病毒载体的包装细胞只要提供 Gag、 Pol和 Env蛋白就可以， 而 HIV载体还需要提供 Tat和 Rev蛋白， 而且其 它辅助基因的功能还不清楚， 而且要产生稳定的包装细胞系也有很 大难度， 分析原因可能与 HIV某些蛋白的毒性肴关。
由于存在上述困难， 最初的 HIV载体的包装系统简单地采用含 HIV-1病毒的核心蛋白、 酶、 辅助基因以及 VSV-G基因等的质粒。
VSV的 G蛋白（ VSV-G )等可以扩大慢病毒栽体的可感染细胞的类型。 而且用 VSV-G还可以得到滴度更高、 稳定性更好的慢病毒栽体。 第二代慢病毒载体包装质粒成分减少到只有 HIV-1的 gag、pol、 tat , rev和 VSV-G, 载体质粒只含有 HIV-1转录、 包装、 反转录和 整合相关的顺式元件。 载体质粒所包括的基因元件的顺序从 5，至 3， 依次是: HIV-1 5'LTR, 前导序列、 5， SD位点（ spl ice donor s ite )、 360 bp gag基因部分、 700 bp env(含 RRE和 SA位点 spl ice acceptor s i te ) , 内部启动子（ CMV的 IE增强子 /启动子或 gk ( phosphogly- cerate kinase )磷酸甘油酸激酶启动子） 、 外源基因和 HIV-1的 3， LTR。重组慢病毒载体的生产方式是将携带了各种必需元件的三种质 粒共转染到 293T细胞中。该慢病毒载体只在有 Tat和 Rev蛋白的条 件下， 才能激活 LTR的转录活性和未切割的基因组 RNA在胞浆中的 聚集。
第三代慢病毒载体是在第二代的基础上， 进一步删除了一些病 毒序列，使包装质粒成分进一步减少到只有 gag、 poK rev和 VSV-G。 比如， 5， LTR的 U3被 RSV ( Rous肉瘤病毒）增强子 /启动子代替， 使病毒 mRNA的转录不受 Tat蛋白的限制， Tat基因及其作用位点被 分别从包^ 粒和载体质粒中删除； 另外， 3，LTR中的 U3区也被完 全删除， 而后者是 LTR转录的模板， U3区的缺失导致载体基因组感 染细胞后， 不会产生新的子代病毒， 成为 "自灭活栽体"
( self-inact ivating vector, SIN ) ， 安全性得到了进一步保障。
慢病毒载体目前还只用于 HIV复制机制和对慢病毒载体系统的 研究等方面，在基因治疗临床应用上仍落后于致瘤逆转录病毒载体。 其中主要原因有二： 一是人们对 HI V栽体安全性的关注和担心远大 于对致瘤逆转录病毒的， 前者含有的某些顺式先件， 比如 RRE序列， 在载体质粒和辅助质粒上都存在， 因此存在重组出野生型病毒的可 能。 另外， HI V的包装信号延伸到了 gag基因中， 因此 HIV载体中包 含部分 gag基因序列， 同样有可能导致载体与辅助质粒的同源重组； 另一个限制因素是 VSV-G对细胞有毒性， 因此难以构建 HIV载体包 装细胞株。 有专利 （US PATENT 6218181 )用可调控启动子， 如四环 素启动子来启动 VSV- G的表达， 从而构建出 HIV载体包装细胞株， 再用转染的方式将载体质粒导入该细胞株中， 生产出 HIV载体。 但 目前通用的方法仍是多个（2— 4个）质粒共转染， 因此载体滴度低， 而且能产生重组的可复制型载体。 人 1型单纯疱疹病毒：人 1型单纯疱疹病毒 HSV1基因组的全长 152kb, 共 72个基因， 其中约 1/3是非必需基因， 即这些基因缺失 或失活不影响 HSV1载体细胞中的感染和繁殖。 HSV1基因组中插入 15kb以下的外源基因， 不影响其基因组的包装和病毒的感染性。 另 外， HSV1对大部分动物或人的细胞都有较高水平的感染， 其携带的 外源基因能在这些细胞中以较高水平表达。 因此， HSV1成为用来携 带外源基因到细胞中比较理想的载体。 发明内容
