WO2020125423A1 - Virus de l'herpès simplex recombinant, kit et utilisation de celui-ci - Google Patents

Virus de l'herpès simplex recombinant, kit et utilisation de celui-ci Download PDF

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WO2020125423A1
WO2020125423A1 PCT/CN2019/123221 CN2019123221W WO2020125423A1 WO 2020125423 A1 WO2020125423 A1 WO 2020125423A1 CN 2019123221 W CN2019123221 W CN 2019123221W WO 2020125423 A1 WO2020125423 A1 WO 2020125423A1
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reagent
herpes simplex
simplex virus
surface antigen
detecting
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赵长云
唐维
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重庆点检生物科技有限公司
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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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • C12N2710/16621—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention relates to the field of in vitro diagnosis, in particular to the detection of circulating prostate cancer cells, in particular to recombinant herpes simplex virus, kits and uses thereof.
  • Prostate cancer refers to an epithelial malignant tumor that occurs in the prostate.
  • the incidence of prostate cancer in tumor registration areas in my country was 9.92 per 100,000, ranking sixth in the incidence of male malignant tumors.
  • the age of onset is at a low level before the age of 55, and gradually increases after the age of 55.
  • the incidence rate increases with age.
  • the peak age is 70 to 80 years.
  • prostate cancer tumors are the second leading cause of male death after lung cancer.
  • the incidence of prostate cancer in my country has increased significantly.
  • PSA is the most commonly used method for detecting prostate cancer
  • benign prostatic hyperplasia and prostatitis can also result in positive PSA, and the false positive rate of screening is high.
  • the most common diagnostic method for prostate cancer is ultrasound-guided transrectal prostate puncture, but this method has many limitations.
  • Most prostate cancers can not be displayed on ultrasound, so the puncture is random "blind puncture", it is difficult to accurately locate the lesion, the diagnostic sensitivity is not enough, and the risk of missed diagnosis is high.
  • the rate of missed diagnosis is higher.
  • Circulating tumor cells are tumor cells that detach from tumor lesions and spread into the blood. These CTC cells are the cause of metastasis and recurrence of malignant tumors. CTC detection is the most non-invasive diagnosis and real-time curative effect monitoring method with great development potential. It is of great significance for the screening and diagnosis of early metastasis and recurrence of prostate cancer patients.
  • the methods for detecting CTC can be divided into two categories: immunological method capture and physical method separation.
  • Immunological method capture is the use of leukocyte-specific surface antigens (such as CD45, CD14, CD56, etc.) to remove the negative selection of leukocytes or the use of epithelial cell adhesion molecules, such as EpCAM and other antibodies for positive enrichment.
  • leukocyte-specific surface antigens such as CD45, CD14, CD56, etc.
  • epithelial cell adhesion molecules such as EpCAM and other antibodies for positive enrichment.
  • the sensitivity of Cellsearch is not high. For some patients with fewer CTCs, the sensitivity of detection is insufficient.
  • the physical method is based on the size of tumor cells and directly enriches CTC through the filter.
  • the disadvantage is that CTCs with diameters smaller than the pore size of the filter and poor or alive survival conditions are missed, and the CTC enrichment ability is poor and the background is high.
  • there are some methods that combine the two to improve a certain capturing ability but its essential shortcomings have not been solved to a certain extent.
  • the existing methods have the defects of insufficient sensitivity and low specificity for the capture and separation of prostate cancer CTC, and a rapid, high sensitivity and specific detection method for prostate cancer CTC is urgently needed.
  • the present invention provides a circulating tumor cell with stable detection results, high accuracy, high sensitivity and good precision, which is suitable for clinical promotion, preferably a detection kit for prostate cancer circulating tumor cells and its use.
  • the detection result obtained by the detection kit of the present invention is stable, with good repeatability, high accuracy, high sensitivity and good precision.
  • the invention provides a recombinant herpes simplex virus.
