WO2008014668A1 - Salmonelle atténuée portant un plasmide effectif et son utilisation antitumorigène - Google Patents

Salmonelle atténuée portant un plasmide effectif et son utilisation antitumorigène Download PDF

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WO2008014668A1
WO2008014668A1 PCT/CN2007/002157 CN2007002157W WO2008014668A1 WO 2008014668 A1 WO2008014668 A1 WO 2008014668A1 CN 2007002157 W CN2007002157 W CN 2007002157W WO 2008014668 A1 WO2008014668 A1 WO 2008014668A1
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stat3
grim
recombinant plasmid
plasmid
sirna
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PCT/CN2007/002157
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French (fr)
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Xuejian Zhao
Ling Zhang
Deqi Xu
Lifang Gao
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Jilin University
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an attenuated Salmonella carrying siRNA-Stat3, siRNA-Survivin and co-expressing siRNA-Stat3 and GRIM-19 plasmids, and a preparation method thereof and application thereof in treating tumors. Background technique
  • Salmonella is a facultative anaerobic bacterium with tumorigenic activity. It can reproduce well under hypoxic or hypoxic conditions and produce oncolytic effects. Many reports indicate that the specificity of Salmonella reproduction in tumor tissues is comparable to that of normal tissues. Can be more than 1000 times, so the use of attenuated Salmonella for tumor treatment has a very promising application prospects.
  • the oncogene Stat3 is an important member of the Signal Transducer and Activator of Transcription (STAT) family.
  • the Stat3 signal transduction pathway is closely related to cell proliferation, differentiation and apoptosis. Abnormal proliferation and malignant transformation of cells.
  • the activation of Stat3 ' is a key part of this regulatory abnormality. Blocking the oncogene Stat3 signal transduction pathway in tumor cells may play a role in the treatment of tumors.
  • RNA interference is a post-transcriptional gene silencing.
  • Small or short inference RNA can trigger a post-transcriptional monitoring program that recognizes mRNAs with homologous sequences and produces specificity. Cut, thereby blocking its translation function.
  • RNAi does not completely block the expression of genes, especially genes that are abnormally highly expressed. So there is still a need to find better treatments to enhance the efficacy.
  • the cell death regulator GRIM-19 is a member of the Grim (gene associated with retinoid-IFN-induced mortality) family, and its overexpression leads to apoptosis.
  • GRIM-19 is a negative regulator of Stat3, which binds to the transcriptional activation region (TAD) of StaG and inhibits its transcriptional activity, as down-regulation of transcription of anti-apoptotic genes downstream of Stat3 may further damage mitochondria and allow mitochondria to recombine.
  • GRIM-19 of substance I is released into the cytosol in a large amount, thereby increasing the progress of apoptosis.
  • the invention provides a GRIM-19 gene co-expression system and a preparation method thereof for attenuating Salmonella typhimurium as a carrier carrying Stat3 and Survivin specific siRNA, and antagonizing Stat3, and the application thereof in gene therapy, Significant effects have been achieved in the treatment of various tumors, such as prostate cancer, liver cancer, lung cancer, gastric cancer, laryngeal cancer, breast cancer and melanoma. At the same time, a comprehensive target for deep tumors with attenuated Salmonella as a co-expression plasmid carrier was also explored. To the treatment, it also showed significant prevention and inhibition of the occurrence of metastatic cancer.
  • Antisense chain 5 AGCTTTTCCAAAAAAGCAGCAGCTGAACAACATGTCT
  • Stat3 siRNA connection annealed template oligonucleotide to the linearized p & 7e "Expression cer TM neo 3.1-H1 siRNA vector.
  • the ligation product was transformed into E. coli.
  • the recombinant clones were screened positive.
  • the p & 7em er TM neo 3.1- Hl-Stat3 siRNA plasmid (hereinafter referred p5HlSi-Stat3) with restriction enzymes (BamH I, Hind III) double digestion, the reaction conditions were as follows:? Plasmid 8 ⁇ 1; BamH I ⁇ ⁇ ; Hind III ⁇ ⁇ ; ⁇ Buffer 2 ⁇ 1 ; ddH 2 0 8 ⁇ 1; mixed in a 37 ° C water bath for 2 h. The 5 ⁇ l digestion product was electrophoresed on a 2% agarose gel, and the plasmid from which the target fragment was released was a positive recombinant plasmid.
