WO2014036807A1 - 重组单纯疱疹病毒、其制备方法及应用 - Google Patents
重组单纯疱疹病毒、其制备方法及应用 Download PDFInfo
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- WO2014036807A1 WO2014036807A1 PCT/CN2013/001040 CN2013001040W WO2014036807A1 WO 2014036807 A1 WO2014036807 A1 WO 2014036807A1 CN 2013001040 W CN2013001040 W CN 2013001040W WO 2014036807 A1 WO2014036807 A1 WO 2014036807A1
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- icp4
- herpes simplex
- simplex virus
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
Definitions
- the present invention relates to a recombinant herpes simplex virus, and more particularly to a herpes simplex virus which has improved antitumor activity and tumor targeting properties. Background technique
- Herpes s implex virus has been proposed in the prior art for the treatment of cancer by oncolytic therapy, and the herpes simplex virus is improved by eliminating the ICP34. 5 gene and the ICP47 gene, so that it can be selectively Tumor cells replicate in the cells without harming normal cells.
- this improvement does not effectively allow herpes simplex virus to specifically replicate only in tumor cells, but also herpes simplex virus replicates in certain normal cells. This causes damage to these normal cells. How to achieve herpes simplex virus specifically killing only tumor cells without destroying normal cells has been a key subject of research by those skilled in the art.
- Telomeres are a special structure at the end of a chromosome in eukaryotes. Their role is to maintain chromosome structural stability, including preventing chromosome end fusion, protecting chromosome structural genes, and avoiding the loss of genetic information in replication. Telomerase is a reverse transcriptase composed of small RNAs and proteins. It can synthesize telomere DNA using its own RNA as a template to compensate for telomeres that are gradually shortened with mitosis.
- telomerase RNA telomerase-associated ted prote in (TP1 /TLP1)
- human telomerase reverse human telomerase reverse Transcr iptase
- hTERT human telomerase reverse transcriptase
- Telomerase RNA is expressed in most cells
- human telomerase reverse transcriptase is the rate-limiting component of telomerase, expressed only in telomerase-positive cells and is associated with telomerase activity.
- ALT telomerase length telomeres
- ALT telomerase length telomeres
- hTERT Expressed only in a very small number of normal somatic cells.
- ICPs Infec ted cel l proteins expressed by herpes simplex virus are classified into three grades: immediate ear ly (IE), early (early), and late (late). Among them, IE protein is a key protein affecting viral replication, including: ICP0, ICP4, ICP22, ICP27 and ICP47, among which ICP4 is the most important replication-related protein.
- Pan-tumor-specific promoters which are promoters that exert their functions in various tumor cells, such as the hTERT promoter and the survivin promoter.
- Tissue-specific promoters such as the prostate specific antigen (PSA) promoter.
- PSA prostate specific antigen
- Promoters specific to certain tumors such as the adenine globulin (AFP) promoter unique to liver cancer cells, and the carcinoembryonic antigen (CEA) promoter of epithelial tumors.
- AFP adenine globulin
- CEA carcinoembryonic antigen
- the newly recombinant herpes simplex virus can only express its early replication protein ICP4 in tumor cells with high expression of hTERT, thereby replicating and proliferating, killing Tumor cells are expected to solve the above technical problems.
- Another object of the present invention is to provide a method for producing the above recombinant herpes simplex virus.
- a further object of the present invention is to provide a pharmaceutical composition and its use in the preparation of a medicament for treating cancer, and the use of the above recombinant herpes simplex virus for the preparation of a medicament for treating cancer.
- Another object of the present invention is to provide another recombinant herpes simplex virus, its use in the preparation of a medicament for diagnosing a tumor, and a tumor diagnostic kit.
- the object of the present invention and solving the technical problems thereof are achieved by the following technical solutions.
- the recombinant herpes simplex virus according to the present invention replaces the ICP4 gene promoter in the herpes simplex virus genome containing the ICP4 gene with the human telomerase reverse transcriptase promoter hTERTp or other tumor-specific promoter.
- the aforementioned recombinant herpes simplex virus has a biological deposit number of CGMCC No. 6397.
- herpes simplex virus excludes one or both of the ICP34.5 gene and the ICP47 gene.
- a method for preparing the recombinant herpes simplex virus according to the present invention comprising the steps of: replacing the ICP4 gene promoter in the herpes simplex virus containing the ICP4 gene with a human telomerase reverse transcriptase promoter hTERTp, and constructing the recombinant simple Herpesvirus HSV-hTERTp_ICP4:
- the herpes simplex virus containing the ICP4 gene is cultured with BHK cells, and the gene is purified.
- step b Amplification of the upstream flanking sequence of the ICP4 gene: Using the viral genomic DNA obtained in step a as a template, the ICP4USf forward primer and the ICP4USr reverse primer are used:
- ICP4USf forward primer CCCTCC AGACGC ACCGGAGTCGGGGG
- ICP4USr Reverse Primer AAGTCGACTCTAGAGGATCGATCTCTGACCTG
- ICP4DSf forward primer AAAAGTCGACCTGC AGGC ATGCTAACGAGGAA
- ICP4DSr reverse primer AAAAAAGCTTGCATGCCCACGTGCGCGGGGCC
- the upstream and downstream flanking sequences were cloned into the pSP73 plasmid to construct the pICP4del and pICP4del-eGFP plasmids: the upstream flanking sequence of the amplified ICP4 gene and the Sall/Hindlll double-digested amplified ICP4 gene were digested with Sail.
- the downstream flanking sequences were ligated and ligated into the EcoRV/Hindlll site of pSP73 to obtain pICP4del; the eGFP expression cassette controlled by CMV promoter was excised from pcDNA3.
- eGFP with EcoRI/XhoI and the end was filled with T4 DNA polymerase and inserted into pICP4del.
- the EcoRV site obtaining pICP4del-eGFP;
- ICP4-l st forward primer TTTTTTGAATTCATGGCGTCGGAGAACAAGCAGCGCC
- ICP4-l st reverse primer TGGAGCCACCCCATGGCCTCCGCGT
- ICP4-2 nd forward primer CGACGCCGCGC AGC AGT ACGCCCTG
- ICP4-2 nd reverse primer CGGCGGGGGCGGGCCCGGCGCACCG
- ICP4-3 rd forward primer CCTC ATGTTTGACCCGCGGGCCCTG
- ICP4- 3 rd reverse primer TTTTTTCTCGAGTTACAGCACCCCGTCCCCCTCGAAC
- ICP4-2 nd and ICP4-3 three gene fragments ICP4-l st , ICP4-2 nd and ICP4-3" 3 were amplified, respectively, and then the three segments were respectively
- the gene fragment was inserted into the EcoRV site of the pSP73 plasmid to construct the following three plasmids: pSP73-ICP4-l st pSP73-ICP4-2 nd , pSP73-ICP4-3 rd , used from the three plasmids EcoRI and BsrGI cut out ICP4-l st , cut out ICP4-2 nd with BsrGI and Pvul, and cut out ICP-3" with Pvul and Xhol" 1 for use;
- telomere reverse transcriptase promoter hTERTp with Nrul and Hindlll
- CMV promoter excised from Npul and Hindlll on pcDNA3-NHN was replaced to obtain the plasmid pcDNA3-NHN-hTERTp.
- pcDNA3-NHN is obtained by inserting a Nhel-Hapl-Nhel restriction site sequence at the Nhel site of pcDNA3;
- step e Mix the ICP4-l st , ICP4-2 nd and ICP4-3" 1 in step c and connect to the EcoRI and Xhol sites of the pcDNA3-NHN-hTERTp in step d to obtain plasmid pcDNA3-NHN- hTERTp_ICP4;
- the plasmid pICP4del containing the upstream and downstream flanking sequences of the ICP4 gene in step b was digested with Sail, and the terminal was used for excision.
- the plasmid pcDNA3-NHN-hTERTp-ICP4 obtained from step e was used to cut hTERTp with Pmel and Hpal.
- BHK-ICP4 helper cells The ICP4 gene was digested with the plasmid pcDNA3-NHN-hTERTp_ICP4 obtained from step e using EcoRI and Xhol, and cloned into the EcoRI and Xhol positions downstream of the CMV promoter in pcDNA3 to obtain pcDNA3- CMV-ICP4 plasmid; The pcDNA3-CMV-ICP4 plasmid was transfected into BHK cells, and the pcDNA3-CMV-ICP4 plasmid DNA was recombined into the BHK cell genome, and some BHK recombinant cells were resistant to neomycin and expressed ICP4. The anti-recombinant BHK cells were killed by antibiotic G418. After several rounds of subcloning screening, BHK-ICP4 helper cells expressing ICP4 were screened by RT-PCR;
- step A The viral genomic DNA in step A is co-transferred with the plasmid pICP4del-eGFP in step (1) b into the BHK-ICP4 helper cell in step (1) g, and homologously recombined, the plasmid pICP4del-eGFP
- the green fluorescent protein GFP expression cassette replaces the ICP4 gene of the herpes simplex virus HSV containing the ICP4 gene, so that the plaque of the recombinant virus is green-fluorescent, and after several rounds of plaque purification, green fluorescent venom is selected, and the recombinant can be purified.
- Virus HSV-d4GFP ;
- Step C The genomic DNA of the recombinant virus HSV-d4GFP and the DNA of the step (1) f of the plasmid pICP4del-hTERTp_ICP4 were co-transformed into the BHK-ICP4 helper cell, which was replaced by homologous recombination, hTERTp-ICP4 cassette
- the green fluorescent protein expression cassette GFP of the recombinant virus HSV-d4GFP prevents the venom of the new recombinant virus from emitting green fluorescence. After several rounds of plaque purification and selection of non-fluorescent plaques, the recombinant herpes simplex virus HSV- can be purified.
- the aforementioned method for producing a recombinant herpes simplex virus wherein the method further comprises the step of sequencing all of the involved plasmids to confirm that no mutation occurs.
- a pharmaceutical composition according to the present invention wherein the pharmaceutical composition comprises the aforementioned recombinant herpes simplex virus, and a pharmaceutically acceptable carrier or excipient.
- the pharmaceutical composition according to the above, wherein the pharmaceutical composition is an injection the injection comprises a pharmaceutically acceptable carrier and the aforementioned recombinant herpes simplex virus, and the injection contains 10 2 to 10 1 Q of the reconstitution per ml of the injection.
- Simple disease virus .
- the aforementioned pharmaceutical composition wherein the pharmaceutically acceptable carrier is a phosphate buffer having a pH of from 4.0 to 9.0.
- the object of the present invention and solving the technical problems thereof are also achieved by the following technical solutions.
- Another recombinant herpes simplex virus according to the present invention is a fluorescent protein expression inserted in the aforementioned recombinant herpes simplex virus genome.
- the fluorescent protein expression cassette is any one of a green fluorescent protein expression cassette, a cyan fluorescent protein expression cassette, a red fluorescent protein expression cassette, a yellow fluorescent protein expression cassette, and other indicator protein expression cassettes.
- the object of the present invention and solving the technical problems thereof are also achieved by the following technical solutions.
- a tumor diagnostic kit according to the present invention wherein the tumor diagnostic kit comprises the aforementioned recombinant herpes simplex virus.
- the aforementioned tumor diagnostic kit is characterized in that the sample detected by the tumor diagnostic kit is selected from the group consisting of whole blood, plasma, lymphocyte suspension, bone marrow, pleural effusion or peritoneal effusion.
- the tumor diagnosis kit of the present invention characterized in that the tumor diagnosis kit further comprises: RPMI-1640 medium, a red blood cell lysate having a pH of 7, and a phosphate buffer having a pH of 7.2 to 7.4, wherein the red blood cell lysate Containing 0.15M ammonium chloride, ⁇ potassium hydrogencarbonate and InM ethylenediaminetetraacetic acid; or RPMI-1640 medium, specific gravity of 1.077 ⁇ 0.001 kg / liter of polysucrose-diatrizoate and phosphoric acid with pH 7.2 ⁇ 7.4 Buffer.