  • the recombinant herpes simplex virus is a human telomerase reverse transcriptase in which the promoter of the ICP4 gene in the genome of the herpes simplex virus containing the infected cell protein 4 (infected cell protein 4) ICP 4 gene is replaced.
  • the herpes simplex virus of the promoter hTERTp or the recombinant herpes simplex virus deposited under the accession number CGMCC NO.
  • the recombinant herpes simplex virus ICP47 gene is deleted, and an expression cassette controlled by the prostate specific membrane antigen promoter (PSMAp) that produces a detectable protein gene is inserted into the deleted ICP47 gene position.
  • PSMAp prostate specific membrane antigen promoter
  • the recombinant herpes simplex virus ICP 34.5 gene is deleted.
  • the protein that generates detectable light is green fluorescent protein, yellow fluorescent protein, orange fluorescent protein, and/or cyan fluorescent protein; preferably, the prostate-specific membrane antigen promoter contains SEQ ID NO. 1. The sequence shown.
  • the position in the ICP4 gene corresponding to 127235-131131 of the 17+ strain is deleted, and the human telomerase reverse transcriptase promoter hTERTp is inserted into the corresponding position.
  • the position in the ICP 47 gene corresponding to 145316-145582 of the 17+ strain is deleted, and the expression cassette is inserted into the deleted position.
  • the position in the ICP 34.5 gene corresponding to 513-1259 and/or 125115-125861 of the 17+ strain is deleted.
  • the present invention also provides a kit for detecting circulating prostate cancer cells in a subject, which comprises the recombinant herpes simplex virus according to the present invention, and optionally reagents for detecting leukocyte surface antigens and detecting circulating prostate cancer cell surface antigens Of reagents;
  • the kit contains erythrocyte lysate, washing solution and/or transfection reagent;
  • the reagent for detecting the surface antigen of leukocytes and/or the reagent for detecting the surface antigen of circulating prostate cancer cells are labeled, preferably the reagent for detecting the surface antigen of leukocytes and/or the detection of circulating prostate cancer cells
  • the reagent of surface antigen is antibody;
  • the reagent for detecting the surface antigen of leukocytes and/or the reagent for detecting the surface antigen of circulating prostate cancer cells are labeled with different detectable labels.
  • the detectable label is selected from PE, APC, PE-CY5, PerCP-CY5.5, FITC, PerCP, CY-7, APC-CY7, eFluor/Super Bright series fluorescent dyes, and Alexa Fluor series fluorescent dye.
  • the leukocyte surface antigen is a CD45 molecule and/or the circulating prostate cancer cell surface antigen is PSMA.
  • the present invention also provides the use of a reagent in the preparation of a kit for detecting circulating prostate cancer cells in a subject, wherein the reagent is the recombinant herpes simplex virus of the present invention or contains the recombinant herpes simplex virus of the present invention, detection Leukocyte surface antigen reagent and reagent for detecting circulating prostate cancer cell surface antigen, optionally including erythrocyte lysate, washing solution and/or transfection reagent, wherein preferably, the reagent for detecting leukocyte surface antigen and/or the detection
  • the reagent for circulating prostate cancer cell surface antigen is labeled, preferably the reagent for detecting leukocyte surface antigen and/or the reagent for detecting circulating prostate cancer cell surface antigen is an antibody;
  • the reagent for detecting the surface antigen of leukocytes and/or the reagent for detecting the surface antigen of circulating prostate cancer cells are labeled with different detectable markers; the detectable marker is selected from PE, APC, PE-CY5, PerCP-CY5.5, FITC, PerCP, CY-7, APC-CY7, eFluor/Super Bright series fluorescent dyes, and Alexa Fluor series fluorescent dyes.
  • the leukocyte surface antigen is a CD45 molecule and/or the circulating prostate cancer cell surface antigen is PSMA.
  • FIG. 1 Construction of plasmid pH2dI34.5-GFP.