本发明描述了一种重组人 1型单纯疱疹病毒（Herpes Simplex Virus Type 1, HSV1 ) "重组 HSVl-Lent i-helper病毒" , 它的基 因组中插入了慢病毒载体包装所需的各种反式蛋白的表达盒（ gag、 pol、 VSV-G、 Rev等） 。 用该重组人 HSV1病毒感染栽体细胞株， 能 高水平表达 gag、 poK VSV-G、 Rev等蛋白， 从而将载体细胞株染色 体中以 provirus形式存在的慢病毒载体基因组拯救出来， 包装出慢 病毒载体。 用亲和层析、 离子吸附层析或超离等方法， 可以将慢病 毒载体与 HSV1分离， 达到纯化的目的。 本方法 ^特点是用病毒感染 的方式代替质粒转染方式生产慢病毒载体， 载体滴度高， 成本低， 有利于大规模生产。
在本发明中， 插入到 HSV1基因组的外源 DNA片段包括：
( 1 ) gag/pol : 为逆转录病毒、特别是人免疫缺陷病毒 1型（ HIV-1 ) 的病毒核心蛋白 /复制及整合酶的编码基因。 gag/pol基因上游的启 动子是真核强启动子， 比如含人 bet a-g lob in intron增强子的人巨 细胞病毒 CMV启动子等。 gag/pol基因下游是 HIV- 1的 Rev效应元 件 （RRE) ， RRE在 Rev的作用下， 表达 Gag蛋白和 Pol蛋白。 RRE 下游是真核转录终止和加尾信号， 比如人 beta-globin polyA。 gag/pol的 DNA片段组成的示意图参见说明书附图中的图 1。
( 2 ) Rev: 为逆转录病毒、特别是人免疫缺陷病毒 1型（ HIV-1 ) 的调控蛋白的编码基因。其上游是真核强启动子， 比如 Rous肉瘤病 毒 RSV的增强子 /启动子。 Rev基因下游是真核转录终止和加尾信号， 比如 HIV- 1的 LTR的 polyA。 Rev的 DNA片段组成的示意图参见说 明书附图中的图 2。
(3)非 HIV-1的包膜蛋白： 包括、 但不局限于以下病毒的包膜 蛋白的编码基因： Moloney鼠白血病病毒（MoMuLV) 、 人免疫缺陷 病毒（HIV) 、 人水泡口炎病毒的 G蛋白 （VSV- G)等。 基因上游的 启动子是真核强启动子， 比如含人 beta- globin intron增强子的人 巨细胞病毒 CMV启动子。 下游是真核转录终止和加尾信号， 比如人 beta-globin polyA. 非 HIV-1的包膜蛋白的 DNA片段组成的示意图 参见说明书附图中的图 3。
上述（1)、 （2)、 （3)中的三种 DNA片段分别通过酶切插入 或同源臂重组等方法插入或取代 HSV1的非必需基因区。
上述（1)、 （2)、 （3)中的三种 DNA片段可以分别插入三株 单纯疱疹病毒基因组中， 也可以将（1) 、 （2) 、 （3)插入同一株 单纯疱疹病毒基因组中， 或是（1) 、 （3)插入同一株单纯疱疹病 毒基因组中， （2)插入另一株单纯疱疹病毒基因组中。
所谓 "酶切插入" 方法是指： 利用一套粘粒系统 SetC， 通过酶 切、 连接、 共转染的方法重组出含上述（1)、 （2)、 （3)三种 DNA 片段的重组单纯疱疹病毒。 Set C粘粒按 HSVl病毒全基因顺序由依次分载了 HSVl病 17 株）全基因组的 5个粘粒组成 ： cos6、 cos28、 cosl4、 cos56、 cos48。 为 Davi s ion AJ赠送 (Cormingham C， Davi s ion AJ. Vi rology, 1993, 197: 116-124)。该套粘粒中，从 cos6开始，按顺序至 cos28、 cos l4、 cos56,最后到 cos48，其中所装载的 HSVl病毒基因组的各片段的 3， 末端与装载于下一粘粒中的 HSV1片段的 5，末端序列重复，即： cos6 中所装载的 HSV1病毒基因组的片段的 3' 末端与 cos28中所装载的 HSV1病毒基因组的片段的 5，末端序列重复， cos28中所装载的 HSV1 病毒基因组的片段的 3' 末端与 cosl4中所装载的 HSV1病毒基因组 的片段的 5， 末端序列重复， 依次类推， cosl4中所装载的 HSV1病 毒基因组的片段的 3， 末端与 cos56中所装载的 HSV1病毒基因组的 片段的 5， 末端序列重复， cos56中所装载的 HSV1病毒基因组的片 段的 3， 末端与 cos48中所装载的 HSV1病毒基因组的片段的 5' 末 端序列重复， cos48中所装载的 HSV1病毒基因组的片段的 3， 末端 与 cos6中所装载的 HSV1病毒基因组的片段的 5，末端序列重复。