- the present invention has significant advantages and advantageous effects over the prior art. According to the above technical solution, the recombinant herpes simplex virus of the present invention, the preparation method and application thereof can achieve considerable technical progress and practicability, and have extensive industrial use value, and at least have the following advantages:
- the recombinant herpes simplex virus of the present invention can more effectively selectively grow in human tumor cells than the existing herpesvirus, and does not multiply in human normal cells, thereby killing cancer cells more effectively. , protect normal cells;
- Another recombinant herpes simplex virus of the present invention which inserts a fluorescent protein expression cassette into the genome of the former recombinant herpes simplex virus, which can emit light in tumor cells, so that it can be faster, more accurate, more sensitive and Early diagnosis of tumors and diagnosis of tumor metastasis are widely performed.
- herpes simplex virus type I The recombinant herpes simplex virus obtained by the present invention is classified as herpes simplex virus type I, and the Latin literature name is Herpes Simplex Virus Type 1, which was deposited on August 14, 2012 at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The China Microbial Culture Collection Management Committee General Microbiology Center (CGMCC), the deposit number is CGMCC No. 6397.
- CGMCC General Microbiology Center
- oHSVl-hTERTp_ICP4 strain The preserved biological material oHSVl-hTERTp_ICP4 strain, the meaning of its forest number is: oHSVl Refers to herpes simplex virus type 17+ strain (HSV1); hTERTp-ICP4 refers to the replacement of the ICP4 gene promoter with the human telomerase reverse transcriptase promoter hTERTp.
- Fig. 1 is a schematic view showing the steps of a method for replacing the ICP4 gene promoter in the genome of the wild-type herpes simplex virus 17+ strain with the human telomerase reverse transcriptase promoter hTERTp according to Example 1 of the present invention.
- Fig. 2 shows the results of a comparative experiment of recombinant herpes simplex virus oHSVl-hTERTp_ICP4 prepared in Example 1 and wild type herpes simplex virus (17+) infecting normal cells (human fibroblasts).
- Figure 3 shows the recombinant herpes simplex virus oHSV1-d34.5-d47-hTERTp- ICP4 prepared in Example 2 and the ICP34.5 gene and ICP47 i-infected virus (oHSVl) which only exclude the wild-type herpes simplex virus genome in the prior art.
- oHSVl ICP47 i-infected virus
- Fig. 5A-Fig. 5D are the results of comparison between the tumor inhibition effect and the survival time of the animals after the injection of oHSVl-d34.5-d47 and oHSVl-d34.5-d47-hTERTp_ICP4. The best way to achieve the invention
- the present invention provides a recombinant herpes simplex virus, wherein the recombinant herpesvirus replaces the ICP4 gene promoter in the herpes simplex virus genome containing the ICP4 gene with a human telomerase reverse transcriptase promoter hTERTp or other tumor-specific promoter child.
- the herpes simplex virus containing the ICP4 gene may be a wild-type herpes simplex virus, a virus in which any gene fragment in the wild-type herpes simplex virus genome (except the ICP4 gene and the ICP4 gene promoter) is removed, but is not limited thereto. Any herpes simplex virus containing the ICP4 gene obtained by a person skilled in the art using conventional techniques can be used.
- the present invention provides a recombinant herpes simplex virus having a microorganism deposit number of CGMCC ⁇ 6397.
- the herpes simplex virus excludes one or both of the ICP34.5 gene and the ICP47 gene, and the ICP34.5 gene (neurotoxic gene) is eliminated, which makes the oncolytic virus safer; and the ICP47 gene is eliminated. Promotes immune response and enhances oncolytic activity.
- the two genes ICP34.5 and ICP47 are deleted.
- the present invention also provides a method for preparing the recombinant herpes simplex virus, wherein the method comprises the steps of: replacing the ICP4 gene promoter in the herpes simplex virus genome containing the ICP4 gene with a human telomerase reverse transcriptase promoter hTERTp, Construction of the recombinant herpes simplex virus HSV-hTERTp-ICP4:
- the ICP4 gene 3 ⁇ 4J herpes simplex virus is cultured with BHK cells, and the gene is purified.
- ICP4USf forward primer CCCTCC AGACGC ACCGGAGTCGGGGG
- ICP4USr Reverse Primer AAGTCGACTCTAGAGGATCGATCTCTGACCTG
- Amplification of the downstream (DS) flanking sequence of the ICP4 gene Using the viral genomic DNA obtained in step a as a template, the following ICP4DSf forward primer and ICP4USr reverse primer were used:
- ICP4DSf forward primer AAAAGTCGACCTGC AGGC ATGCTAACGAGGAA
- ICP4DSr reverse primer AAAAAAGCTTGCATGCCCACGTGCGCGGGGCC
- the upstream and downstream flanking sequences were cloned into the pSP73 plasmid to construct the pICP4del and pICP4del-eGFP plasmids: the upstream flanking sequence of the amplified ICP4 gene digested by Sail and the previously amplified ICP4 gene digested with Sall/Hindlll.
- the downstream flanking sequences were ligated and ligated into the EcoRV/Hindlll site of pSP73 to obtain pICP4del; the EGFP expression cassette controlled by CMV promoter was excised from pcDNA3.1-eGFP with EcoRI/XhoI, and the end was inserted by T4 DNA polymerase. Go to the EcoRV site of pICP4del to obtain pICP4del-eGFP;
- ICP4- 1 st forward primer TTTTTTGAATTCATGGCGTCGGAGAACAAGCAGCGCC
- ICP4-l st reverse primer TGGAGCCACCCCATGGCCTCCGCGT
- ICP4-2 nd forward primer CGACGCCGCGC AGC AGT ACGCCCTG
- ICP4-2 nd reverse primer CGGCGGGGGCGGGCCCGGCGC ACCG
- ICP4-3 rd forward primer CCTCATGTTTGACCCGCGGGCCCTG
- ICP4-3 rd reverse primer TTTTTTCTCGAGTTACAGCACCCCGTCCCCCTCGAAC
- the three genomic DNA fragments ICP4- 1 51 , ICP4-2 nd and ICP4-3 rd were amplified by using the viral genomic DNA obtained in step a as a template, and then the three-segment genes were respectively amplified.
- the fragment was inserted into the EcoRV site of the pSP73 plasmid to construct the following three plasmids: pSP73-ICP4-l st , pSP73-ICP4-2 nd , pSP73-ICP4-3 rd , from which ICP4 was cleaved with EcoRI and BsrGI -l st , cut out ICP4-2 nd with BsrGI and Pvul and cut out ICP-3" with Pvul and Xhol" 1 for use;
- telomere reverse transcriptase promoter hTERTp with Nrul and Hindlll
- CMV promoter excised from Npul and Hindlll on pcDNA3-NHN was replaced to obtain plasmid pcDNA3-NHN-hTERTp.
- pcDNA3-NHN is obtained by inserting a sequence of N el-Hapl-Nhel cleavage site at the Nhel site of pcDNA3;
- step e Mix the ICP4-l st , ICP4-2 nd ⁇ ICP4-3 rd in step c and connect to the EcoRI and Xhol sites of the pcDNA3-NHN-hTERTp in step d (the three gene fragments ICP4-
- the order of l st , ICP4-2 nd and ICP4-3 rd is determined by the sequence of the terminal sequence and the sequence of the EcoRI and Xhol sites of the pcDNA3-NHN-hTERTp, which can be automatically matched and ligated to obtain a plasmid.
- the plasmid pICP4dd containing the upstream and downstream flanking sequences of the ICP4 gene in step b was digested with Sail, and the plasmid was blunt-ended, and the plasmid pcDNA3-NHN-hTERTp_ICP4 obtained from step e with Pmel and Hpal was used.
- the hTERTp_ICP4 expression cassette fragment was cleaved and ligated into the pICP4del plasmid which was digested to construct the plasmid pICP4del-hTERTp_ICP4;
- BHK-ICP4 helper cells The ICP4 gene was digested with the plasmid pcDNA3-NHN-hTERTp_ICP4 obtained from step e using EcoRI and Xhol, and cloned into the EcoRI and Xhol sites downstream of the CMV promoter in pcDNA3 to obtain pcDNA3. -CMV-ICP4 plasmid; The pcDNA3-CMV-ICP4 plasmid was transfected into BHK cells, and the pcDNA3-CMV-ICP4 plasmid DNA was recombined into the BHK cell genome to obtain resistance and expression of neomycin to some BHK recombinant cells. ICP4, anti-recombinant BHK cells were killed by antibiotic G418, and after several rounds of subcloning screening, BHK-ICP4 helper cells expressing ICP4 were screened by RT-PCR;
- step A The viral genomic DNA in step A is co-transferred with the plasmid pICP4del-eGFP in step (1) b into the BHK-ICP4 helper cell in step (1) g, and homologously recombined, the plasmid pICP4del-eGFP
- the green fluorescent protein expression cassette GFP replaced the ICP4 gene of the herpes simplex virus HSV containing the ICP4 gene, and the plaque of the recombinant virus was green-fluorescent. After several rounds of plaque purification, green fluorescent venom was selected to purify the recombinant.
- Virus HSV-d4GFP (d4 indicates knockout of ICP4 gene);
- step B culturing the HSV-d4GFP virus in step B, and extracting genomic DNA
- Step C The genomic DNA of the recombinant virus HSV-d4GFP and the DNA of the step (1) f of the plasmid pICP4del-hTERTp_ICP4 were co-transformed into the BHK-ICP4 helper cell, and the recombinant virus was replaced by homologous recombination, hTERTp_ICP4 cassette.
- the green fluorescent protein expression cassette GFP of HSV-d4GFP prevents the venom of the new recombinant virus from emitting green fluorescence. After several rounds of plaque purification, the fluorescent herpes simplex virus HSV-hTERTp can be purified. ICP4.
- the preparation method of the recombinant herpes simplex virus wherein one or both of the ICP34.5 gene and the ICP47 gene of the herpes simplex virus containing the ICP4 gene can be first removed, and then the human telomere reverse transcription is reversed.
- the enzyme promoter hTERTp replaces the ICP4 gene promoter; or the recombinant herpes simplex virus HSV-hTERTp-ICP4 is obtained by replacing the ICP4 gene promoter with the human telomerase reverse transcriptase promoter hTERTp, and then the recombinant herpes simplex virus HSV- is eliminated.
- hTERTp - ICP44.5 gene and ICP47 base of ICP4! One or two of 3 ⁇ 4.
- the method for preparing the recombinant herpes simplex virus further comprises the step of sequencing all the plasmids involved to confirm that no mutation occurs. For the monitoring of the plasmid sequence, the accuracy of the entire preparation process can be guaranteed.
- the preparation method of the recombinant herpes simplex virus is not limited to using only the green fluorescent protein expression cassette, and can also be expressed by a cyan fluorescent protein expression cassette, a red fluorescent protein expression cassette, a yellow fluorescent protein expression cassette and other indicator proteins. Replace any of the boxes.
- the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the aforementioned recombinant herpesvirus, and a pharmaceutically acceptable carrier or excipient.
- the pharmaceutical composition is an injection comprising a pharmaceutically acceptable carrier and the aforementioned recombinant herpes simplex virus, which contains 10 2 to 10 1 Q of the recombinant herpes simplex virus per ml of the injection.
- the pharmaceutically acceptable carrier is a phosphate buffer having a pH of from 4.0 to 9.0.
- the injection solution further contains a protective agent and/or an osmotic pressure adjusting agent;
- the protective agent is contained in an amount of 0.01 to 30% by weight based on the injection, and the protective agent is selected from the group consisting of muscles One or more of alcohol, sorbitol and sucrose; 200 to 700 mg of the osmotic pressure adjusting agent per kg of the injection, the osmotic pressure adjusting agent being sodium chloride and/or potassium chloride.
- herpes simplex virus and pharmaceutical compositions described above can be used to prepare a medicament for treating tumors.
- the present invention also provides another recombinant herpes simplex virus, which inserts a fluorescent protein expression cassette into the recombinant herpes simplex virus genome, and the fluorescent protein expression cassette can be a green fluorescent protein expression cassette, a cyan fluorescent protein expression cassette, Any one of a red fluorescent protein expression cassette, a yellow fluorescent protein expression cassette, and other fluorescent protein expression cassettes; since the fluorescent protein expression cassette can emit light in tumor cells, a tumor can be realized more quickly, accurately, sensitively, and broadly. Early diagnosis and diagnosis of tumor metastasis.
- the present invention provides a tumor diagnostic kit comprising the aforementioned another recombinant herpes simplex virus,
- the tumor diagnostic kit detects a sample selected from the group consisting of whole blood, plasma, lymphocyte suspension, bone marrow, pleural effusion, and peritoneal effusion.