  • FIG. 1 Construction of plasmid pdICP47PSMApGFP.
  • FIG. 1 Construction of 17+hTERTpICP4PSMApGFP.
  • Figure 4 Comparison of the detection results of herpes simplex virus 17+hTERTpICP4PSMApGFP and 17+hTERTpICP4d34.5GFP on lung cancer A549 cells and prostate cancer PC-3 cells.
  • Figure 5 Sensitivity-specific analysis, ROC analysis of the detection results of healthy people and patients with prostate cancer.
  • Figure 6 Diagram of linear analysis of the detection method.
  • Figure 7 Schematic diagram of the cell population to be analyzed. The location of the tumor cells on the flow chart is shown as the position of the monocyte population. Because the number is very small, it is necessary to circle the cell population at the location during analysis.
  • Figure 8 Schematic diagram of CD45 negative cells.
  • the CD45-negative cell population was analyzed for the circled cell population to be analyzed because the general marker of immune cell population in peripheral blood was CD45.
  • the CD45 negative cell population is the non-immune cell population in the blood.
  • FIG. 9 Schematic diagram of CTC test results.
  • Figure 10 ROC curve analysis chart.
  • Figure 11 17+hTERTpICP4-d34.5-GFP performance detection in the three-label method.
  • Figure 12 Performance test of 17+hTERTpICP4PSMApGFP in the three-label method. The results of further analysis after circled the location map of the cell population to be analyzed, that is, the monocyte population is obtained first, and the monocyte population is analyzed, and the CD45 negative population is analyzed, and GFP positive in this part of the negative population , PSMA-positive Shuangyang cells are the upper right corner.
  • Subject refers to mammals including humans, chimpanzee primates, dogs, cats and other pet animals, cattle, horses, sheep, goats and other livestock animals, rodents such as mice and rats, etc. animal.
  • sensitivity refers to the value of (number of true positives)/(number of true positives + number of false negatives). If the sensitivity is high, prostate cancer can be detected early, resulting in a complete resection of the cancerous part and a reduction in the recurrence rate.
  • specificity refers to (the number of true negatives)/(the number of true negatives + the number of false positives). If the specificity is high, the implementation of an unhelpful additional examination caused by misdiagnosing the normal body as a prostate cancer patient is prevented, resulting in a reduction in the burden on the patient and a reduction in medical expenses.
  • tumor refers to a disorder characterized by excessive cell growth.
  • circulating tumor cells may be circulating tumor cells of any type of cancer.
  • the circulating tumor cells are prostate cancer circulating tumor cells.
  • CD45 The CD45 molecule is expressed on all leukocytes and is called leukocyte common antigen (LCA).
  • CD45 consists of a class of structurally similar transmembrane proteins with large molecular weights, which are widely present on the surface of leukocytes.
  • Prostate-specific membrane antigen (prostate-specific membrane antigen) (PSMA) is a multifunctional type II transmembrane protein that exists in the prostate cell membrane and consists of 750 amino acid residues.
  • CD45 monoclonal antibodies can be combined with leukocyte CD45 molecules in vitro to label leukocytes in human peripheral blood samples with the APC-CY7 fluorescent group carried by the antibodies.
  • cells can be further labeled with antibodies to the human prostate cancer cell surface marker PSMA carrying the APC fluorophore.
  • PSMA human prostate cancer cell surface marker carrying the APC fluorophore.
  • GFP-positive and PSMA-positive and CD45 molecule-negative cells are defined as circulating prostate cancer cells in the peripheral blood of prostate cancer patients.
  • the inventor constructed a new recombinant herpes simplex virus, which can distinguish prostate cancer cells from other types of cancer cells, by adding human telomerase reverse transcriptase promoter hTERTp and prostate specificity to recombinant herpes simplex virus at the same time Membrane antigen promoter.