  • the positive clones were subjected to cDNA sequencing by an automated sequencer from Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • PCR was performed using normal human placenta tissue as a template to amplify the full-length sequence of human GRIM-19:
  • reaction cycle conditions were: pre-denaturation at 94 ° C for 5 min; 94 ° C 30 Sec, 62 ° C 45 Sec, 72 ° C lmin, 30 cycles; 72 ° C extension for 10 min.
  • the PGR product was electrophoresed and recovered using the QIAquick Gel Extration Kit.
  • the pMD18-T-GRIM-19 recombinant plasmid and the pcDNA3.1 vector were digested with Kpnl and EcoRI, respectively, and ligated to construct a pcDNA3.1-GRIM-19 recombinant plasmid (hereinafter abbreviated as pGRIM-19).
  • the ligation product was transformed into Escherichia coli JM109, and positive clones were selected using Amp-containing culture plates. '
  • a single colony was picked from the culture plate and the plasmid was extracted. Double digestion with Kpnl and EcoRI.
  • Primer design - Primers P3 and P4 were designed with reference to the pcDNA3.1 map, and Bgl II and Nm l restriction sites were introduced upstream and downstream according to the primer design principle: the length was 213 bp.
  • the pSHlSi-Stat3 vector was used as a template for PCR amplification of the HI promoter and siRNA-Stat3 sequences:
  • the reaction cycle conditions were: pre-denaturation at 94 °C for 5 min; 94 °C at 30 Sec, 55. C45Sec, 72 °C lmin, 30 cycles; 72 °C extension for 10 min.
  • the PCR product was electrophoresed and recovered with the QIAquick Gel Extration Kit.
  • the pMD18-T vector was ligated, transformed and sequenced.
  • the pMD18-T-HI Si-Stat3 recombinant plasmid, pcDNA3.1 plasmid and pcDNA3.1-GRIM-19 recombinant plasmid were digested with Bgl II Si Nru I and ligated to construct pcDNA3.1- HI Si-Stat3 recombinant plasmid. (hereinafter abbreviated as pH1Si-Stat3) and pcDNA3.1-HI-Stat3-G IM-19 (hereinafter abbreviated as p GRIM-19-Si-Stat3) recombinant plasmid.
  • pH1Si-Stat3 pH1Si-Stat3
  • pcDNA3.1-HI-Stat3-G IM-19 hereinafter abbreviated as p GRIM-19-Si-Stat3
  • a single colony was picked from the culture plate and the plasmid was extracted.
  • Monoclonal attenuated Salmonella colonies were picked with a pipette tip and placed in a 50 ml centrifuge tube containing 5 ml of LB liquid medium. (At the same time, the blank of the culture medium and the tip of the gun) was cultured at 37 ° C, 220 rpm for 14-16 hours. On the next day, the 5 ml of the bacterial liquid was poured into 500 ml of LB liquid medium at a ratio of 1:100, shaken at 37 ° C, 220 rpm for 2-3 h, and the OD value was measured every half hour. When the OD value reached 0.3- At 0.4, stop the culture.
  • the bacterial solution was pre-cooled on ice for 30 minutes, and then the bacterial liquid was dispensed into a 100 ml pre-cooled centrifuge tube, and centrifuged at 4200 rpm for 10 minutes at 4 °C. Discard the supernatant, add 1mmol/L ice pre-cooled HEPES (pH7.0) lml to the centrifuge tube, resuspend the pellet, and add 1mmol/L ice-cold HEPES (pH7.0) 49ml, 4°C , centrifuge at 4200 rpm for 10 min. Repeat 2 times.
  • Discard the supernatant add a small amount of 10% glycerol to the centrifuge cup (sterilized, pre-cooled), resuspend the cells, add 10% glycerol, 4 ° C, 4200 rpm, centrifuge for 10 min. Discard the supernatant, add 500W 10% glycerol to each centrifuge tube, and suspend the sediment. Place the bacterial solution on a 500 ⁇ l tube in a 1.5ml centrifuge tube and add liquid nitrogen for 1min, -80°C. save.
  • Attenuated Salmonella has tumor-forming properties, and the specificity of reproduction in tumor tissues can exceed that of normal tissues. More than 1000 times, 1 can be expressed by attenuated Salmonella to express siRNA-Stat3.
  • GRIM19 can transport effector plasmids to deep tumors and can significantly inhibit tumor growth and metastasis, and can significantly prolong the survival time of mice.
  • the oncogene Stat3 is an important member of the transcriptional signal transducer and activator family. It is found in many human and murine malignancies such as head and neck squamous cell carcinoma, multiple melanoma, breast cancer, prostate cancer, and lung cancer. Excessive activation and expression, activated Stat3 plays an important role in the regulation of tumor cell formation, growth, apoptosis inhibition, etc., suggesting that its regulatory abnormalities are closely related to tumorigenesis and development. Therefore, blocking the oncogene Stat3 signal transduction pathway in tumor cells may play a role in the treatment of tumors.