- the tumor diagnostic kit further includes: RPMI-1640 medium, pH 7 Red blood cell lysate and phosphate buffer having a pH of 7.2 to 7.4, wherein the red blood cell lysate comprises 0.15 M ammonium chloride, ⁇ potassium hydrogencarbonate and InM ethylenediaminetetraacetic acid; or RPMI-1640 medium, specific gravity Is 1.077 ⁇ 0.001 kg/L of sucrose-diatrizoate and phosphate buffer pH 7.2-7.4.
- the method disclosed in Chinese Patent No. CN102220292 A Publication Date: October 19, 2011
- Chinese Patent No. CN102220292 A Publication Date: October 19, 2011
- the following example uses the standard virulence forest of type I herpes simplex virus: 17+ strain (Genebank JN555585.1), purchased from the UK HPA (Health Protection Agency). The 17+ strain full gene sequence is known. Unless otherwise stated, the enzymes and plasmids used are purchased.
- the ICP4 gene promoter in the genome of the wild-type herpes simplex virus 17+ strain is replaced with the human telomerase reverse transcriptase promoter hTERTp, see Figure 1, the steps are:
- step b Amplification of the upstream flanking sequence of the ICP4 gene: using the 17+ viral genomic DNA obtained in step a as a template, using ICP4USf (forward primer: CCCTCCAGACGCACCGGAGTCGGGGG) and ICP4USr (reverse primer: AAGTCGACTCTAGAGGATCGATCTCTGACCTG
- Amplification of the downstream flanking sequence of the ICP4 gene using the 17+ viral genomic DNA obtained in step a as a template, using ICP4DSf (forward primer: AAAAGTCGACCTGCAGGCATGCTAA)
- CATGCCCACGTGCGCGGGGCCAGACGGGCT Amplification of the downstream flanking sequence of the ICP4 gene; the upstream and downstream flanking sequences were cloned into the pSP73 plasmid (purchased from Promega) to construct the pICP4del and pICP4del-eGFP plasmids: Sail-cleaved the aforementioned amplified ICP4 gene The upstream flanking sequence and the downstream flanking sequence of the amplified ICP4 gene of Sall/Hindlll double digestion were mixed and ligated into the EcoRV/Hindlll site of pSP73 to obtain pICP4del.
- Three-stage PCR amplification of the three-stage sequence of the ICP4 gene First, using the primers shown in Table 1, the 17+ viral genomic DNA obtained in step a was used as a template to amplify the three-segment gene fragment ICP4-l st ICP4-2 nd and ICP4-3 rd , and then the three gene fragments were inserted into the EcoRV site of pSP73 to construct the following three plasmids: pSP73-ICP4-l st , pSP73-ICP4-2 nd , pSP73-ICP4- 3 rd .
- the hTERTp fragment was excised from the plasmid containing the human telomerase reverse transcriptase promoter hTERTp with Nrul and Hindlll, and the substitution was obtained from pcDNA3-NHN (pcDNA3 was purchased from Invitrogen, and Nel-Hapl was inserted at the Nhel site of pcDNA3).
- pcDNA3 was purchased from Invitrogen, and Nel-Hapl was inserted at the Nhel site of pcDNA3).
- -Nhel cleavage site sequence constructed into pcDNA3-NHN) CMV promoter excised with Nrul and Hindlll to obtain plasmid pcDNA3-NHN-hTERTp;
- step e Mix the ICP4-l st , ICP4-2 nd and ICP4-3 rd in step c and connect to the EcoRI and Xhol sites of the pcDNA3-NHN-hTERTp in step d to obtain plasmid pcDNA3-NHN-hTERTp_ICP4 f.
- the plasmid pICP4del containing the upstream and downstream flanking sequences of the ICP4 gene was digested with Sail, and the end of the plasmid was blunt-ended.
- hTERTp-ICP4 The expression of hTERTp-ICP4 was cleaved from the plasmid pcDNA3-NHN-hTERTp-ICP4 obtained from step e using Pmel and Hpal. The cassette fragment was ligated to the pICP4del 3 ⁇ 4 body after digestion and the plasmid pICP4del-hTERTp_ICP4 was constructed.
- BHK-ICP4 helper cells The ICP4 gene was digested with the plasmid pcDNA3-NHN-hTERTp_ICP4 obtained from step e using EcoRI and Xhol, and cloned into the downstream EcoRI and Xhol sites initiated by CMV in pcDNA3 to obtain pcDNA3- CMV-ICP4 plasmid; The pcDNA3-CMV-ICP4 plasmid was transfected into BHK cells, and the pcDNA3-CMV-ICP4 plasmid DNA was recombined into the BHK cell genome, and some BHK recombinant cells were resistant to neomycin. And express ICP4. Unreacted BHK cells were killed with antibiotic G418. After several rounds of subcloning screening, BHK-ICP4 helper cells expressing ICP4 were screened by RT-PCR.
- step A The viral genomic DNA in step A is co-transferred with the plasmid pICP4del-eGFP in step (1) b into the BHK-ICP4 helper cell in step (1) g, and homologously recombined, the plasmid pICP4del-eGFP
- the green fluorescent protein expression cassette replaces the ICP4 gene of the herpes simplex virus HSV containing the ICP4 gene, so that the plaque of the recombinant virus is green-fluorescent, and after several rounds of plaque purification, green fluorescent venom is selected, and the recombinant virus can be purified.
- step B culturing the oHSV1-d4GFP virus in step B, and extracting genomic DNA;
- Example 2 This example is similar to Example 1, except that the ICP4 gene promoter in the genome of wild-type herpes simplex virus in which the ICP47 gene and the ICP34.5 gene are deleted is replaced with human telomerase reverse transcriptase. SubhTERTp, the specific steps are as follows:
- Amplification of the upstream flanking sequence of the ICP47 gene using the 17+ viral genomic DNA obtained in step (1) as a template, using ICP47USf (forward primer: AAAAGAATTCGATTGGGTTCGAT)
- AACTAGCGCGGACCGGTCG Amplifies ICP47 DS FLR.
- the upstream and downstream flanking sequences were cloned into pBSK (purchased from Stratagene) plasmid to construct pdICP47 and pdICP47-eGFP plasmids: EcoRI/Spel double-digested the upstream flanking sequence of the amplified ICP47 gene, Hindlll/Sall Double-cleavage of the downstream flanking sequence of the amplified ICP47 gene and a complementary ligation sequence with a Spel Hindlll double-cleavage site (Linker 1)
- d Co-transfecting the 17+ virus genomic DNA obtained in step (1) with pdICP47-eGFP into BHK cells. After homologous recombination, after several rounds of plaque purification and selection of green fluorescent plaques, the pure recombinant virus oHSVl-d47-GFP can be obtained.
- the GFP expression cassette in oHSVl-d47-GFP was knocked out by pdICP47 in the same manner to obtain oHSV1-d47.
- ICP34.5USr reverse primer: GCGGCCGCAGCGCTGCGGCCGCCG
- ICP34.5DSr reverse primer: TTCTTCCCTCTTCTCCCGCCCTCCA amplified the downstream flanking sequence of ICP34.5.
- the upstream and downstream flanking sequences were cloned into the pSP72 (purchased from Promega) plasmid to construct the pdICP34.5 and pdICP34.5-eGFP plasmids: the overlapping PCR34.5 upstream and downstream flanking sequences were ligated by overlapping polymerase chain reaction (over PCR).
- the pSP72 vector was ligated with a pSP72 vector double-cut and filled with BamHI/Xhol to obtain pdICP34.5.
- the eGFP expression cassette was excised from pcDNA3.1-eGFP using EcoRI/XhoI, and the ends were blunted with T4 DNA polymerase and inserted into the Afel site of pdICP34.5 to obtain pdICP34.5-eGFP.
- oHSV1-d47 genomic DNA obtained in step (2) was co-transfected into BHK cells with pdICP34.5-eGFP obtained in step D. After homologous recombination, after several rounds of plaque purification and selection of green fluorescent plaques, a pure recombination virus oHSVl-d47-d34.5-GFP can be obtained.
- the GFP expression cassette in oHSV d47-d34.5-GFP was knocked out by pdICP34.5 in the same manner to obtain oHSV d34.5-d47.
- the human telomerase reverse transcriptase promoter hTERTp was substituted for the ICP4 gene promoter of oHSV1-d34.5-d47 obtained above to construct a recombinant herpes simplex virus.
- oHSV d34.5-d47-hTERTp - ICP4 the ICP47 gene and the ICP34.5 gene can also be eliminated by methods known in the art.
- the ICP47 gene and the ICP34.5 gene can also be deleted after the recombinant herpes simplex virus oHSV1-hTERTp-ICP4 obtained in Example 1.
- the method of eliminating the ICP47 gene and the ICP34.5 group may be the method of Example 2, or other methods in the prior art may be employed.
- the recombinant herpes simplex virus oHSVl-hTERTp-ICP4 of the present invention can kill a variety of tumor cells and has a broad-spectrum anti-tumor effect.
- gastric cancer human BGC823 gastric adenocarcinoma
- HuH7 human hepatocellular carcinoma
- Figures 5A and 5C show that both virus groups have significant tumor suppressor effects compared to the control group and there is little difference between the two.
- the animal survival time in Figures 5B and 5D showed that the oHSVl-d34.5-d47-hTERTp_ICP4 treatment group was significantly better than the oHSVl-d34.5-d47 treatment group.
- the median survival time of the oHSV d34.5-d47-hTERTp_ICP4 group in the BGC823 model was 100 days, which was significantly longer than the oHSVl-d34.5-d47 group (27 days) and the control group (61 days).
- the median survival time of the oHSVl -d34.5-d47-hTERTp_ICP4 group in the HuH7 model was 62 days, which was also significantly longer than the oHSVl-d34.5-d47 group (48 days) and the control group (47 days). Therefore, the in vivo anti-tumor experiments of both models showed that oHSVl-d34.5-d47-hTERTp_ICP4 was superior to oHSVl-d34.5-d47 in terms of therapeutic effect and safety.
- herpes simplex virus type 17+ strain Although the above two examples are exemplified by the herpes simplex virus type 17+ strain, the present invention is not limited to the 17+ strain, and is also applicable to the herpes simplex virus type I strain F, KOS strain, and JS1 strain. The same applies to the herpes simplex virus type II, such as the HG52 strain, which is applicable to any herpes simplex virus containing the ICP4 gene.
- telomerase reverse transcriptase promoter hTERTp which replaces the ICP4 promoter of the herpes simplex virus genome containing the ICP4 gene with other tumor-specific promoters known to those skilled in the art, such as: survivin promoter, prostate specific antigen (PSA) Promoter, adenoma globulin (AFP) promoter unique to liver cancer cells, carcinoembryonic antigen (CEA) promoter of epithelial tumors, and the like.