  • the recombinant herpes simplex virus of the present invention does not produce detectable light when only the human telomerase reverse transcriptase promoter hTERTp is activated, and only when the human telomerase reverse transcriptase promoter hTERTp and the prostate-specific membrane antigen promoter are both Detectable light can only be generated when activated.
  • the inventors also found that compared with the recombinant herpes simplex virus constructed by the existing method, the recombinant herpes simplex virus of the present invention is very useful in the method of detecting prostate cancer cells, especially the method of detecting prostate cancer cells in combination with CD45 antibody and PSMA antibody High sensitivity.
  • 17+ strain (Genebank JN555585.1), which was purchased from the British HPA (Health Protection Agency) company. The entire gene sequence of 17+ strains is known. Unless otherwise specified, the enzymes and plasmids used are purchased. pSP73 is from Promega; pcDNA3 is from Invitrogen; pcDNA3.1-eGFP is from YRGGENE.
  • Example 1 Construction of recombinant herpes simplex virus type I oHSV1d4-GFP excluding the ICP4 gene
  • BHK (hamster kidney) cells purchased from ATCC, commodity number CCL-10 TM cultivated wild-type 17+ virus, and purified the virus DNA using DNAzol TM Genomic DNA Isolation Kit (Helena Biosciences Cat. No. DN127200).
  • BHK cells were grown in 175 square centimeter culture flasks, and the culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin. The culture conditions are 37°C and 5% carbon dioxide. When the cells grow to 90% saturation, the virus is inoculated. Continue to incubate for 24-48 hours. When more than 90% of the cells show cytopathy, remove the culture solution and add 10ml of DNAzol.
  • Amplify the upstream flanking sequence of the ICP4 gene Using the 17+ viral genomic DNA obtained in step a as a template, use the ICP4USf (forward primer: CCCTCCAGACGCACCGGAGTCGGGGG) and ICP4USr (reverse primer: AAGTCGACTCTAGAGGATCGATCTCTGACCTGAGATTGGCGGCACTGAGGTA) to amplify the upstream flanking sequence of the ICP4 gene;
  • ICP4DSf forward primer: AAAAGTCGACCTGCAGGCATGCTAACGAGGAACGGGCAGGGGGC
  • ICP4DSr reverse primer: AAAAAAGCTTGCATGCCCACGTGCGCGGGGCCAGACGGGCT
  • the upstream and downstream flanking sequences were cloned into the pSP73 plasmid (purchased from Promega) to construct the pICP4del and pICP4del-eGFP plasmids: the upstream flanking sequence of the previously amplified ICP4 gene digested with SalI and the SalI/HindIII double digested
  • the downstream flanking sequence of the aforementioned amplified ICP4 gene is mixed and ligated to the EcoRV/HindIII site of pSP73 to obtain pICP4del. Sequence analysis confirmed that the pICP4del plasmid had no mutations.
  • the eGFP expression cassette controlled by the CMV promoter was cut from pcDNA3.1-eGFP (purchased from YRGENE, Beijing) with EcoRI/XhoI, and the ends were filled in with T4DNA polymerase and inserted into pICP4del's EcoRV Site, get pICP4del-eGFP.
  • CMC carboxymethyl cellulose
  • Three PCRs were used to amplify the three sequences of the ICP4 gene: First, using the primers shown in Table 1, using the 17+ viral genomic DNA obtained in step a as a template, the three gene fragments ICP4-1st and ICP4- were amplified respectively 2nd and ICP4-3rd, and then insert the three gene fragments into the EcoRV site of pSP73 to construct the following three plasmids: pSP73-ICP4-1st, pSP73-ICP4-2nd, pSP73-ICP4-3rd.