  • GRIM-19 gene (gene associated with retinoid-IFN-induced mortality 19) is a member of the Grim family, a novel cell death regulator induced by IFN-e in combination with RA (Retinoic acid).
  • GRIM-19 is a 16-kDa protein distributed in the nucleus and cytoplasm and widely expressed in most tissues. Zhang et al.
  • GRIM-19 used yeast two-hybrid library screening to confirm that GRIM-19 can bind to the proto-oncogene Stat3 and block Depending on the gene expression of Stat3, it is speculated that the TAD of Stat3 may be a direct site of GRIM-19 binding, binding to Stat3, forming a point-like dense structure co-localized in the perinuclear, thereby inhibiting the transcriptional activity of Stat3, so GRIM-19 is Active inhibitor of Stat3, GRIM-19 was listed as an anti-cancer gene series. Binding of GRIM-19 to Stat3 and down-regulating the binding of these anti-apoptotic proteins to other pro-apoptotic regulators may further damage mitochondria, leading to disruption of oxidative phosphorylation, thereby increasing the apoptotic response process.
  • GRIM-19 expression is inhibited or expressed in some tumor tissues, whereas GRIM-19 expression is normal in adjacent normal tissues. It is shown that GRIM-19 may be necessary to maintain normal tissues or inhibit cancer.
  • This study used co-expression of the si-Stat3 and GRIM-19 gene eukaryotic recombinant plasmids, which both down-regulated Stat3 expression by RNAi and provided exogenous GRIM-19 to enhance the therapeutic goal of inhibiting prostate cancer.
  • the invention simulates the pathophysiological process of complete prostate cancer metastasis from primary tumor formation to metastasis formation through a plurality of tumor models such as prostate cancer in situ tissue mass transplantation model, and uses attenuated Salmonella typhimurium as a transport
  • the vector for gene therapy has achieved remarkable results in the treatment of prostate cancer.
  • tissue bacterial colony formation assays and fluorescence microscopy attenuated Salmonella typhimurium exhibits preferential aggregation and replication in tumor tissues.
  • the hypoxic environment not only allows the growth of facultative anaerobic bacteria, but also invades and finally kills macrophages and neutrophils. At the same time, typhoid bacteria that enter macrophages are more likely to invade with macrophages.
  • the tumor cells are rich in nutrients, and the rapidly growing tumor tissue tends to form hypoxic regions and tumor necrotic regions, which makes the tumor tissue environment different from normal tissues;
  • Recent studies have found on the surface of tumor cells. There is a complement inhibitory factor.
  • irregular vascular distribution and intra-tumor pressure prevent the penetration of antibodies and serotypes of Salmonella, and few are found in tumor tissues.
  • Salmonella finds a safe in tumor tissue.
  • TOF e tumor necrosis factor beta
  • Salmonella Due to the facultative anaerobic properties of Salmonella, it can settle in small metastatic tumor cells with a certain oxygen content, and can also settle in the center of deep hypoxia in large tumor tissues, and Showing the function of dissolving and killing tumor tissue;
  • SPI2 pathogenicity island 2
  • the present invention is the first to use an attenuated Salmonella carrying Stat3 or Survivin-specific siR A, and a GRIM-19 gene co-expression system that antagonizes Stat3, and conducts in vitro and in vivo studies against various tumors.
  • Local injection of tumor or tail vein injection of Stat3 sequence-specific siRNA and GRIM-19 combined expression vector system have obvious synergistic therapeutic effects.
  • the results showed that the attenuated Salmonella had tumor-forming characteristics, and the specificity of reproduction in tumor tissues was more than 1000 times higher than that of normal tissues.
  • the expression level in normal tissues was significantly decreased after 15 days, and the system had significant effects on experimental tumors. The therapeutic effect can prolong the survival time of the mice.