这 是 5个 HSV1基因组片段在细胞中发生同源重组从而产生重组 HSV1 的^ ftij。 SetC粘粒图谱的示意图参见说明书附图中的图 4。
在 Set C中的 cos6上的 HSV1的非必需基因 UL2和 cos56上的 HSV1的非必需基因 UL44中各有一个 Xbal单切点， cos28上的 HSV1 的非必需基因 TK中有一个 Ns i l单切点。 这三个单酶切位点被用于 将外源基因插入其中，并通过将 5个粘粒共转染至 HSV1敏感的真核 细胞（如 BHK-21 ) ， 通过 5个 HSV1基因组片段的重组产生插入了 外源基因的重组 HSV1病毒， 本重组 HSV1病毒被命名为 "重组
HSVl-Lent i - he lper病毒" 。
上述（1 ) 、 （2 )、 ( 3 )中的三种 DNA片段可以分别插入 cos6 的 UL2的 Xbal单切点、 cos56的 UL44的 Xbal单切点或 cos28的 TK 的 Ns i l单切点中， 位置不限。 所谓 "同源臂重组方法" 是指： 分别在要插入（1) 、 （2) 、 (3)三种 DM片段的 HSV1非必需基因的上、 下游各找一^ ^列， 长约 100—2000bp,用 DNA合成或 PCR方法将其调出，分别放在（1)、 ( 2 ) 、 （ 3 )三种 DNA片段的两端， 将这样构建好的 DNA构建体与 HSV1病毒或含相应非必需基因的 SetC粘粒共同导入对 HSV1敏感的 真核细胞（如 BHK-21) 中， 筛选出含有 （1) 、 （2) 、 (3)三种 DNA片段的 HSV1重组病毒， 或筛选出含有 （1) 、 （2) 、 （3)三 种 DNA片段的粘粒， 与 SetC的其它几种粘粒共转染对 HSV1敏感的 真核细胞（如 BHK-21)， 同样将得到含有（1) 、 （2) 、 （3)三种 DM片段的 HSV1重组病毒 "重组 HSV1- Lent i- helper病毒" 。
上述（1)、 （2)、 （3)中的三种 DNA片段可以分别用同源重 组的方法插入 HSV1的任何非必需基因区， 位置不限。
上述（ 1 ) 、 (2) 中提到的逆转录病毒可以是除 HIV-1外的其 它逆转录病毒。
用以上方法得到的重组 HSV1病毒 "重组 HSVl-Lenti-helper 病毒" 可以用对 HSV1敏感的真核细胞（如 BHK-21)制备并进行稳 定地长期传代， 或- 70°C保存。 本发明提出的重组单纯疱疹病毒的用途
以上方法得到的含有 （ 1 ) 、 （ 2 ) 、 （ 3 )三种 DNA片段的重组 HSV1病毒 "重组 HSVl-Lenti-helper病毒" 主要用途是生产携带外 源基因的' it病毒载体， 特别是 HIV-1载体。
具体方法是， 先构建一个载体质粒， 该质粒含有以下成分： 1、 两端带有' f曼病毒、 特别是 HIV-1的长末端重复序列（LTR)的外源基 因表达盒， 该外源基因的启动子是强真核启动子， 如人巨细胞病毒 CMV的 IE启动子， 外源基因下游是真核转录终止和加尾信号， 比如 SV40病毒的 polyA; 2、 两个 LTR之间还含有慢病毒、 特别是 HIV-1 基因组包装和定位所需的两个顺式元件 ps i (包装信号）和 RRE (将 完整的病毒 RNA基因组带出细胞核）； 3、 在真核细胞中表达的抗性 基因表达盒， 如 neor、 blas t icidinr, kanr等； 4、 使该质粒在原核 细胞， 如大肠杆菌中复制的复制起点序列， 如 pUC ori以及抗性筛 选基因， 如 ampr等。