- PSA prostate specific antigen
- AFP adenoma globulin
- CEA carcinoembryonic antigen
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Abstract
本发明提供了一种重组单纯疱疹病毒及其制备方法,其是将含有ICP4基因的单纯疱疹病毒基因组中的ICP4基因启动子替换为人端粒酶逆转录酶启动子hTERTp或其他肿瘤特异性启动子,从而更有选择性地在人肿瘤细胞内生长繁殖,有效杀伤癌细胞。本发明还提供了该重组单纯疱疹病毒在制备治疗肿瘤药物中的应用。本发明还提供了另一种重组单纯疱疹病毒,其是在前述重组单纯疱疹病毒基因组中插入荧光蛋白表达盒,使其能在肿瘤细胞内发光,可实现肿瘤的早期诊断及肿瘤转移的诊断。
Description
重组单纯疱疹病毒、 其制备方法及应用
技术领域
本发明涉及一种重组单纯疱疹病毒, 特别是涉及一种在抗肿瘤活性及肿瘤靶向性上 有所改进的单纯疱疹病毒。 背景技术
现有技术中已经提出单纯疱疹病毒(herpes s implex vi rus , HSV)可用于溶瘤法治 疗癌症, 并通过剔除 ICP34. 5基因和 ICP47基因对单纯疱疹病毒进行改进, 使其可选择 性地在肿瘤细胞内复制, 而不伤害正常细胞, 然而这种改进并不能有效地使单纯疱疹病 毒特异性地只选择在肿瘤细胞内复制, 而是也会有单纯疱疹病毒在某些正常细胞内复 制,从而对这些正常细胞造成伤害。如何实现使单纯疱疹病毒特异性地只杀伤肿瘤细胞, 而不破坏正常细胞一直是本领域技术人员研究的重点课题。
端粒是真核生物染色体末端的一种特殊结构, 其作用是维护染色体结构稳定, 包括 防止染色体末端融合, 保护染色体结构基因和避免遗传信息在复制中丢失。 端粒酶是由 小分子 RNA和蛋白质组成的逆转录酶,能利用自身 RNA为模板合成端粒 DNA,弥补随细胞 有丝分裂逐渐缩短的端粒。其有三个主要组成部分:人端粒酶 RNA (human telomerase RNA, hTR)、 端粒酶相关蛋白(te lomerase— associa ted prote in, TP1 /TLP1)、 人端粒酶逆转 录醉 (human telomerase reverse transcr iptase, hTERT)。 端粒酶 RNA在大多数细胞 中都表达,而人端粒酶逆转录酶是端粒酶的限速成分, 仅在端粒酶阳性的细胞中表达, 与端粒酶活性相关。 尽管一些肿瘤细胞在重组时通过替代性端粒延长( al ternat ive lengthening of telomeres, ALT) 机制来维持端粒长度,但仍有 85 %以上的肿瘤细胞通 过上调 hTERT来活化端粒酶, 而且 hTERT仅在极少数正常体细胞中表达。 这就为肿瘤治 疗提供了一个很好的契机。单纯疱疹病毒表达的感染细胞蛋白(infec ted cel l proteins, ICPs)分为三个等级: 初始(immediate ear ly, IE) 、 早期(ear ly)和晚期(late)。 其中 IE蛋白是影响病毒复制的关键蛋白, 包括: ICP0、 ICP4、 ICP22、 ICP27和 ICP47 , 而其 中 ICP4是最关键的复制相关蛋白。
现已发现多种肿瘤特异性启动子。 根据其特点, 可分为以下几种: (1 ) 泛肿瘤特 异启动子, 指在多种肿瘤细胞内发挥其功能的启动子, 如 hTERT 启动子、 存活素 ( surviv in )启动子等。 (2 )组织特异性启动子, 如前列腺特异性抗原(PSA )启动子。 ( 3 )某些肿瘤特有的启动子, 如肝癌细胞特有的曱胎球蛋白 (AFP )启动子、 上皮细胞 肿瘤的癌胚抗原 (CEA ) 启动子等。 这些肿瘤特异性启动子已广泛应用于肿瘤基因治疗 及肿瘤诊断领域。
若将 ICP4启动子替换为 hTERT启动子 (hTERTp )或其它肿瘤特异性启动子, 使 新重组单纯疱疹病毒只能在 hTERT 高表达的肿瘤细胞中表达其早期复制蛋白 ICP4, 从 而进行复制增殖, 杀伤肿瘤细胞, 有望解决上述技术难题。 发明内容
本发明的目的在于, 提供一种重组单纯疱疹病毒, 该重组单纯疱疹病毒能选择性地 在人肿瘤细胞内生长繁殖, 而不在人正常细胞中繁殖, 从而杀伤肿瘤细胞, 且不伤害正 常细胞。
本发明的另一目的在于, 提供一种上述重组单纯疱疹病毒的制备方法。
本发明的再一目的在于, 提供一种药物组合物及其在制备治疗癌症的药物中的应 用, 以及上述重组单纯疱疹病毒在制备治疗癌症的药物中的应用。
本发明的另一目的在于, 提供另一种重组单纯疱疹病毒、 其在制备诊断肿瘤的药物 中的应用以及一种肿瘤诊断试剂盒。
本发明的目的及解决其技术问题是采用以下技术方案来实现的。依据本发明提出的 重组单纯疱疹病毒,其是将含有 ICP4基因的单纯疱疹病毒基因组中的 ICP4基因启动子 替换为人端粒酶逆转录酶启动子 hTERTp或其它肿瘤特异性启动子。
本发明的目的及解决其技术问题还可采用以下技术措施进一步实现。
前述的重组单纯疱疹病毒, 其^!生物保藏号为 CGMCC No.6397。
前述的重组单纯疱疹病毒, 其中该单纯疱疹病毒剔除了 ICP34.5基因和 ICP47基因 中的一种或两种。
本发明的目的及解决其技术问题还采用以下技术方案来实现。依据本发明提出的一 种制备前述重组单纯疱疹病毒的方法, 其包括以下步骤: 用人端粒酶逆转录酶启动子 hTERTp取代含有 ICP4基因的单纯疱疹病毒中的 ICP4基因启动子, 构建该重组单纯疱 疹病毒 HSV-hTERTp_ICP4:
( 1 ) 构建穿梭; ί粒 pICP4del-hTERTp_ICP4和 pICP4del- eGFP:
a. 用 BHK细胞培养该含有 ICP4基因的单纯疱疹病毒, 并纯化其基因
组 DNA;
b. 扩增 ICP4基因上游侧翼序列: 以步骤 a中所得病毒基因组 DNA为模板, 用以 下 ICP4USf正向引物和 ICP4USr反向引物:
ICP4USf正向引物: CCCTCC AGACGC ACCGGAGTCGGGGG
ICP4USr反向引物: AAGTCGACTCTAGAGGATCGATCTCTGACCTG
AGATTGGCGGCACTGAGGTA
扩增出 ICP4基因上游侧翼序列;
扩增 ICP4基因下游侧翼序列: 以步骤 a中所得病毒基因组 DNA为模板, 用以下 ICP4DSf正向引物和 ICP4USr反向引物:
ICP4DSf正向引物: AAAAGTCGACCTGC AGGC ATGCTAACGAGGAA
CGGGCAGGGGGC
ICP4DSr反向引物: AAAAAAGCTTGCATGCCCACGTGCGCGGGGCC
AGACGGGCT
扩增出 ICP4基因下游侧翼序列;
将上下游侧翼序列克隆到 pSP73质粒上,构建 pICP4del及 pICP4del-eGFP质粒: 将 Sail酶切的前述扩增出的 ICP4基因的上游侧翼序列及 Sall/Hindlll双酶切的前述扩增出 的 ICP4基因的下游侧翼序列混合并连接到 pSP73的 EcoRV/Hindlll位点,得到 pICP4del; 用 EcoRI/XhoI从 pcDNA3. eGFP切下 CMV启动子控制的 eGFP表达盒, 经 T4 DNA 聚合酶补平末端后插入到 pICP4del的 EcoRV位点, 得到 pICP4del-eGFP;
c. 分三次 PCR扩增出 ICP4基因中三段序列: 首先, 使用以下引物:
ICP4-lst正向引物: TTTTTTGAATTCATGGCGTCGGAGAACAAGCAGCGCC ICP4-lst反向引物: TGGAGCCACCCCATGGCCTCCGCGT
ICP4-2nd正向引物: CGACGCCGCGC AGC AGT ACGCCCTG
ICP4-2nd反向引物: CGGCGGGGGCGGGCCCGGCGCACCG
ICP4-3rd正向引物: CCTC ATGTTTGACCCGCGGGCCCTG
ICP4- 3rd反向引物: TTTTTTCTCGAGTTACAGCACCCCGTCCCCCTCGAAC 以步骤 a 中所得病毒基因组 DNA 为模板, 分别扩增出三段基因片段 ICP4-lst、 ICP4-2nd和 ICP4-3"3, 而后分别将该三段基因片段插入 pSP73质粒的 EcoRV位点构建出 以下三种质粒: pSP73-ICP4-lst pSP73-ICP4-2nd 、 pSP73-ICP4-3rd, 从该三种质粒中用
EcoRI和 BsrGI剪切出 ICP4-lst、 用 BsrGI和 Pvul剪切出 ICP4-2nd及用 Pvul和 Xhol剪 切出 ICP-3"1待用;
d.从含人端粒醉逆转录酶启动子 hTERTp的质粒中用 Nrul和 Hindlll切下 hTERTp 片段, 取代从 pcDNA3-NHN 上用 Nrul 和 Hindlll 切除的 CMV 启动子, 得到质粒 pcDNA3-NHN-hTERTp, 其中, pcDNA3-NHN 是在 pcDNA3 的 Nhel 位点插入 Nhel-Hapl-Nhel酶切位点序列所得;
e. 将步骤 c 中的该 ICP4-lst、 ICP4-2nd和 ICP4-3"1混合并连接到步骤 d 中的该 pcDNA3-NHN-hTERTp的 EcoRI及 Xhol位点,得到质粒 pcDNA3-NHN-hTERTp_ICP4; f. 用 Sail酶切步骤 b中含 ICP4基因上下游侧翼序列的质粒 pICP4del, 并补 末端 待用, 用 Pmel和 Hpal从步骤 e获得的该质粒 pcDNA3-NHN-hTERTp— ICP4剪切出 hTERTp— ICP4表达盒片段, 并将其与该酶切后待用的 pICP4del质粒连接, 构建出质粒 pICP4del-hTERTp_ICP4;
g. 构建 BHK-ICP4 辅助细胞: 用 EcoRI 和 Xhol 从步骤 e 获得的该质粒 pcDNA3-NHN-hTERTp_ICP4中酶切出 ICP4基因,并克隆到 pcDNA3中 CMV启动子的 下游 EcoRI和 Xhol位 , 得到 pcDNA3-CMV-ICP4质粒; 将该 pcDNA3-CMV-ICP4质 粒转染 BHK细胞, 该 pcDNA3-CMV-ICP4质粒 DNA可重组到 BHK细胞基因组中, 使 有些 BHK重组细胞获得对新霉素的抗性和表达 ICP4,用抗菌素 G418杀死未重组的 BHK 细胞, 经过几轮的亚克隆筛选, 用 RT- PCR方法筛选出表达 ICP4的 BHK-ICP4辅助细 胞;
( 2 )剔除基因组中 ICP4基因启动子并插入端粒酶逆转录酶启动子 hTERTp启动子:
A. 用 BHK细胞培养该含有 ICP4基因的单纯疱疹病毒, 并提取病毒基因组 DNA;
B. 将步骤 A中该病毒基因组 DNA与步骤( 1 ) b中该质粒 pICP4del-eGFP共转入 步骤( 1 ) g中该 BHK-ICP4辅助细胞内, 经同源重组, 该质粒 pICP4del-eGFP中绿色荧 光蛋白 GFP表达盒置换了该含有 ICP4基因的单纯疱疹病毒 HSV的 ICP4基因, 使得重 组病毒的毒斑发绿色荧光, 经过几轮的噬斑纯化, 挑选绿色荧光毒斑, 就能纯化出重组 病毒 HSV-d4GFP;
C 培养步骤 B中该 HSV-d4GFP病毒, 并提取基因组 DNA;
D. 将步骤 C 该重组病毒 HSV-d4GFP 的基因组 DNA 和步骤 ( 1 ) f 该质粒 pICP4del-hTERTp_ICP4 的 DNA 共转入该 BHK-ICP4 辅助细胞, 经同源重组, hTERTp— ICP4表 盒置换了该重组病毒 HSV-d4GFP的绿色荧光蛋白表达盒 GFP,使新 重组病毒的毒斑不发绿色荧光, 经过几轮的噬斑纯化, 挑选无荧光毒斑, 就能纯化出该 重组单纯疱疹病毒 HSV-hTERTp_ICP40
本发明的目的及解决其技术问题还采用以下技术措施进一步实现。
前述的重组单纯疱疹病毒的制备方法, 其中先剔除该含有 ICP4基因的单纯疱疹病 毒的 ICP34.5基因和 ICP47基因中的一种或两种,再以人端粒酶逆转录酶启动子 hTERTp 替换 ICP4基因启动子;或在以人端粒酶逆转录酶启动子 hTERTp替换 ICP4基因启动子 得到该重组单纯疱疹病毒 HSV- hTERTp—ICP4 后, 再剔除该重组单纯疱疹病毒 HSV-hTERTp_ICP4的 ICP34.5基因和 ICP47基因中的一种或两种。
前述的重组单纯疱疹病毒的制备方法, 其中以其它肿瘤特异性启动子替代该人端粒 酶逆转录酶启动子 hTERTp。
前述的重组单纯疱疹病毒的制备方法, 其中该方法还包括对所有涉及的质粒进行测 序以证实无突变发生的步骤。
前述的重组单纯疱疹病毒的制备方法, 其中该绿色荧光蛋白表达盒可由青色荧光蛋 白表达盒、 红色荧光蛋白表达盒、 黄色荧光蛋白表达盒或其它指示蛋白表达盒所替换。
本发明的目的及解决其技术问题还采用以下技术方案来实现。依据本发明提出的药 物组合物, 其中该药物组合物包含前述的重组单纯疱疹病毒, 以及药物可接受的载体或 赋形剂。
本发明的目的及解决其技术问题还采用以下技术措施进一步实现。
前述的药物组合物, 其中该药物组合物为注射液, 所述注射液包括药学上可接受的 载体以及前述的重组单纯疱疹病毒, 每毫升所述注射液中含有 102 ~ 101Q个该重组单纯 疾渗病毒。 .