  • Figure 1 Construction of the co-expression plasmid pGRIM-19-Si-Stat3;
  • Figure 2 (A) The results of restriction enzyme digestion of pSHlSi-Stat3 expression plasmid
  • Figure 2 (B) Amplification of the full length of GRIM-19 using the pCXN2mycAGRIM-19 expression plasmid as a template; ' Figure 2 (C) Kpnl and EcoRI double digestion to identify pMD18-T-GRIM-19 plasmid;
  • FIG. 3 (A) Cellular immunochemical staining showed that the expression of GRIM-19 was enhanced in the P GRIM-19-Si-Stat3 group;
  • Figure 4 MTT assay to detect the inhibition of tumor cell proliferation by each group of plasmids
  • Annexin V-CY3 apoptosis kit detects co-expression of pGRIM-19-Si-Stat3 plasmid can induce tumor cell apoptosis;
  • Figure 8 co-expressing plasmid has growth inhibitory effect on laryngeal carcinoma of nude mice
  • Figure 10 A total of plasmids have growth inhibition on lung cancer in nude mice
  • T4 DNA ligase was purchased from Promega, USA; BamH I, Hind III, Nru I, Kpn I and other endonucleases were purchased from Dalian Bao Bioengineering Co.; DNA purification system Wizard plus SV Minipreps was purchased from Promega, USA; Yeast extract was purchased from OXOID Company; agarose and agar powder were purchased from Dalian Bao Bioengineering Co.; DNA gel recovery kit was purchased from Dalian Bao Bioengineering Co.; DNA Marker DL2000, 1Kb Ladder DNA marker were products of Takara. Primer synthesis and sequencing were completed by Shanghai Shenggong Bioengineering Technology Service Company.
  • Ethidium bromide, agarose, SDS, TEMED, acrylamide, N, N-dimethyl bis acrylamide, MTT, DTT, DMSO, PI, PMSF were purchased from Sigma, USA; ampicillin, kanamycin were purchased from Beijing Dingguo Company; DEPC was purchased from Merk, Germany; high-fidelity Taq DNA polymerase, DAB, Triton X-100, dNTP, MMV reverse transcriptase and plasmid extraction and purification kits were purchased from Promega; RA enzyme and proteinase K were purchased. In the United States Ambion company.
  • Transwell cell culture chamber and matrigel are products of BD Biosciences, USA; Lipofetion2000 and Trizol are products of Invitrogen, USA; trypsin and IMDM medium are products of Hyclone Company of USA; Newborn bovine serum is purchased from Hangzhou Sijiqing Company; Annexin V-CY3 withered The dead kit was purchased from SIGMA Corporation; other conventional chemical reagents were of analytical grade.
  • PCR amplification instrument GeneAmp, USA; ultra-clean workbench (YZ-875 Suzhou purification equipment factory); . automatic high pressure steam sterilizer (SONY, Japan); low temperature refrigerator (-80 °C SANYO, Japan); constant temperature water bath Box (Jiangsu Changzhou Guohua Instrument Factory); High-speed cryogenic centrifuge (TOMY GRX-220, Japan); Electric blast drying oven (Shanghai Experimental Instrument Factory); Electronic balance (OHAUS, USA); Electrophoresis (Bio-rad) , USA); gel imaging system (Shanghai Tianneng Technology Co., Ltd.); phase contrast microscope (OLYMPUS, Japan); fully automatic microscope digital camera system (OLYMPUS, Japan); microplate reader (TAKARA, Japan); fluorescence microscope (OLYMPUS , Japan); Flow Cytometry (CC ⁇ LTER, USA); Deionized Water Unit (Japan); PCR Amplifier (GeneAmp, USA); Visible, UV Spectrophotometer and Analytic
  • the pSilencerTM nGO 3.1-H1 siRNA expression vector was purchased from Ambion. Plasmid with U6 promoter and GFP pGCsilencerTM U6/Neo/GFP was purchased from Shanghai Jikai Chemical Company. Attenuated Salmonella was purchased from Berna Biotech, USA. The PMD-18T vector was purchased from Protec. The pcDNA3.1 expression vector and Escherichia coli JM109 were purchased from Invitrogen, USA. '
  • Mouse prostate cancer RM-1 cell line was purchased from Shanghai Institute of Cell. Each cell line was cultured in an IMDM medium containing 10% newborn calf serum in an incubator at 37 ° C, 5 % C02, and passaged with 0.25% trypsin.
  • mice The experimental animals were purchased from the Laboratory Animal Research Institute of the Chinese Academy of Medical Sciences.
  • BALB/C nu/nu male nude mice 150, 4 to 6 weeks old, weighing 18 to 20 g, raised in a constant temperature (22-25 °C), constant humidity (40%-50%) SPF laminar flow chamber Medium, autoclaved standard feed and water for free consumption by animals.
  • 50 male C57BL6 inbred mice aged 8 weeks, weighing 18 ⁇ 20g, constant temperature, constant humidity, clean, no special pathogens, regular replacement of litter, clean drinking water and feeding for free photography In.