  • ICP4-1st was cut out of the three plasmids with EcoRI and BsrGI, ICP4-2nd was cut out with BsrGI and PvuI, and ICP-3rd was cut out with PvuI and XhoI for use;
  • HTERTp hTERT promoter from the plasmid containing pcDNA3-NHN-hTERTp (with hTERTp fragment (hTERT gene promoter sequence (SEQ ID No.1) at HindIII and NruI cut: tcgcgagtttaaactggcccctcctcgggttaccccacagcctaggccgattcgacctctctcccgctggggccctcgctggcgtccctgcaccctgggagcgcgcgggcggggaagcgcggcccagaccccccgggtccgcccggagcagctgcgctgtcggggccaggccgggctcccagtggattcgcgggcacagacgcccaggaccgcgctccccacgtggcggagggactggacccgcgcccacgt
  • the plasmid pICP4del containing the upstream and downstream flanking sequences of the ICP4 gene was digested with SalI, and the ends were filled in for use.
  • the hTERTp_ICP4 expression cassette fragment was cut from the plasmid pcDNA3-NHN-hTERTp_ICP4 with PmeI and HpaI, and then cut with the enzyme for later Using the pICP4del vector, the plasmid pICP4del-hTERTp_ICP4 was constructed.
  • BHK-ICP4 helper cells EcoRI and XhoI were used to digest the ICP4 gene from the obtained plasmid pcDNA3-NHN-hTERTp_ICP4, and cloned into the EcoRI and XhoI sites downstream of the CMV promoter in pcDNA3 to obtain the pcDNA3-CMV-ICP4 plasmid ; Transfect the pcDNA3-CMV-ICP4 plasmid into BHK cells, and the pcDNA3-CMV-ICP4 plasmid DNA can be recombined into the genome of BHK cells, so that some BHK recombinant cells acquire resistance to neomycin and express ICP4. The anti-recombinant BHK cells were killed with antibiotic G418. After several rounds of subcloning screening, BHK-ICP4 helper cells expressing ICP4 were screened by RT-PCR.
  • BHK-ICP4 cells Prepare BHK-ICP4 cells with a density of 80-90% using a 6-well culture plate.
  • the above-mentioned oHSV1d4-GFP viral DNA and pICP4del-hTERTp-ICP4 plasmid DNA were co-transfected into BHK-ICP4 cells, and after homologous recombination, the oHSV1d4-GFP fluorescent protein expression cassette and human telomerase on the pICP4del-hTERTp-ICP4 plasmid were reversed Homologous recombination occurs when the promoter of the enzyme-expressed gene is connected to the ICP4 gene expression cassette, and the plaque of the recombinant virus occurs without fluorescence.
  • the recombinant virus used in the examples may also be a virus strain purchased from the General Microbiology Center of the China Microbiological Culture Collection Committee under the deposit number CGMCC NO. 6397.
  • the recombinant virus (17+hTERTpICP4) was amplified by culture, and finally 10 10 pfu of recombinant virus solution was obtained, and the solvent was DMEM medium.
  • Example 2 Construction of recombinant herpes simplex virus type 1 with ICP34.5 gene deleted
  • PCR 50ul reaction volume
  • the following reaction conditions are used: 20ng17+ viral genomic DNA; 30mM Tris-HCl (pH9.2); 10mM magnesium sulfate; 15mM sodium chloride; 100uM dNTPs; 50pMol forward Primers; 50pMol reverse primer; 1U (enzyme reaction unit) TaqDNA polymerase.
  • 35 cycles of amplification, the temperature and time of each cycle are: 95°C, 60 seconds; 62°C, 20 seconds; 72°C, 120 seconds.
  • the upstream and downstream flanking sequences were cloned into pSP72 (purchased from Promega) plasmids to construct pdICP34.5 and pdICP34.5-eGFP plasmids: using overlapping polymerase chain reaction (72°C, 10 minutes, 4°C, 10 minutes) (overlapping PCR) ligated the upstream and downstream flanking sequences of ICP34.5, and ligated with the pSP72 vector double-cut and filled with BamHI/Xhol to obtain pdICP34.5.
  • the eGFP expression cassette was cut from pcDNA3.1-eGFP with EcoRI/XhoI, the ends were filled in with T4 DNA polymerase, and inserted into the AfeI site of pdICP34.5 to obtain pdICP34.5-eGFP.