将该载体质粒通过脂质体等转染试剂转导到对 HSV1敏感的真核 细胞（如 BHK-21 ) 中， 再在培养液中加入合适浓度的抗生素， 经过 一定时间的培养， 筛选出载体质粒被稳定携带和或整合到细胞染色 体中的细胞株。
该细胞株感染含有（1 )、 （2 )、 （3 )三种 DNA片段的重组 HSV1 病毒 "重组 HSVl-Lent i-helper病毒" （三种 DNA片段在同一重组 HSV1病毒基因组上）后， 表达 gag/pol、 VSV-G和 Rev蛋白， 将整 合到细胞染色体中的载体 DNA反转录成慢病毒 RM基因组， 并包装 出慢病毒载体。
该细胞株以一定比例感染两种分别含有（ 1 )、 （ 3 )两种 DNA片 段和（2 )—种 DNA片段的重组 HSV1病毒后， 表达 gag/pol、 VSV-G 和 Rev蛋白，将整合到细胞染色体中的载体 DNA反转录成慢病毒 RNA 基因组， 并包装出慢病毒载体。
用本发明提出的重组单纯疱疹病毒（ "重组 HSVl-Lent i-helper 病毒" ）感染载体细胞株的方法生产的慢病毒载体的细胞裂解液中 混杂有重组单纯疱疹病毒（ "重组 HSV1- Lent i-helper病毒" ） ， 后者对动物或人细胞是有细胞毒性，需要除去。可以采用针对 VSV-G 蛋白的亲和层析、 离子吸附层析或 CsCl密度梯 离心等方法将其去 除。 附图说明
说明书附图之图 1是 gag/pol的 DNA片段组成的结构示意图， 它的上游（即 5，端）是人巨细胞病毒 CMV启动子（ CMV promoter )， 其后依次是： beta-globin intron增强子、 gag/po K RRE、 beta-g lobin po lyA。
说明书附图之图 2是 Rev的 DNA片段组成的结构示意图， 它的 5，端从上游至下游依次是 RSV启动子（RSV enhancer/promoter ) 、 Rev、 HIV的 LTR polyA„
说明书附图之图 3是非 HIV- 1的包膜蛋白的 D 片段組成的结 构示意图， 它的 5，端从上游至下 次是人巨细胞病毒 CMV启动子 ( CMV promoter ) 、 beta-globin intron增强子、 VSV_G、 beta-g lobin polyA。
说明书附图之图 4是 SetC 的结构示意图图语， Set C 粘粒按 HSV1病毒全基因顺序由依次分载了 HSV1病毒（17株）全基因组且 相邻的粘粒首位 DNA序列有部分完全相同的 5个粘粒 cos6、 cos28、 cos l4、 cos56、 cos48组成。 具体实施方式
以下实施例对本发明的用于重组慢病毒载体生产的全功能辅助 病毒（ "重组 HSVl-Lent i-helper病毒" ：） 的制备和用途作了详细 说明， 但并不意味着限制本发明的内容。
实施例 1 cos6- VSVG的构建 将质粒 pLP/VSVG (Invi trogen 公司生产）中的 VSV-G表达盒（CMV启动子 /beta- g lobin增强子 +VSV-G+beta-globin polyA )用 PCR的方式调出 （长约 3770 bp ) , 引物是： 上游 5， TCTAGACTTGGCCCATTGCATA 3， ( SEQ ID NO: 1 )； 下游 5， TCTAGAACTGCCATGTCGAGGG 3， ( SEQ ID NO: 2 ) 。 两端是 Xba l位点。 反应糾是： 94'C， 30秒； 56°C， 30秒； 72 °C， 4分钟。
PCR产物用 Xba l酶切后， 装入 cos6的 Xba l位点中， 得到粘粒 cos 6-VSVG。 实施例 2 cos56-gag/pol的构建
将质粒 pLPl (Invitrogen公司生产）中的 gag/pol表达盒（ CMV 启动子 /beta- globin增强子 +gag/pol+RRE+beta-globin polyA)用 PCR的方式调出 （长约 6837 bp) ， 引物是： 上游 5，