  • the oligonucleotide chain sequence is GCAGCAGCTGAACAACATG, synthetically encoded siRNA DNA template:
  • Antisense chain 5 AGCTTTTCCAAAAAAGCAGCAGCTGAACAACATGTCT
  • the oligonucleotide was diluted to a final concentration of 1 ⁇ £/ ⁇ 1.
  • Stat3 siRNA connection annealed template oligonucleotide to 3.1-H1 siRNA expression vector linearized p3 ⁇ 4 / encer TM neo.
  • the 5 ⁇ l annealed Stat3 siRNA template oligonucleotide was diluted with 45 ⁇ l of denuclease water to a final concentration of 8 ⁇ ⁇ / ⁇ 1; 10 ⁇ of the ligation reaction system was established: One negative control was set at 4 ° C overnight.
  • the reaction system is as follows: Dilute-annealed siRNA template oligonucleotide ⁇ ⁇ ; denuclease water 6 ⁇ l; 10xT4 DNA ligase buffer ⁇ ⁇ ; p&'/eMCerTMneo 3.1-H1 siRNA vector ⁇ ⁇ ; ⁇ 4 DNA ligase (5 ⁇ / ⁇ 1) 1 ⁇ 1.
  • the host strain JM109 was inoculated on LB solid medium and cultured overnight. On the next day, single colonies were picked from LB plates, inoculated in 3 ml of LB liquid medium, and shake cultured at 37 ° C for about 12 hours until the late logarithmic growth.
  • the bacterial suspension was inoculated in 100 ml of LB liquid medium at a ratio of 1:50, and cultured at 37 ° C for 2-3 hours until the OD600 reached 0.45 "0.55.
  • the ice bath liquid was incubated for 10 minutes, and 50 ml of the cells were collected by centrifugation at 4000 g for 10 min. Resuspend in 10 ml of ice pre-cooled 0.1 M CaCl 2 .
  • the pellet was resuspended in 2 ml of ice pre-cooled 0.1 M CaCI 2 and placed at 4 ° C. After 16 h, ⁇ was taken for the conversion test, or glycerol was added to a final concentration of 10%, and stored at -70 ° C for later use.
  • a single colony grown on the selection plate was picked, added to 5 ml of LB liquid medium containing ampicillin (100 g/mL), and cultured overnight at 37 ° C with shaking.
  • the plasmid was prepared in small amounts by alkaline lysis. The procedure was as follows: 1.5 ml of the culture was poured into an Ep tube, centrifuged at 7 000 g for several seconds, and the culture solution was aspirated, and the previous step was repeated to make the bacterial precipitate as dry as possible.
  • the bacterial pellet was resuspended in 100 ⁇ l ice-cold solution so l u tion I (50mmol / L glucose, 25mmol / L Tris.Cl pH 8.0, In 10 mmol/L EDTA pH 8.0), it was shaken vigorously and left at room temperature for 5 min. Force 200 ⁇ l of the newly prepared solution solution II (0.2 mol/L NaOH, 1% SDS), cap the tube, and quickly invert the tube several times to mix the contents, then place the tube on ice for 5 min.
  • l u tion I 50mmol / L glucose, 25mmol / L Tris.Cl pH 8.0, In 10 mmol/L EDTA pH 8.0
  • Ice-cold solution solutionlll (5 mol/L potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml dd3 ⁇ 40) was added, the tube was tightly closed, the tube was inverted for 20 sec, and the tube was placed on ice for 5 min. Centrifuge at 12 000 g for 10 min, transfer the supernatant to another Ep tube, add an equal amount of phenol: chloroform (25: 24), mix by shaking, centrifuge at 2 ° C for 12 min for 2 min, transfer the supernatant to another Ep tube. in. Add two volumes of ethanol, mix by shaking, and let stand for 2 min at room temperature.
  • the p & 7ewcer TM neo 3.1-Hl -Stat3 siRNA plasmid (hereinafter referred pSHlSi-Stat3) with restriction enzymes (BamH I, Hind III) double digestion, the reaction conditions were as follows: Plasmid 8 ⁇ 1; BamH I ⁇ ⁇ ; Hind III ⁇ ⁇ ; ⁇ Buffer 2 ⁇ 1; ddH 2 0 8 ⁇ 1; mixed in a 37 ° C water bath for 2 h. The 5 ⁇ l digestion product was electrophoresed on a 2% agarose gel, and the plasmid from which the target fragment was released was a positive recombinant plasmid.