  • BHK-ICP4 cells were co-transfected with 17+hTERTpICP4 genomic DNA obtained in the above steps and the resulting pdICP34.5-eGFP. After homologous recombination, after several rounds of plaque purification and selection of green fluorescent plaques, pure recombinant virus 17+hTERTpICP4-d34.5-GFP can be obtained.
  • the GFP expression cassette in 17+hTERTpICP4-d34.5-GFP was removed by pdICP34.5 in the same manner as described above to obtain 17+hTERTpICP4d34.5.
  • co-transfection of pdICP34.5 with 17+hTERTpICP4-d34.5-GFP in BHK-ICP4 cells produces no fluorescence of the plaques of the recombinant virus, and selection of no green fluorescent plaques can purify the recombinant virus 17+hTERTpICP4d34 .5.
  • Example 3 Construction of recombinant herpes simplex virus type 17 17+hTERTpICP4PSMApGFP
  • the recombinant herpes simplex virus type I of the present invention removes the ICP34.5 gene and the ICP47 gene, and replaces the viral genome ICP4 wild-type promoter with the promoter of human telomerase reverse transcriptase and adds it at the position of ICP47
  • the fluorescent protein expression cassette regulated by the prostate-specific membrane antigen promoter regulates growth and proliferation in prostate cancer cells specifically.
  • the promoter sequence of the human prostate-specific membrane antigen-expressing gene was obtained by querying the National Center for Biotechnology Information (NCBI) (GenBank accession number: AF007544.1, SEQ ID NO. 2), and the NruI was obtained by base synthesis.
  • NCBI National Center for Biotechnology Information
  • the single-stranded DNA of the sense strand and anti-sense strand at /HindIII site anneals the single-stranded DNA to form double-stranded DNA.
  • Annealing (50ul reaction volume) system and reaction conditions 50uMol forward primer; 50uMol reverse primer; 30mM Tris-HCl (pH9.2); 95°C for 5 minutes, 70°C for 10 minutes, and gradually cooled to room temperature.
  • the nucleotide sequence of the promoter of human prostate specific membrane antigen expression gene (SEQ ID NO. 2)
  • the pcDNA3.1-eGFP plasmid was cleaved with the restriction enzymes NruI and HindIII to cleave the CMV fragment on the original vector, and the double-stranded DNA of the promoter of the human prostate specific membrane antigen expression gene was inserted into the NruI and HindIII positions of pcDNA3CMVGFP, respectively. Point to form pcDNA3PSMApGFP plasmid
  • step b Insert the sequence of the upstream flanking region of the PCR product amplified in step a into the SmaI site of the pSP73 plasmid to obtain the pSP73ICP47US plasmid;
  • the virus was cultured and the virus genomic DNA was prepared and extracted as described above.
  • the purified 17+hTERTpICP4d34.5 recombinant virus DNA and pdICP47PSMApGFP plasmid DNA were transfected into BHK cells together, and the ICP47 site on 17+hTERTpICP4d34.5 was recombined and then eliminated to obtain the 17+hTERTpICP4PSMApGFP recombinant virus.
  • the virus plaques formed by this round of recombinant virus fluoresce green. Therefore, the virus plaques with green fluorescence are selected and purified by picking five times, and then the recombinant virus is cultured and the virus DNA is extracted.
  • Example 4 Herpes simplex virus 17+hTERTpICP4PSMApGFP and Comparison of 17+hTERTpICP4-d34.5-GFP
  • the lung cancer A549 cells and the prostate cancer PC-3 cells were cultured to the logarithmic growth phase, when the growth state was good, they were digested with trypsin to make a single cell suspension, and centrifuged at 800rpm-1000rpm (120g-190g) for 5 minutes to complete After discarding the supernatant, add appropriate amount of RPMI medium containing 10% FBS to resuspend the cells and count.