前述的药物组合物,其中该药学上可接受的载体为 pH值为 4.0 - 9.0的磷酸緩沖液。 前述的药物组合物,其中该注射液还含有保护剂和 /或渗透压调节剂; 以该注射液为 基准, 所述保护剂的含量为 0.01 ~ 30% (重量) , 所述保护剂选自肌醇、 山梨醇和蔗糖 中的一种或一种以上; 每千克该注射液含有 200 - 700毫克所述渗透压调节剂, 所述渗 透压调节剂为氯化钠和 /或氯化钾。
本发明的目的及解决其技术问题还采用以下技术方案来实现。依据本发明提出的前 述的重组单纯疱疹病毒, 或前述的药物组合物在制备治疗肿瘤的药物中的应用。
本发明的目的及解决其技术问题还采用以下技术方案来实现。依据本发明提出的另 一种重组单纯疱疹病毒, 其是在前述的重组单纯疱疹病毒基因组中插入荧光蛋白表达 。
本发明的目的及解决其技术问题还采用以下技术措施进一步实现。
前述的另一种重组单纯疱疹病毒, 其中该荧光蛋白表达盒为绿色荧光蛋白表达盒、 青色荧光蛋白表达盒、 红色荧光蛋白表达盒、 黄色荧光蛋白表达盒及其它指示蛋白表达 盒中任一种。
本发明的目的及解决其技术问题还采用以下技术方案来实现。依据本发明提出的前 述的另一种重组单纯疱疹病毒在制备诊断肿瘤的药物中的应用。
本发明的目的及解决其技术问题还采用以下技术方案来实现。依据本发明提出的一 种肿瘤诊断试剂盒, 其中该肿瘤诊断试剂盒包含前述的另一种重组单纯疱疹病毒。
本发明的目的及解决其技术问题还采用以下技术措施进一步实现。
前述的肿瘤诊断试剂盒, 其特征在于该肿瘤诊断试剂盒检测的样品选自受试者全 血、 血浆、 淋巴细胞悬浮液、 骨髓、 胸腔积液或腹腔积液。
前述的肿瘤诊断试剂盒,其特征在于该肿瘤诊断试剂盒还包括: RPMI-1640培养基、 pH为 7的红细胞裂解液和 pH为 7.2 ~ 7.4的磷酸緩冲液, 其中, 所述红细胞裂解液包含 0.15M氯化铵、 ΙΟηΜ碳酸氢钾和 InM 乙二胺四乙酸; 或 RPMI-1640培养基、 比重为 1.077 ~ 0.001千克 /升的聚蔗糖 -泛影葡胺和 pH为 7.2 ~ 7.4的磷酸緩沖液。
本发明与现有技术相比具有明显的优点和有益效果。 借由上述技术方案,本发明重 组单纯疱疹病毒、 其制备方法及应用可达到相当的技术进步性及实用性, 并具有产业上 的广泛利用价值, 其至少具有下列优点:
( 1 )本发明的重组单纯疱疹病毒相对于现有的疱疹病毒而言, 能更有效地选择性地 在人肿瘤细胞内生长繁殖, 而不在人正常细胞中繁殖, 从而更有效地杀伤癌细胞, 保护 正常细胞;
( 2 )本发明的另一种重组单纯疱疹病毒, 其是在前一种重组单纯疱疹病毒基因组中 插入了荧光蛋白表达盒, 其能在肿瘤细胞内发光, 所以可更快速、 准确、 灵敏并广谱地 实现肿瘤的早期诊断以及肿瘤转移的诊断。
本发明获得的重组单纯疱疹病毒的分类命名为 I 型单纯疱疹病毒, 拉丁文学名为 Herpes Simplex Virus Type 1 , 于 2012年 8月 14日保藏于位于北京市朝阳区北辰西路 1 号院 3号的中国微生物菌种保藏管理委员会普通微生物中心 (CGMCC ) , 保藏编号为 CGMCC No.6397。被保藏的生物材料 oHSVl-hTERTp_ICP4株,其林号的含义为: oHSVl
指 I 型单纯疱疹病毒 17+株(HSV1 ) ; hTERTp— ICP4 指以人端粒酶逆转录酶启动子 hTERTp替换 ICP4基因启动子。
上述说明仅是本发明技术方案的概述, 为了能够更清楚了解本发明的技术手段, 而 可依照说明书的内容予以实施, 并且为了让本发明的上述和其他目的、 特征和优点能够 更明显易懂, 以下特举较佳实施例, 并配合附图,详细说明如下。 附图的简要说明
图 1为本发明实施例 1将野生型单纯疱疹病毒 17+毒株的基因组中的 ICP4基因启 动子替换为人端粒酶逆转录酶启动子 hTERTp的方法步骤示意图。
图 2显示实施例 1制备的重组单纯疱疹病毒 oHSVl-hTERTp_ICP4与野生型单纯疱 疹病毒(17+ )感染正常细胞(人成纤维细胞) 的对比实验结果。
图 3显示实施例 2制备的重组单纯疱疹病毒 oHSVl- d34.5-d47-hTERTp— ICP4与现 有技术中只剔除了野生型单纯疱疹病毒基因组的 ICP34.5 基因和 ICP47 i因的病毒 ( oHSVl-d34.5-d47 )感染正常人白细胞的对比实验结果。
图 4A-图 4F显示重组单纯疱疹病毒 oHSVl-hTERTp— ICP4对肝癌、肺癌、头颈鳞癌、 黑色素瘤、 结肠癌和前列腺癌的杀伤作用试验结果(ΜΟΙ=1 ) 。
图 5A- 图 5D 为 棵 鼠 瘤 内 分 另' J 注 射 oHSVl-d34.5-d47 和 oHSVl-d34.5-d47-hTERTp_ICP4后其抑瘤效果及动物存活时间的对比实验结果。 实现发明的最佳方式
为更进一步阐述本发明为达成预定发明目的所采取的技术手段及功效,以下结合附 图及较佳实施例, 对依据本发明提出的重组单纯疱疹病毒、 其制备方法及应用其具体实 施方式、 结构、 特征及其功效, 详细说明如后。
本发明提供了一种重组单纯疱疹病毒, 其中, 该重组疱疹病毒是将含有 ICP4基因 的单纯疱疹病毒基因组中的 ICP4基因启动子替换为人端粒酶逆转录酶启动子 hTERTp 或其它肿瘤特异性启动子。该含有 ICP4基因的单纯疱疹病毒可为野生型单纯疱疹病毒、 剔除了野生型单纯疱疹病毒基因组中任何基因片段(除了 ICP4基因和 ICP4基因启动子) 的病毒, 但不局限于此, 可为本领域技术人员利用常规技术对其进行变化所得的任何含 有 ICP4基因的单纯疱疹病毒。
更具体地, 本发明提供了一种重组单纯疱疹病毒, 其微生物保藏号为 CGMCC Νο·6397。
优选地, 该单純疱疹病毒剔除了 ICP34.5基因和 ICP47基因中的一种或两种, 剔除 ICP34.5基因 (神经毒基因) , 可使溶瘤病毒更安全; 而剔除 ICP47基因, 以促进免疫 应答及增强溶瘤活性。 优选剔除 ICP34.5和 ICP47两种基因。
本发明还提供了一种制备所述重组单纯疱疹病毒的方法, 其中该方法包括以下步 骤: 用人端粒酶逆转录酶启动子 hTERTp取代含有 ICP4基因的单纯疱疹病毒基因组中 的 ICP4基因启动子, 构建该重组单纯疱疹病毒 HSV-hTERTp一 ICP4:
( 1 ) 构建穿梭质粒 pICP4del-hTERTp— ICP4和 pICP4del-eGFP:
a. 用 BHK细胞培养该含有 ICP4基因 ¾J单纯疱疹病毒, 并纯化其基因
组 DNA;
b. 扩增 ICP4基因上游(US )侧翼序列 ( FlankingRegion, 筒称 FLR ): 以步骤 a 中所得病毒基因组 DNA为模板, 用以下 ICP4USf正向引物和 ICP4USr反向引物:
ICP4USf正向引物: CCCTCC AGACGC ACCGGAGTCGGGGG
ICP4USr反向引物: AAGTCGACTCTAGAGGATCGATCTCTGACCTG
AGATTGGCGGCACTGAGGTA
扩增出 ICP4基因上游侧翼序列;
扩增 ICP4基因下游( DS )侧翼序列: 以步骤 a中所得病毒基因组 DNA为模板, 用以下 ICP4DSf正向引物和 ICP4USr反向引物:
ICP4DSf正向引物: AAAAGTCGACCTGC AGGC ATGCTAACGAGGAA
CGGGCAGGGGGC
ICP4DSr反向引物: AAAAAAGCTTGCATGCCCACGTGCGCGGGGCC
AGACGGGCT
扩增出 ICP4基因下游侧翼序列;
将上下游側翼序列克隆到 pSP73质粒上,构建 pICP4del及 pICP4del-eGFP质粒:将 Sail酶切的前述扩增出的 ICP4基因的上游侧翼序列及 Sall/Hindlll双酶切的前述扩增出 的 ICP4基因的下游侧翼序列混合并连接到 pSP73的 EcoRV/Hindlll位点,得到 pICP4del; 用 EcoRI/XhoI从 pcDNA3.1- eGFP切下 CMV启动子控制的 eGFP表达盒, 经 T4 DNA 聚合酶补平末端后插入到 pICP4del的 EcoRV位点, 得到 pICP4del- eGFP;
c. 分三次 PCR扩增出 ICP4基因中三段序列: 首先, 使用以下引物:
ICP4- 1 st正向引物: TTTTTTGAATTCATGGCGTCGGAGAACAAGCAGCGCC
ICP4-lst反向引物: TGGAGCCACCCCATGGCCTCCGCGT
ICP4-2nd正向引物: CGACGCCGCGC AGC AGT ACGCCCTG
ICP4-2nd反向引物: CGGCGGGGGCGGGCCCGGCGC ACCG
ICP4-3rd正向引物: CCTCATGTTTGACCCGCGGGCCCTG
ICP4-3rd反向引物: TTTTTTCTCGAGTTACAGCACCCCGTCCCCCTCGAAC 以步骤 a 中所得病毒基因组 DNA 为模板, 分别扩增出三段基因片段 ICP4- 151、 ICP4-2nd和 ICP4-3rd, 而后分别将该三段基因片段插入 pSP73质粒的 EcoRV位点构建出 以下三种质粒: pSP73-ICP4-lst、 pSP73-ICP4-2nd 、 pSP73-ICP4-3rd, 从该三种质粒中用 EcoRI和 BsrGI剪切出 ICP4-lst、 用 BsrGI和 Pvul剪切出 ICP4-2nd及用 Pvul和 Xhol剪 切出 ICP-3"1待用;
d.从含人端粒酶逆转录酶启动子 hTERTp的质粒中用 Nrul和 Hindlll切下 hTERTp 片段, 取代从 pcDNA3- NHN 上用 Nrul 和 Hindlll 切除的 CMV 启动子, 得到质粒 pcDNA3-NHN-hTERTp , 其中, pcDNA3-NHN 是在 pcDNA3 的 Nhel 位点插入 N el-Hapl-Nhel酶切位点序列所得;
e. 将步骤 c 中的该 ICP4-lst、 ICP4-2nd ^ ICP4-3rd混合并连接到步骤 d 中的该 pcDNA3-NHN-hTERTp的 EcoRI及 Xhol位点 (该三种基因片段 ICP4-lst、 ICP4-2nd和 ICP4-3rd的连接次序是由其端位序列与该 pcDNA3-NHN- hTERTp的 EcoRI及 Xhol位点 的序列所决定的, 其可自动进行匹配连接) , 得到质粒 pcDNA3-NHN-hTERTp— ICP4; f. 用 Sail酶切步骤 b中含 ICP4基因上下游側翼序列的质粒 pICP4dd, 并 平末端 待用, 用 Pmel和 Hpal从步骤 e获得的该质粒 pcDNA3-NHN-hTERTp_ICP4 剪切出 hTERTp_ICP4表达盒片段, 并将其与该酶切后待用的 pICP4del质粒连接, 构建出质粒 pICP4del-hTERTp_ICP4;