  • pSHlSi-Stat3 siRNA plasmid (hereinafter referred pSHlSi-Stat3) with restriction enzymes (BamH I, Hind III) double digestion
  • the positive clones were subjected to cDNA sequencing by an automated sequencer from Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • PCR was performed using normal human placenta tissue as a template to amplify the full-length sequence of human GRIM-19:
  • reaction cycle conditions were: pre-denaturation at 94 ° C for 5 min; 94 ° C 30 Sec, 62 ° C 45 Sec, 72 ° C lmin, 30 cycles; 72 ° C extension for 10 min.
  • the PCR product was electrophoresed and recovered using the QIAquick Gel Extration Kit:
  • the gel containing the DNA fragment was excised from the agarose gel using a surgical blade, placed in an Ep tube, and crushed; the gel volume was added to the tube.
  • the pMD18-T-GRIM-19 recombinant plasmid and the pcDNA3.1 vector were digested with Kpnl and EcoRI, respectively, and ligated to construct a pcDNA3.1-GRIM-19 recombinant plasmid (hereinafter abbreviated as pGRIM-19).
  • the ligation product was transformed into Escherichia coli JM109, and positive clones were selected using Amp-containing culture plates.
  • a single colony was picked from the culture plate and the plasmid was extracted. Double digestion with Kpnl and EcoRI.
  • primers P3 and P4 were designed with reference to pcDNA3.1 map, and Bgl II and Nru I restriction sites were introduced upstream and downstream: 213 bp in length.
  • PCR was carried out using the pH1Si-Stat3 vector as a template to amplify the HI promoter and the siRNA-Stat3 sequence:
  • the reaction cycle conditions were: pre-denaturation at 94 °C for 5 min; 94 °C 30 Sec, 55 °C 45 Sec, 72 °C lmin, 30 cycles; 72 °C extension for 10 min.
  • the PCR product was electrophoresed and recovered with the QIAquick Gel Extration Kit.
  • the pMD18-T vector was ligated, transformed and sequenced.
  • the pMD18-T-HI Si-Stat3 recombinant plasmid, pcDNA3.1 plasmid and pcDNA3.1-GRIM-19 recombinant plasmid were digested with Bgl II and Nru I, respectively, and ligated to construct pcDNA3.1-HI Si-Stat3 recombinant plasmid. (hereinafter abbreviated as pH1Si-Stat3) and pcDNA3.1-HI-Stat3-GRIM-19 (hereinafter abbreviated as p GRIM-19-Si-Stat3) recombinant plasmid.
  • pH1Si-Stat3 pH1Si-Stat3
  • pcDNA3.1-HI-Stat3-GRIM-19 hereinafter abbreviated as p GRIM-19-Si-Stat3
  • a single colony was taken from the culture plate and the plasmid was extracted.
  • Monoclonal attenuated Salmonella colonies were picked with a pipette tip and placed in a 50 ml centrifuge tube containing 5 ml of LB liquid medium. (At the same time, the blank of the culture medium and the tip of the gun) was cultured at 37 ° C, 220 rpm for 14-16 hours. On the next day, the 5 ml of the bacterial liquid was poured into 500 ml of LB liquid medium at a ratio of 1:100, shaken at 37 ° C, 220 rpm for 2-3 h, and the OD value was measured every half hour. When the OD value reached 0.3- At 0.4, stop the culture.
  • the bacterial solution was pre-cooled on ice for 30 minutes, and then the bacterial liquid was dispensed into a 100 ml pre-cooled centrifuge tube, and centrifuged at 4200 rpm for 10 minutes at 4 °C. Discard the supernatant, add 1mmol/L ice pre-cooled HEPES (pH7.0) lml to the centrifuge tube, resuspend the pellet, and add 1mmol/L ice-cold HEPES (pH7.0) 49ml, 4°C. Centrifuge at 4200 rpm for 10 min. Repeat 2 times.
  • Discard the supernatant add a small amount of 10% glycerol to the centrifuge cup (sterilized, pre-cooled), resuspend the cells, add 10% glycerol, 4 ° C, 4200 rpm, centrifuge for 10 min. Discard the supernatant, add 500 ⁇ 10% glycerol to each centrifuge tube, and suspend the pellet.
  • the bacteria solution is placed in a 1.5 ml centrifuge tube at 300 W/tube on ice, and liquid nitrogen is added for 1 min, -80 °C. save.
  • the tumor cells were trypsinized into a cell suspension, and the cell pellet was blown off with IMDM culture solution by centrifugation. The cell viability was ⁇ 95% by trypan blue assay, and the final cell concentration was 2 ⁇ 10 7 /ml. 2 ⁇ 10 ⁇ (0.1 ml) of tumor cells were inoculated into the subcutaneous tissue of the proximal forearm of the left back of nude mice. The caliper was used to measure the next day, and the nude mice were randomly grouped for experimentation when the tumor was grown to a diameter of 5 mm.