  • KHCO 3 1.0g, NH 4 CL 8.3g, EDTA-Na 2 0.037g, adjust the pH value of the solution to 7.2, and finally add distilled water to bring the volume to 1L.
  • Transfection Take out the transfection reagent (recombinant 17+hTERTpICP4PSMApGFP virus), pipette the transfection reagent several times and mix well. Add 100uL of transfection reagent to each well to infect the cells and shake gently. Incubate in a 37°C carbon dioxide cell incubator (5%) for 1 hour, then add 1 mL of complete cell culture fluid to each well. Continue to place and incubate in the cell incubator, and collect the cells after 16-24 hours.
  • Antibody labeled cells To the cell suspension, add CD45 antibody to label leukocytes, and add PSMA antibody to label prostate cells.
  • test results are positive according to the above test procedure.
  • the results determined that the detection method was feasible from a clinical perspective. This detection method can effectively detect peripheral blood circulating tumor cells.
  • x is the detection value of the number of positive quality control products
  • n is the number of detections.
  • Example 6 Detection of healthy volunteers and patients with prostate cancer with the kit
  • test value for healthy people is 0, which proves that the test kit has a very low false positive rate.
  • the mean value for patients with prostate cancer is 2.3, and the detected value range is 0-8 excluding 0.
  • Linear range test results 20 cell test results are 11 and 12; 50 cell test results are 25 and 34; 100 cell test results are 57 and 49; 150 cell test results are 90 and 82; 200
  • the cell detection results were 122 and 122. The result shows that the detection result of this detection method is stable, the detection linear range is wide, and it has good detection efficiency.
  • Example 7 Comparison of CD45-/GFP+ double labeling or gold standard pathology with CD45-/GFP+/PSMA+ triple labeling detection method
  • Transfection Take out the transfection reagent (recombinant 17+hTERTpICP4PSMApGFP virus), pipette the transfection reagent several times and mix well. Add 100uL of transfection reagent to each well to infect the cells and shake gently. Incubate in a 37°C carbon dioxide cell incubator (5%) for 1 hour, then add 1 mL of complete cell culture fluid to each well. Continue to place and incubate in the cell incubator, and collect the cells after 16-24 hours.
  • Antibody labeled cells To the cell suspension, add CD45 antibody to label leukocytes, and add PSMA antibody to label prostate cells (lacking the transfection step in step 8 for the double labeling method).
  • the three-label detection method of the present invention has a lower detection threshold, that is, the detection sensitivity is higher.
  • Q2 is CD45-/GFP+/PSMA+ cells with a cell number of 3
  • Q2+Q3 is CD45-/GFP+ cells with a cell number of 5, indicating that the three markers have higher sensitivity .
  • Table 11 Samples of 20 healthy volunteers and 20 patients with prostate cancer
  • Example 8 17+hTERTpICP4PSMApGFP and 17+hTERTpICP4-d34.5-GFP Performance testing in the three-mark method
  • the transfection reagents are 17+hTERTpICP4PSMApGFP and 17+hTERTpICP4-d34.5-GFP.
  • the detection of prostate cancer cells 17+hTERTpICP4PSMApGFP by the three-label method is more efficient than the detection of 17+hTERTpICP4-d34.5-GFP (shown as oHSV1-hTERTp-eGFP in the figure).
  • the detection rate of 17+hTERTpICP4PSMApGFP in 10 positive quality control cells was 80%, and the detection rate of 17+hTERTpICP4-d34.5-GFP was 60%.
  • 17+hTERTpICP4PSMApGFP is more sensitive than 17+hTERTpICP4-d34.5-GFP.

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Abstract

L'invention concerne un virus de l'herpès simplex recombinant, un kit et l'utilisation de celui-ci.
PCT/CN2019/123221 2018-12-19 2019-12-05 Virus de l'herpès simplex recombinant, kit et utilisation de celui-ci WO2020125423A1 (fr)

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