g. 构建 BHK-ICP4 辅助细胞: 用 EcoRI 和 Xhol 从步骤 e 获得的该质粒 pcDNA3-NHN-hTERTp_ICP4中酶切出 ICP4基因,并克隆到 pcDNA3中 CMV启动子的 下游 EcoRI和 Xhol位点, 得到 pcDNA3-CMV-ICP4质粒; 将该 pcDNA3-CMV-ICP4质 粒转染 BHK细胞, 该 pcDNA3-CMV-ICP4质粒 DNA可重组到 BHK细胞基因组中, 使 有些 BHK重组细胞获得对新霉素的抗性和表达 ICP4,用抗菌素 G418杀死未重组的 BHK 细胞, 经过几轮的亚克隆筛选, 用 RT-PCR方法筛选出表达 ICP4的 BHK-ICP4辅助细 胞;
( 2 )剔除基因组中 ICP4基因启动子并插入端粒酶逆转录酶启动子 hTERTp启动子:
A. 用 BHK细胞培养该含有 ICP4基因的单纯疱疹病毒, 并提取病毒基因组 DNA;
B. 将步骤 A中该病毒基因组 DNA与步骤( 1 ) b中该质粒 pICP4del-eGFP共转入 步骤( 1 ) g中该 BHK-ICP4辅助细胞内, 经同源重组, 该质粒 pICP4del- eGFP中绿色荧 光蛋白表达盒 GFP置换了该含有 ICP4基因的单纯疱疹病毒 HSV的 ICP4基因, 使得重 组病毒的毒斑发绿色荧光, 经过几轮的噬斑纯化, 挑选绿色荧光毒斑, 就能纯化出重组 病毒 HSV-d4GFP (d4表示剔除 ICP4基因) ;
C. 培养步骤 B中该 HSV-d4GFP病毒, 并提取基因组 DNA;
D . 将步骤 C 该重组病毒 HSV-d4GFP 的基因组 DNA 和步骤 ( 1 ) f 该质粒 pICP4del-hTERTp_ICP4 的 DNA 共转入该 BHK- ICP4 辅助细胞, 经同源重组, hTERTp_ICP4表 盒置换了该重组病毒 HSV-d4GFP的绿色荧光蛋白表达盒 GFP,使新 重组病毒的毒斑不发绿色荧光, 经过几轮的噬斑纯化, 挑选无荧光毒斑, 就能纯化出该 重组单纯疱疹病毒 HSV-hTERTp— ICP4。
同理, 所述重组单纯疱疹病毒的制备方法, 其中, 可先剔除该含有 ICP4基因的单 纯疱疹病毒的 ICP34.5基因和 ICP47基因中的一种或两种, 再以人端粒醉逆转录酶启动 子 hTERTp替换 ICP4基因启动子; 或在以人端粒酶逆转录酶启动子 hTERTp替换 ICP4 基因启动子得到该重组单纯疱疹病毒 HSV-hTERTp— ICP4后, 再剔除该重组单纯疱疹病 毒 HSV-hTERTp— ICP4的 ICP34.5基因和 ICP47基! ¾中的一种或两种。
所述的重组^纯疱疹病毒的制备方法, 其中可以其它肿瘤特异性启动子替代该人端 粒酶逆转录酶启动子 hTERTp, 来替换该含有 ICP4基因的单纯疱疹病毒中的 ICP4基因 启动子, 完成基因重组。
优选地, 上述重组单纯疱疹病毒的制备方法还包括对所有涉及的质粒进行测序以证 实无突变发生的步骤。 对于质粒序列的监控, 可以保证整个制备过程的准确性。
另夕卜,所述重组单纯疱疹病毒的制备方法中,不局限于只使用绿色荧光蛋白表达盒, 还可由青色荧光蛋白表达盒、 红色荧光蛋白表达盒、 黄色荧光蛋白表达盒及其它指示蛋 白表达盒中任一种所替换。
本发明还提供了一种药物组合物, 其中, 该药物组合物前述的重组疱疹病毒, 以及 药物可接受的载体或赋形剂。 优选该药物组合物为注射液, 该注射液包括药学上可接受 的载体以及前述的重组单纯疱疹病毒, 每毫升所述注射液中含有 102 ~ 101Q个该重组单 纯疱疹病毒。 较优地, 其中该药学上可接受的载体为 pH值为 4.0 - 9.0的磷酸緩冲液。 另外, 较优地, 该注射液还含有保护剂和 /或渗透压调节剂; 以该注射液为基准, 所述保 护剂的含量为 0.01 ~ 30% (重量) , 所述保护剂选自肌醇、 山梨醇和蔗糖中的一种或一 种以上; 每千克该注射液含有 200 - 700毫克所述渗透压调节剂, 所述渗透压调节剂为 氯化钠和 /或氯化钾。
上述单纯疱疹病毒与药物组合物可用于制备治疗肿瘤的药物。
另外, 本发明还提供了另一种重组单纯疱疹病毒, 其是在前述重组单纯疱疹病毒基 因组中插入荧光蛋白表达盒, 该荧光蛋白表达盒可为绿色荧光蛋白表达盒、 青色荧光蛋 白表达盒、红色荧光蛋白表达盒、黄色荧光蛋白表达盒及其它荧光蛋白表达盒中任一种; 由于该荧光蛋白表达盒能在肿瘤细胞内发光, 所以可以实现更快速、 准确、 灵敏并广谱 地实现肿瘤的早期诊断以及肿瘤转移的诊断。
基于上述在前述重组单纯疱疹病毒基因组中插入荧光蛋白表达盒的另一种重组单 纯疱疹病毒, 本发明提供了一种肿瘤诊断试剂盒, 该肿瘤诊断试剂盒包括前述另一种重 组单纯疱疹病毒, 该肿瘤诊断试剂盒检测的样品选自受试者全血、 血浆、 淋巴细胞悬浮 液、 骨髓、 胸腔积液和腹腔积液, 该肿瘤诊断试剂盒还包括: RPMI-1640培养基、 pH 为 7的红细胞裂解液和 pH为 7.2 ~ 7.4的磷酸緩冲液,其中,所述红细胞裂解液包含 0.15M 氯化铵、 ΙΟηΜ碳酸氢钾和 InM乙二胺四乙酸; 或 RPMI-1640培养基、 比重为 1.077 ~
0.001千克 /升的聚蔗糖 -泛影葡胺和 pH为 7.2 ~ 7.4的磷酸缓沖液。 对于该肿瘤诊断试剂 盒的使用方法可采用中国发明专利 CN102220292 A (公开日: 2011年 10月 19 日) 中 公开的方法。
下面结合实施例进一步说明本发明。
以下实施例选用 I 型单纯疱疹病毒毒林常用标准毒林: 17+株 ( Genebank JN555585.1 ) , 为从英国 HPA ( Health Protection Agency )公司购买。 17+株全基因序列 已知。 如无特別说明, 所用酶及质粒均为购买所得。
实施例 1
本实施例为将野生型单纯疱疹病毒 17+毒株的基因组中的 ICP4基因启动子替换为 人端粒酶逆转录酶启动子 hTERTp, 请参阅图 1, 步骤为:
( 1 ) 构建穿梭质粒 pICP4del-hTERTp— ICP4和 PICP4del-eGFP:
a. 用 BHK (地鼠肾) 细胞(购自 ATCC )培养该野生型单纯疱疹病毒
17+毒株, 并纯化其基因组 DNA。
b. 扩增 ICP4基因上游侧翼序列: 以步骤 a中所得 17+病毒基因组 DNA为模板, 用 ICP4USf (正向引物: CCCTCCAGACGCACCGGAGTCGGGGG )和 ICP4USr (反向引 物: AAGTCGACTCTAGAGGATCGATCTCTGACCTG
AGATTGGCGGCACTGAGGTA )扩增出 ICP4基因上游侧翼序列;
扩增 ICP4基因下游侧翼序列: 以步骤 a中所得 17+病毒基因组 DNA为模板, 用 ICP4DSf (正向引物: AAAAGTCGACCTGCAGGCATGCTAA
CGAGGAACGGGCAGGGGGC )和 ICP4DSr (反向引物: AAAAAAGCTTG
CATGCCCACGTGCGCGGGGCCAGACGGGCT )扩增出 ICP4基因下游侧翼序列; 将上下游侧翼序列克隆到 pSP73质粒(从 Promega公司购得)上, 构建 pICP4del 及 pICP4del-eGFP 质粒: 将 Sail 酶切的前述扩增出的 ICP4 基因的上游侧翼序列及 Sall/Hindlll双酶切的前述扩增出的 ICP4基因的下游侧翼序列混合并连接到 pSP73 的 EcoRV/Hindlll位点,得到 pICP4del。序列分析证实该 pICP4del质粒无突变。用 EcoRI/XhoI 从 pcDNA3.1-eGFP (从 YRGENE购得,北京)切下 CMV启动子(巨细胞病毒早期启动子 ) 控制的 eGFP表达盒, 经 T4 DNA聚合酶补平末端后插入到 pICP4del的 EcoRV位点, 得到 pICP4del-eGFP;
表 1
c. 分三次 PCR扩增出 ICP4基因中三段序列: 首先, 使用表 1所示的引物, 以步骤 a中所得 17+病毒基因组 DNA为模板, 分别扩增出三段基因片段 ICP4-lst、 ICP4-2nd和 ICP4-3rd, 而后分别将该三段基因片段插入 pSP73的 EcoRV位点构建出以下三种质粒: pSP73-ICP4-lst、 pSP73-ICP4-2nd 、 pSP73-ICP4-3rd。 在证实序列无突变后, 从该三种质
粒中用 EcoRI和 BsrGI剪切出 ICP4- lst、 用 BsrGI和 Pvul剪切出 ICP4-2nd及用 Pvul和 Xhol剪切出 ICP- 3Fd待用;
d.从含人端粒酶逆转录酶启动子 hTERTp的质粒中用 Nrul和 Hindlll切下 hTERTp 片段, 取代从 pcDNA3-NHN ( pcDNA3从 Invitrogen公司购得, 在 pcDNA3的 Nhel位 点插入 N el-Hapl-Nhel酶切位点序列, 构建成 pcDNA3-NHN )上用 Nrul和 Hindlll切 除的 CMV启动子, 得到质粒 pcDNA3- NHN-hTERTp;
e. 将步骤 c 中的该 ICP4-lst、 ICP4-2nd和 ICP4-3rd混合并连接到步骤 d 中的该 pcDNA3-NHN-hTERTp的 EcoRI及 Xhol位点,得到质粒 pcDNA3-NHN-hTERTp_ICP4; f. 用 Sail酶切含 ICP4基因上下游侧翼序列的质粒 pICP4del, 并补平末端待用, 用 Pmel和 Hpal从步骤 e获得的该质粒 pcDNA3 -NHN-hTERTp— ICP4剪切出 hTERTp— ICP4 表达盒片段, 并将其与该酶切后待用的 pICP4del ¾体连接, 构建出质粒 pICP4del-hTERTp_ICP4。
g. 构建 BHK- ICP4 辅助细胞: 用 EcoRI 和 Xhol 从步骤 e 获得的该质粒 pcDNA3-NHN-hTERTp_ICP4中酶切出 ICP4基因,并克隆到 pcDNA3中 CMV启动的下 游 EcoRI和 Xhol位点 , 得到 pcDNA3-CMV-ICP4质粒; 将该 pcDNA3-CMV-ICP4质粒 转染 BHK细胞, 该 pcDNA3-CMV-ICP4质粒 DNA可重组到 BHK细胞基因组中, 使有 些 BHK重组细胞获得对新霉素 (neomycin ) 的抗性和表达 ICP4。 用抗菌素 G418杀死 未重组的 BHK 细胞。 经过几轮的亚克隆筛选, 用 RT-PCR 方法筛选到表达 ICP4 的 BHK-ICP4辅助细胞。
( 2 )剔除基因组中 ICP4基因启动子并插入端粒酶逆转录酶启动子 hTERTp启动子:
A. 用 BHK细胞培养该野生型单纯疱疹病毒 17+, 并提取病毒基因组 DNA;
B. 将步骤 A中该病毒基因组 DNA与步骤( 1 ) b中该质粒 pICP4del-eGFP共转入 步骤( 1 ) g中该 BHK-ICP4辅助细胞内, 经同源重组, 该质粒 pICP4del-eGFP中绿色荧 光蛋白表达盒置换了该含有 ICP4基因的单纯疱疹病毒 HSV的 ICP4基因, 使得重组病 毒的毒斑发绿色荧光, 经过几轮的噬斑纯化, 挑选绿色荧光毒斑, 就能纯化出重组病毒 oHSVl-d4GFP;