  • mice in each group were divided into 5 groups, which were grouped as follows: mock group, pHlSi-Sc ble group, pHlSi-Stat3 group, pGRIM-19 group, pGRIM-19-Si-Stat3 group.
  • Each group of tumors was locally injected with attenuated Salmonella carrying the corresponding group of plasmids, injected at 2 points, 50 ⁇ l per point, and the bacterial concentration was 10 ⁇ /100 ⁇ 1.
  • Tumor tissue RNA and protein were extracted, semi-quantitative PCR analysis of Stat3 GRIM-19 expression at the gene level, Western blotting analysis of Stat3 GRIM-19 expression at the protein level.
  • mice Ten C57BL6 inbred mice were sterilized with strong iodine, and the mouse-derived tumor cell solution was inoculated subcutaneously into the back of the mouse.
  • the inoculated cell concentration was 2 ⁇ 10 7 /ml, and each was inoculated with 100 ⁇ l. Tumor growth was observed every other day.
  • Tumor tissue for surgical orthotopic transplantation is derived from the above-described mouse subcutaneous subcutaneous tumor model.
  • the tumor tissue with better condition is close to the periphery, the color is white and shiny, the texture is tough and elastic, and it is not easy to be broken.
  • physiological saline a tumor mass of 1.5 mm in diameter was cut under a microscope of 10 times.
  • C57BL6 inbred mice were anesthetized with sodium pentobarbital (60 mg/Kg), placed in the supine position, routinely disinfected with strong iodine, and covered with sterile holes.
  • the median incision of the lower abdomen is about 1.5-2.0 cm long, revealing the abdominal cavity.
  • a total of 40 C57BL6 mice were transplanted in situ for each tumor. Grouped as above.
  • C57BL6 mice were sacrificed by excessive anesthesia when they were killed or sacrificed. Under the operating microscope, the tumor growth and lymph node metastasis of orthotopic transplantation were observed. The liver, lung, spleen, kidney, vertebral body, lymph nodes and suspicious bones were taken for Fol. Marin fixed, paraffin embedded, sectioned, HE stained to observe the presence or absence of metastases under light microscope.
  • the pGRIM-19-Si-Stat3 plasmid co-expressing the siRNA-Stat3 and GRIM-19 genes was successfully constructed by sequencing and restriction enzyme digestion.
  • the pSHlSi-Stat3 expression plasmid was digested with BamH I and Hind III, and the enzyme fragments of 66 bp and 4.31 cb were observed by 1.5% agarose gel electrophoresis. The results are shown in Fig. 1. DNA sequencing results confirmed Si-Stat3 and Si-Scramble fragments The 3.1-H1 siRNA expression vector was ligated correctly and the results are shown in Figure 2 (A).
  • the pCXN2mycA GRIM-19 plasmid was used as a template for PCR, and the full length of GRIM-19 was 435 bp.
  • the recombinant plasmid was digested with Kpnl and EcoRI to obtain two fragments, the large fragment was a vector fragment of 2692 bp, and the small fragment was a GRIM-19 fragment with a size of 435 bp.
  • the results of the sequencing were completely consistent with the sequence of Genebanl (NM_015965), and the restriction sites were successfully added at both ends.
  • the recombinant plasmid was digested with Kpnl and EcoRI to obtain two fragments.
  • the large fragment was a vector fragment with a size of 5428 bp ; the small fragment was a GRIM-19 fragment with a size of 435 bp .
  • the results are shown in Figure 2 (D).
  • the HI promoter and the Si-Stat3 fragment were amplified using the pH1Si-Stat3 plasmid as a template, and the obtained product was 204 bp in size, and the results are shown in Fig. 2 (E).
  • the recombinant plasmid was digested with Bgl ll and Nral.
  • the large fragment was a vector fragment with a size of 2692 bp.
  • the small fragment was a HlSi-Stat3 fragment with a size of 204 bp.
  • Figure 2 (F) DNA sequencing confirmed that the HlSi-Stat3 fragment was ligated to the pcDNA3.1 expression vector.
  • the recombinant plasmid was digested with Bgl ll and Nm l, and the large fragment was a vector fragment of 5428 bp and 5860 bp, respectively ; the small fragment was a HlSi-Stat3 fragment with a size of 204 bp.