TCTAGATTGGCCCATTGCATAC 3， ( SEQ ID NO: 3) ； 下游 5，
TCTAGAACTGCCATGTCGAGGG 3， ( SEQ ID NO: 4) 。 两端是 Xbal位点。 反应条件是： 94 C, 30 秒； 56Ό, 30 秒； 72 ， 7 分钟。 PCR产 物用 Xbal酶切后， 装入 cos56的 Xbal位点中， 得到粘粒
cos56-gag/pol。 实施例 3 cos6- rev的构建
将质粒 pLP2 (Invitrogen公司生产）中的 Rev表达盒（RSV启动 子 +Rev+HIV polyA)用 PCR的方式调出 (长约 971 bp) , 引物是： 上游 5， TCTAGACAATGTAGTCTTATGC 3， ( SEQ ID NO: 5 ) ； 下游 5， TCTAGACCAGGGTTTTCCTGAT 3， ( SEQ ID NO: 6) 。 两端是 Xbal位点。 反应条件是： 94。C， 30 秒； 56 ， 30秒； 72 ， 1分钟。 PCR产物 用 Xbal酶切后， 装入 cos6的 Xbal位点中， 得到粘粒 cos6-rev。 实施例 4 重组 1型单纯疱疹病毒 rHSVl-VSVG-gag/pol的构建 将 cos6— VSVG、 cos56-gag/pol与 cosl4， cos28， cos48等 5个 粘粒等摩尔混合， 用 Pacl酶切去 cos骨架（不必分离去除），用酚、 酚 /氯仿（ 1: 1 )和氯仿各抽提一次， 吸取上清， 用 2.5倍无水乙醇 沉淀 DNA。用 lipofactamine (GIBCO BRL公司生产） 2 Oul与 lOugDNA 按产品说明书共转染 80%铺满的 BHK-21细胞 （约 2 χ 10ό)细胞， 5 个 HSV1片段将在细胞内发生同源重组而分别产生
rHSVl-VSVG-gag/pol重组病毒。 转染 24h后换用含 2%FBS (胎牛 血清）的 1640培养液 37。C培养， 每天换液一次。 5天后细胞开始出 现病变， 待细胞完全病变后收培养液上清， 2000 r/min离心 5min， 上清分装保存于 -20 。 对获得的重组病毒进行两次空斑纯化， 可得 到纯一的 rHSVl- VSVG - gag/pol重组病毒。 实施例 5 重组 1型单纯疱疹病毒 rHSVl-rev的构建
将 cos6-rev与 cos56、 cosl4, cos28， cos48等 5个粘粒等摩 尔混合， 用 Pacl酶切去 cos骨架（不必分离去除） ， 用酚、 酚 /氯 仿（ 1: 1 )和氯仿各抽提一次，吸取上清，用 2.5倍无水乙醇沉淀 DNA。 用 lipofactamine (GIBCO BRL公司生产）20ul 与 10 ug DNA按产品 说明书共转染 80%铺满的 BHK-21细胞 （约 2χ 10δ)细胞， 5个 HSV1 片段将在细胞内发生同源重组而分别产生 rHSV-rev重组病毒。转染 24h后换用含 2%FBS的 1640培养液 培养， 每天换液一次。 5 天后细胞开始出现病变， 待细胞完全病变后收培养液上清， 2000 r/min离心 5min， 上清分装保存于 -20X。 对获得的重组病毒进行两 次空斑纯化， 可得到纯一的 rHSVl-rev重组病毒。 实施例 6 携带报告基因 EGFP的载体质粒 pLenti-EGFP的构建 用 PCR的方法将 pEGFP (Gibco公司生产）中的 EGFP基因调出 （长度 约 730 bp ) ， 引物是： 上游 5， cacc atg gtg age aag ggc gag 3， ( SEQ ID NO: 7 )； 下游 5， gaattc cct cta gag tcg egg ccg ctt t 3， (SEQ ID NO: 8) 。 PCR反应^ H : 94V, 30秒； 56°C, 30秒； 72°C， 1分钟。 再通过 TOPO酶定向插入慢病毒载体盾粒