C. 培养步骤 B中该 oHSVl-d4GFP病毒, 并提取基因组 DNA;
D . 将步骤 C 该重组病毒 oHSVl- d4GFP 的基因组 DNA 和步骤 f 该质粒 pICP4del-hTERTp_ICP4 的 DNA 共转入 BHK-ICP4 辅助细胞, 经同源重组, hTERTp— ICP4表 ίϊ盒置换了该重组病毒 oHSVl- d4GFP的绿色荧光蛋白表达盒 GFP,使 新重组病毒的毒斑不发绿色荧光, 经过几轮的噬斑纯化, 挑选无荧光毒斑, 就能纯化出 该重组单纯疱疹病毒 oHSV 1 -hTERTp— ICP4。
所有质粒均经序列分析证实无突变。
图 2为实施例 1制备的该重组单纯疱疹病毒 oHSVl-hTERTp_ICP4与野生型单纯疱 疹病毒(17+ )感染正常细胞(人成纤维细胞)的对比实验结果, ^相同感染条件下(用 1个病毒感染 1个细胞, 即 ΜΟΙ=1, 感染 24或 48小时) , 通过该两种病毒对正常细月包 (人成纤维细胞) 杀伤作用的比较可知, 野生型单纯疱疹病毒 17+引起了严重的细胞病 变并杀死正常细胞(人成纤维细胞) , 而该重组单纯疱疹病毒 oHSVl-hTERTp— ICP4则 对正常细包无影响。
实施例 2
本实施例与实施例 1相似, 不同之处为本实施例是将剔除了 ICP47基因与 ICP34.5 基因的野生型单纯疱疹病毒的基因组中的 ICP4基因启动子替换为人端粒酶逆转录酶启 动子 hTERTp, 具体步骤如下:
( 1 )提取 17+病毒的基因组 DNA: 用 BHK (地鼠肾 ) 细胞培养 17+病毒并提取基 因组 DNA。
( 2 ) 剔除 ICP47基因, 构建重组病毒 oHSV d47
a. 扩增 ICP47基因上游侧翼序列: 以步骤 ( 1 ) 中所得 17+病毒基因组 DNA为模 板, 用 ICP47USf (正向引物: AAAAGAATTCGATTGGGTTCGAT
TGGCAATGTTGTCTC )和 ICP47USr (反向引物: AAAAACTAGTGATGTC
CCGGGTACGACCATCACCCGAG )扩增出 ICP47 US FLR。
b. 扩增出 ICP47基因下游 FLR: 以步骤( 1 ) 中所得 17+病毒基因组 DNA为模板, 用 ICP47DSf (正向引物: AAAAAAGCTTCACGACATGCTC
CCCCCCGACGAGC )和 ICP47DSr (反向引物: AAAACAGCTGACGCGG
AACTAGCGCGGACCGGTCG )扩增出 ICP47 DS FLR。
c. 将上下游侧翼序列克隆到 pBSK (从 Stratagene公司购买)质粒上,构建 pdICP47 及 pdICP47-eGFP质粒: 将 EcoRI/Spel双酶切的前述扩增出的 ICP47基因的上游侧翼序 列、 Hindlll/Sall 双酶切的前述扩增出的 ICP47 基因的下游侧翼序列及互补的具有 Spel Hindlll 双 酶 切 位 点 的 连 接 序 列 (Linker 1
AGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGAA TTCA )进行混合并连接到 pBSK的 EcoRI/Sall位点, 得到 pdICP47。 用 EcoRI/XhoI从 pcDNA3.1 -EGFP (从 YRGENE购得,北京)切下 CMV启动子控制的 eGFP表达盒,经 T4 DNA聚合酶补平末端后插入到 pdICP47的 EcoRV位点, 得到 pdICP47-eGFP。
d. 将步骤( 1 )中所得 17+病毒的基因组 DNA与 pdICP47- eGFP共转染 BHK细胞。 同源重组后, 经过几轮的噬斑纯化, 挑选绿色荧光毒斑, 就能得到纯的重组病毒 oHSVl-d47-GFP。 以同样的方法用 pdICP47剔除 oHSVl-d47-GFP中 GFP表达盒, 得到 oHSVl-d47。
( 3 ) 剔除 ICP34.5基因, 构建重组病毒 oHSVl-d34.5-d47
A. 提取 17+病毒的基因组 DNA: 用 BHK细胞培养 oHSVl-d47病毒并提取基因组 DNA。
B. 扩增 ICP34.5基因上游侧翼序列: 以步骤( 1 )所得 17+病毒基因组 DNA为模板, 用 ICP34.5USf (正向引物: CTCTGACCTGAGATTGGCGGC
ACTG )和 ICP34.5USr (反向引物: GCGGCCGCAGCGCTGCGGCCGCCG
CGGGCGCGCTCCTGACCGCGGG )扩增出 ICP34.5 US FLR。
C. 扩增 ICP34.5基因下游侧翼序列: 以步骤( 1 )所得 17+病毒基因组 DNA为模 板, 用 ICP34.5DSf (正向引物: GCGGCCGCAGCGCTGCGGCC
GCCAGCGCGG CGGGGCCCGGCCAACCA ) 和 ICP34.5DSr ( 反 向 引 物 : TTCTTCCCTCTTCTCCCGCCCTCCA )扩增出 ICP34.5下游侧翼序列。
D. 将上下游側翼序列克隆到 pSP72 (从 Promega购得)质粒上, 构建 pdICP34.5 及 pdICP34.5-eGFP质粒: 用重叠聚合酶链反应 ( overlapping PCR )连接 ICP34.5上下游 侧翼序列, 并与用 BamHI/Xhol 双切并补平的 pSP72 载体连接, 得到 pdICP34.5。 用 EcoRI/XhoI从 pcDNA3.1-eGFP切下 eGFP表达盒, 经 T4 DNA聚合酶补平末端后插入 到 pdICP34.5的 Afel位点, 得到 pdICP34.5-eGFP。
E. 将步骤( 2 ) 所得 oHSVl-d47基因组 DNA与步骤 D所得 pdICP34.5-eGFP共转 染 BHK细胞。 同源重组后, 经过几轮的噬斑纯化, 挑选绿色荧光毒斑, 就能得到纯的 重 组 病 毒 oHSVl-d47-d34.5-GFP 。 以 同 样 的 方 法 用 pdICP34.5 剔 除 oHSV d47-d34.5-GFP中 GFP表达盒, 得到 oHSV d34.5-d47。
之后采用实施例 1 的方法, 用人端粒酶逆转录酶启动子 hTERTp取代前述得到的 oHSVl-d34.5-d47 的 ICP4 基 因 启 动 子 , 构 建 重 组 单 纯 疱 疹 病 毒
oHSV d34.5-d47-hTERTp— ICP4。 当然, 在本实施例中, 也可采用现有技术中已知的方 法对 ICP47基因和 ICP34.5基因进行剔除。
本实施例中, 所有质粒均经序列分析证实无突变。
在另一实施例中, 也可在实施例 1所得该重组单纯疱疹病毒 oHSVl-hTERTp— ICP4 后, 再对其 ICP47基因和 ICP34.5基因进行剔除。 剔除 ICP47基因和 ICP34.5基 的方 法可为实施例 2中方法, 也可采用现有技术中的其它方法。
图 3为实施例 2制备的该重组单纯疱疹病毒 oHSVl-d34.5-d47-hTERTp_ICP4与现 有技术中只剔除了野生型单纯疱疹病毒基因组的 ICP34.5 基因和 ICP47 i因的病毒 ( oHSVl-d34.5-d47 )感染正常人白细胞的对比实验结果, 在相同感染条件下 (用 1 个 病毒感染 1个细胞, 即 ΜΟΙ=1 , 感染 24小时) , 流式检测发现 oHSVl-d34.5-d47能感 染白细胞(检测值为 0.8% ) , 而该重组单纯疱疹病毒 oHSVl-d34.5-d47-hTERTp_ICP4 则不感染白细胞(检测值为 0.0% ) , 由此说明后者临床使用更安全。
图 4A-图 4F为该重组单纯疱疹病毒 oHSV hTERTp— ICP4对肝癌、肺癌、头颈鳞癌、 黒色素瘤、 结肠癌和前列腺癌的杀伤作用试验结果(ΜΟΙ=1 ) , 其中对照组为野生型单 纯疱疹病毒。 由该实验结果可知, 本发明的重组单纯疱疹病毒 oHSVl-hTERTp— ICP4能 够杀伤多种肿瘤细胞, 具有广谱的抗肿瘤作用。
图 5A- 图 5D 为 棵 鼠 瘤 内 分 另1 J 注 射 oHSVl-d34.5-d47 和 oHSVl-d34.5-d47-hTERTp_ICP4后其抑瘤效果及动物存活时间的对比实验结果。用胃癌 (人 BGC823胃细胞腺癌)及肝癌 (人肝细胞癌 HuH7 )诱导棵鼠成瘤后, 分 3组, 每 组 6 只动物。 组 1 瘤内注射无血清培养基作为对照, 组 2 和 3 分别瘤内注射 oHSVl-d34.5-d47和 oHSVl-d34.5- d47-hTERTp_ICP4, 病毒注射剂量均为 5x106 Pfu/次, 隔 2天一次, 共 3次。 观察病毒抑瘤效果及动¾ /存活时间。 图 5A和 5C显示: 与对照 组比较, 两病毒组均有明显抑瘤效果且二者之间差别不大。 而动物存活时间方面图 5B 和 5D则显示 oHSVl-d34.5-d47-hTERTp_ICP4治疗组的明显优于 oHSVl-d34.5-d47治疗 组。 BGC823模型中 oHSV d34.5-d47-hTERTp_ICP4组中位生存时间为 100天, 明显长 于 oHSVl-d34.5-d47 组 ( 27 天 ) 及对照组 ( 61 天 )。 HuH7 模型 中 oHSVl -d34.5-d47-hTERTp_ICP4组中位生存时间为 62天,也明显长于 oHSVl-d34.5-d47 组 ( 48 天) 及对照组 ( 47 天)。 因此, 两种模型的体内抑瘤实验均显示 oHSVl-d34.5-d47-hTERTp_ICP4在治疗效果及安全性方面均优于 oHSVl-d34.5-d47。
虽然前述两个实施例是以 I型单纯疱疹病毒毒抹 17+株为例,但本发明不局限于 17+ 株, 同样也适用于 I型单纯疱疹病毒毒抹 F株、 KOS株、 JS1株和 BLI株等, 另外, 同 样适用于 II型单纯疱疹病毒, 如 HG52株, 即适用于含有 ICP4基因的任一单纯疱疹病 毒。
需要说明的是, 虽然前述实施例是将单纯疱疹病毒的基因组中的 ICP4基因启动子 替换为人端粒酶逆转录酶启动子 hTERTp, 但本发明的范围不局限于人端粒酶逆转录酶 启动子 hTERTp, 其可将含有 ICP4基因的单纯疱疹病毒基因组的 ICP4启动子替换为本 领域技术人员所知悉的其它肿瘤特异性启动子, 如: 存活素(survivin )启动子、 前列腺 特异性抗原 (PSA ) 启动子、 肝癌细胞特有的曱胎球蛋白 (AFP ) 启动子、 上皮细胞肿 瘤的癌胚抗原 (CEA ) 启动子等。
以上所述, 仅是本发明的较佳实施例而已, 并非对本发明作任何形式上的限制, 虽 然本发明已以较佳实施例揭露如上, 然而并非用以限定本发明, 任何熟悉本专业的技术 人员, 在不脱离本发明技术方案范围内, 当可利用上述揭示的技术内容作出些许更动或 修饰为等同变化的等效实施例, 但凡是未脱离本发明技术方案的内容, 依据本发明的技 术实质对以上实施例所作的任何简单修改、 等同变化与修饰, 均仍属于本发明技术方案 的范围内。
Claims
1、 一种重组单纯疱疹病毒, 其特征在于将含有 ICP4基因的单純疱疹病毒基因组中 的 ICP4基因启动子替换为人端粒酶逆转录酶启动子 hTERTp或其它肿瘤特异性启动子。
2、 根据权利要求 1 所述的重组单纯疱疹病毒, 其特征在于其微生物保藏号为 CGMCC Νο·6397。
3、 根据权利要求 1 所述的重组单纯疱疹病毒, 其特征在于该单纯疱疹病毒剔除了 ICP34.5基因和 ICP47基因中的一种或两种。
4、 一种制备如权利要求 1或 2所述重组单纯疱疹病毒的方法, 其特征在于其包括 以下步骤:
用人端粒酶逆转录酶启动子 hTERTp取代含有 ICP4基因的单纯疱疹病毒中的 ICP4 基因启动子, 构建该重组单纯疱疹病毒 HSV-hTERTp— ICP4:
( 1 ) 构建穿梭质粒 pICP4del-hTERTp— ICP4和 pICP4del-eGFP:
a. 用 BHK细胞培养该含有 ICP4基因的单纯疱疹病毒, 并纯化其基因
组 DNA;
b. 扩增 ICP4基因上游侧翼序列: 以步骤 a中所得病毒基因组 DNA为模板, 用以 下 ICP4USf正向引物和 ICP4USr反向引物:
ICP4USf正向引物: CCCTCC AGACGC ACCGGAGTCGGGGG
ICP4USr反向引物: AAGTCGACTCTAGAGGATCGATCTCTGACCTG
AGATTGGCGGCACTGAGGTA
扩增出 ICP4基因上游侧翼序列;
扩增 ICP4基因下游侧翼序列: 以步骤 a中所得病毒基因组 DNA为模板, 用以下 ICP4DSf正向引物和 ICP4USr反向引物:
ICP4DSf正向引物: AAAAGTCGACCTGC AGGC ATGCTAACGAGGAA
CGGGCAGGGGGC
ICP4DSr反向引物: AAAAAAGCTTGCATGCCC ACGTGCGCGGGGCC
AGACGGGCT
扩增出 ICP4基因下游侧翼序列;
将上下游侧翼序列克隆到 pSP73质粒上,构建 pICP4del及 pICP4del-eGFP质粒:将 Sail酶切的前述扩增出的 ICP4基因的上游侧翼序列及 Sall/Hindlll双醉切的前述扩增出 的 ICP4基因的下游侧翼序列混合并连接到 pSP73的 EcoRV/Hindlll位点,得到 pICP4del; 用 EcoRI/XhoI从 pcDNA3.1-eGFP切下 CMV启动子控制的 eGFP表达盒, 经 T4 DNA 聚合酶补平末端后插入到 pICP4del的 EcoRV位点, 得到 PICP4del-eGFP;
c. 分三次 PCR扩增出 ICP4基因中三段序列: 首先, 使用以下引物:
ICP4- 1 st正向引物: TTTTTTGAATTC ATGGCGTCGGAGAAC AAGC AGCGCC
ICP4-lst反向引物: TGGAGCCACCCCATGGCCTCCGCGT
ICP4-2nd正向引物: CGACGCCGCGC AGC AGTACGCCCTG
ICP4-2nd反向引物: CGGCGGGGGCGGGCCCGGCGC ACCG
ICP4-3rd正向引物: CCTCATGTTTGACCCGCGGGCCCTG
ICP4-3rd反向引物: TTTTTTCTCGAGTTACAGCACCCCGTCCCCCTCGAAC 以步骤 a中所得病毒基因组 DNA为模板, 分别扩增出三段基因片段 ICP4-lst、 ICP4-2nd 和 ICP4-3rd,而后分别将该三段基因片段插入 pSP73质粒的 EcoRV位点构建出以下三种 质粒: pSP73-ICP4-lst、 pSP73-ICP4-2nd 、 pSP73-ICP4-3rd, 从该三种质粒中用 EcoRI和 BsrGI剪切出 ICP4-lst、用 BsrGI和 Pvul剪切出 ICP4- 2nd及用 Pvul和 Xhol剪切出 ICP-3rf 待用;
d.从含人端粒酶逆转录酶启动子 hTERTp的质粒中用 Nrul和 Hindlll切下 hTERTp 片段, 取代从 pcDNA3-NHN 上用 Nrul 和 Hindlll 切除的 CMV 启动子, 得到质粒 pcDNA3-NHN-hTERTp , 其中, pcDNA3-NHN 是在 pcDNA3 的 Nhel 位点插入 Nhel-Hapl-Nhel酶切位点序列所得;
e. 将步骤 c 中的该 ICP4-lst、 ICP4-2nd和 ICP4-3"5混合并连接到步骤 d 中的该 pcDNA3-NHN-hTERTp的 EcoRI及 Xhol位点,得到质粒 pcDNA3-NHN-hTERTp— ICP4; f. 用 Sail酶切步骤 b中含 ICP4基因上下游側翼序列的质粒 pICP4del, 并补平末端 待用, 用 Pmel和 Hpal从步骤 e获得的该质粒 pcDNA3-NHN-hTERTp— ICP4 剪切出 hTERTp_ICP4表达盒片段, 并将其与该酶切后待用的 pICP4del质粒连接, 构建出质粒 pICP4del-hTERTp_ICP4;
g. 构建 BHK-ICP4 辅助细胞: 用 EcoRI 和 Xhol 从步骤 e 获得的该质粒 pcDNA3-NHN-hTERTp_ICP4中酶切出 ICP4基因,并克隆到 pcDNA3中 CMV启动子的 下游 EcoRI和 Xhol位点, 得到 pcDNA3-CMV-ICP4质粒; 将该 pcDNA3-CMV-ICP4质 粒转染 BHK细胞, 该 pcDNA3-CMV-ICP4质粒 DNA可重组到 BHK细胞基因组中, 使 有些 BHK重组细包获得对新霉素的抗性和表达 ICP4,用抗菌素 G418杀死未重组的 BHK 细胞, 经过几轮的亚克隆筛选, 用 RT-PCR方法筛选出表达 ICP4的 BHK-ICP4辅助细 胞;
( 2 )剔除基因组中 ICP4基因启动子并插入端粒酶逆转录酶启动子 hTERTp启动子:
A. 用 BHK细胞培养该含有 ICP4基因的单纯疱疹病毒 HSV, 并提取病毒基因组 DNA;
B. 将步骤 A中该病毒基因组 DNA与步骤( 1 ) b中该质粒 pICP4del-eGFP共转入 步骤( 1 ) g中该 BHK-ICP4辅助细胞内, 经同源重组, 该质粒 pICP4del-eGFP中绿色荧 光蛋白表达盒 GFP置换了该含有 ICP4基因的单纯疱疹病毒 HSV的 ICP4基因, 使得重 组病毒的毒斑发绿色荧光, 经过几轮的噬斑纯化, 4兆选绿色荧光毒斑, 就能纯化出重组 病毒 HSV-d4GFP;
C. 培养步骤 B中该 HSV-d4GFP病毒, 并提取基因组 DNA;
D . 将步骤 C 该重组病毒 HSV- d4GFP 的基因组 DNA 和步骤 ( 1 ) f 该质粒 pICP4del-hTERTp_ICP4 的 DNA 共转入该 BHK-ICP4 辅助细月包, 经同源重组, hTERTp— ICP4表^ Ϊ盒置换了该重组病毒 HSV-d4GFP的绿色荧光蛋白 GFP表达盒, 使 新重组病毒的毒斑不发绿色荧光, 经过几轮的噬斑纯化, 挑选无荧光毒斑, 就能纯化出 该重组单纯疱疹病毒 HSV-hTERTp— ICP4。
5、 根据权利要求 4所述的重组单纯疱疹病毒的制备方法, 其特征在于先剔除该含 有 ICP4基因的单纯疱疹病毒的 ICP34.5基因和 ICP47基因中的一种或两种, 再以人端 粒酶逆转录酶启动子 hTERTp替换 ICP4基因启动子, 或在以人端粒酶逆转录酶启动子 hTERTp替换 ICP4基因启动子得到该重组单纯疱疹病毒 HSV-hTERTp— ICP4后,再剔除 该重组单纯疱疹病毒 HSV-hTERTp— ICP4的 ICP34.5基因和 ICP47基因 的一种或两种。
6、 根据权利要求 4或 5所述 重组单纯疱疹病毒的制备方法, 其特征在于以其它 肿瘤特异性启动子替代该人端粒酶逆转录酶启动子 hTERTp。
7、 根据权利要求 4或 5所述的重组单纯疱疹病毒的制备方法, 其特征在于该方法 还包括对所有涉及的质粒进行测序以证实无突变发生的步骤。
8、 根据权利要求 4或 5所述的重组单纯疱疹病毒的制备方法, 其特征在于该绿色 荧光蛋白表达盒可由青色荧光蛋白表达盒、 红色荧光蛋白表达盒、 黄色荧光蛋白表达盒 或其它指示蛋白表达盒所替换。
9、 一种药物组合物, 其特征在于该药物组合物包含权利要求 1或 2所述的重组单 纯疱疹病毒, 以及药物可接受的载体或赋形剂。
10、 根据权利要求 9所述的药物组合物, 其特征在于该药物组合物为注射液, 所述 注射液包括药学上可接受的载体以及权利要求 1至 3任一权利要求所述的重组单纯疱疹 病毒, 每毫升所述注射液中含有 102 ~ 101Q个该重组单纯疱疹病毒。
1 1、 根据权利要求 10所述的药物组合物, 其特征在于该药学上可接受的载体为 pH 值为 4.0 ~ 9.0的磷酸緩沖液。
12、 根据权利要求 10所述的药物组合物, 其特征在于该注射液还含有保护剂和 /或 渗透压调节剂; 以该注射液为基准, 所述保护剂的含量为 0.01 ~ 30% (重量) , 所述保 护剂选自肌醇、 山梨醇和蔗糖中的一种或一种以上; 每千克该注射液含有 200 - 700毫 克所述渗透压调节剂, 所述渗透压调节剂为氯化钠和 /或氯化钾。
13、 权利要求 1至 3任一权利要求所述的重组单纯疱疹病毒, 或 9至 12任一权利 要求所述的药物组合物在制备治疗肿瘤的药物中的应用。
14、 一种重组单纯疱疹病毒, 其特征在于在如权利要求 1至 3任一权利要求所述的 重组单纯疱疹病毒基因组中插入荧光蛋白表达盒或其它指示蛋白表达盒。
15、 根据权利要求 14所述的重组单纯疱疹病毒, 其特征在于该荧光蛋白表达盒为 绿色荧光蛋白表达盒、 青色荧光蛋白表达盒、 红色荧光蛋白表达盒、 黄色荧光蛋白表达 盒及其它指示蛋白表达盒中任一种。
16、 权利要求 14或 15所述的重组单纯疱疹病毒在制备诊断肿瘤的药物中的应用。
17、 一种肿瘤诊断试剂盒, 其特征在于该肿瘤诊断试剂盒包含如权利要求 14或 15 所述的重组单纯疱疹病毒。
18、 根据权利要求 17所述的肿瘤诊断试剂盒, 其特征在于该肿瘤诊断试剂盒检测 的样品选自受试者全血、 血浆、 淋巴细胞悬浮液、 骨髓、 胸腔积液或腹腔积液。
19、 根据权利要求 17所述的肿瘤诊断试剂盒, 其特征在于该肿瘤诊断试剂盒还包 括:
RPMI-1640培养基、 pH为 Ί的红细胞裂解液和 pH为 7.2 ~ 7.4的磷酸緩沖液,其中, 所述红细胞裂解液包含 0.15M氯化铵、 ΙΟηΜ碳酸氢钾和 InM乙二胺四乙酸; 或
RPMI-1640培养基、 比重为 1.077 ~ 0.001千克 /升的聚蔗糖 -泛影葡胺和 pH为 7.2 ~ 7.4的磷酸緩冲液。
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