pLenti6/D-T0P0 ( Invitrogen公司生产） 中， 得到重组载体质粒 pLenti - EGFP。 实施例 7 稳定整合载体质粒 pLenti-EGFP的载体细胞株的构建 将栽体质粒 pLenti- EGFP用 1 ipofactamine (GIBCO BRL公司生 产）转染至 80%铺满的 BHK-21细胞 （约 2 χ 10δ)细胞。 24h小时后， 在 1640培养液中加入终浓度为 50 g/ml的抗生素 "Blasticdin" (杀稻瘟菌素）， 约 10天后， 挑选出单细胞克隆， 分别在荧光显微 镜下观察， 挑选出 GFP表达较高的细胞株。 成为稳定整合载体盾粒 pLenti-EGFP的载体细胞株。 实施例 8 慢病毒载体 Lenti-EGFP的产生
用实施例 4产生的 rHSVl-VSVG-gag/pol和实施例 5产生的 rHSVl-rev两个重组单纯疱疹病毒按一定滴度比例 （比如 10: 1 )感 染上述载体细胞株 pLenti-EGFP (Μ0Ι=1) , 37 X：继续培养 2-3 天， 至细胞病变完全。反复冻融 3次后， 离心得到上清， 即是混有重组 1 型单纯疱疹病毒的慢病毒载体 Lenti-EGFP。 实施例 9 慢病毒载体 Lenti-EGFP的纯化
上述混有重组 1型单纯疱疹病毒的慢病毒载体 Lenti-EGFP的上 清加到含有 VSV-G单克隆抗体的亲和层析柱中， 通过提高盐离子浓 度的梯度洗脱方式， 将慢病毒载体 Lenti-GFP洗脱下来， 得到纯化 了的' 病毒载体 Lent i- EGFP。 实施例 10 慢病毒载体 Lenti-EGFP的滴度测定
将用 PBS进行 10倍比稀释的慢病毒载体 Lenti-EGFP分别感染 80%铺满的 BHK-21细胞， 24小时后， 在培养液 ( 10%FBS 1640即含 10%胎牛血清的 1640培养液） 中加入终浓度为 5() g/ml的抗生素 "Blasticidin" (杀稻瘟菌素），约 10 天后， 分别在荧光显微镜下 观察， 计算细胞克隆数， 即得到慢病毒载体 Lenti-EGFP的滴度。

Claims

权 利 要 求
1. 一种重组人 1型单纯疱疹病毒 HSV1 ,其在基因组中包含可 提供慢病毒载体包装所需的反式蛋白的基因元件。
2. 权利要求 1的重组人 1型单纯疱疹病毒， 其中， 所述反式 蛋白为选自逆转录病毒的 Gag蛋白、 Pol蛋白、 Rev蛋白、及非 HIV-1 的包膜蛋白和它们的功能等价物的任一种， 或其组合。
3. 权利要求 2的重组人 1型单纯疱疹病毒， 其中所述逆转录 病毒为 HIV- 1。
4. 权利要求 2的重组人 1型单纯疱疹病毒， 其中所述非 HIV 的包膜蛋白为 VSV-G蛋白。
5. 权利要求 1-4之任一项的重组人 1型单纯疱疹病毒， 其中 所述基因元件为能表达所述反式蛋白的表达盒。
6. 权利要求 1-5之任一项的重组人 1型单纯疱疹病毒， 其中 所述基因元件位于基因组的非必需基因区。
7. 权利要求 1-6之任一项的重组人 1型单纯疱疹病毒， 其用 于制备慢病毒载体。
8. 权利要求 1-7之任一项的重组人 1型单纯疱疹病毒在制备 慢病毒载体中的用途。
9. 制备权利要求 1-7之任一项的重组人 1型单纯疱疹病毒的 方法， 包括步骤： 通过例如酶切插入或同源臂重组， 将权利要求 1-6 中定义的基因元件导入病毒基因组中。
10. 权利要求 7的制备方法， 其特征在于使用一套粘粒系统 Se tC进行。
11. 一种制备 it病毒载体的方法，其包括步骤：用权利要求 1-7 之任一项的重组人 1型单纯疱疹病毒感染载体细胞株， 包装出慢病 毒载体， 和任选地分离慢病毒载体。
12. 权利要求 11的方法，其中所述细胞包含以原病毒形式整合 在所述细胞的染色体中的外源基因， 优选所述细胞可以通过将包含 载体质粒导入细胞获得。
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