  • the pGRIM-19-Si-Stat3 recombinant plasmid was digested with Kpnl and EcoRI to obtain two fragments.
  • the large fragment was a vector fragment with a size of 5428 bp.
  • the small fragment was a GRIM-19 fragment with a size of 435 bp.
  • the cells in the control group grew adherently, in good condition, mostly fusiform, moderate in size, clear in nucleoli, visible mitotic phase, good cell refraction, and vigorous cell proliferation; pHlSi-Stat3 and pGRIM-19 groups prolonged transfection time
  • the cells grow slowly, the shape is irregular, the cells shrink, the particles increase, and the cell debris increases.
  • the pGRIM-19-Si-Stat3 group the intracellular particulate matter increases, the original morphology is lost, the nucleus is pyknotic, and the lysis is surrounded by the plasma membrane. Fragments, the number of cells was significantly reduced compared to the control group, and some cells floated in the culture medium.
  • the three groups of recombinant plasmids significantly inhibited the proliferation of PC-3M cells, the inhibition rates were 51.7%, 42.3% and 20.9%, respectively, compared with the control group, the difference was significant (P ⁇ 0.01). There was no significant difference in the proliferation activity between the liposome control group and the pH1Si-Scramble control group (P>0.05).
  • the expression of the siRNA-Stat3 and GRIM-19 gene eukaryotic sputum group was significantly enhanced compared with the single use. ( ⁇ 0.05).
  • PC-3M cells in the recombinant plasmid transfection group showed obvious apoptosis at 72 h after transfection (P ⁇ 0.01) (Table 1).
  • the PC-3M cells in the liposome control group and the pH1Si-Scramble group were uniformly green by laser confocal microscopy, indicating that early apoptosis was observed in PC-3M cells transfected with the recombinant plasmid.
  • pGRIM-19, pHlSi-Stat3, pGRIM-19-Si-Stat3 transfection group showed different degrees of annexin V positive expression, and apoptotic cells were yellow with both AnnCy3 and 6-CF red-green fluorescent markers.
  • tumor tissue 100 mg of tumor, spleen, liver and lung were aseptically taken, ground, diluted 5 times with cold PBS, inoculated into Amp-containing LB plates, overnight at 37 °C, and the number of individual clones formed was calculated the next day (Fig. 6A) ).
  • the number of clones formed by tumor tissue was 1890 ⁇ 98.2; the formation of spleen tissue was 10 ⁇ 3.5; the formation of liver tissue was 8 ⁇ 4.3; the formation of lung tissue was 2 ⁇ 0.3. Tumors were statistically significant compared to the rest of the organs ( ⁇ 0.01).
  • the nude mouse subcutaneous xenograft model proved that the P GRIM-19-Si-Stat3 recombinant plasmid had a synergistic effect, and the average tumor weight compared with the control group. Both the volume and the volume are reduced.
  • pHl Si-Scramble 25.36 ⁇ 2.58 2.18 ⁇ 0.64 1100.62 ⁇ 235.21
  • pGRIM-19 24.31 ⁇ 2.36 1.22 ⁇ 0.25 357.27 ⁇ 70.07* pHlSi-Stat3 25.12 ⁇ 2.77 0.94 ⁇ 0.3l” 271.46 ⁇ 65.11* pGRIM-19-Si-Stat3 27.17 ⁇ 2.92 0.26 ⁇ 0.11* 84.19 ⁇ 24.33*
  • the model group can have various organs.
  • the metastasis is a good simulation of the progression and metastasis of advanced cancer ( Figure 14). Tumor cells developed apoptosis, and the metastatic site was lower than that of the control group (Table 4).
  • the activity of MMP-2 in the tumor tissues of the liposome control group and the pH1Si-Scramble control group was significantly higher than that of the pGRIM-19, pH1Si-Stat3 and pGRIM-19-Si-Stat3 transfection groups.
  • Figure 26 shows the results of Western blot analysis.
  • the expression of GRIM-19 protein was enhanced in pGRIM-19 group and pGRIM-19-Si-Stat3 group; Stat3 protein expression was weakened in pHlSi-Stat3 group and pGR!M-19-Si-Stat3 group.
  • Hematoxylin-eosin (HE) staining was observed under ordinary light microscope: pGRIM-19, P HlSi-Stat3 and PGRIM-19-Si-Stat3 transfected group, the cell nucleus was contracted, blue-black, and cytoplasm was light. Red (apoptotic cells), the normal nuclei are evenly light blue or blue, and the necrotic nuclei disappear in a very pale blue or blue color.
  • the present invention was formulated into a 3 X 10 8 /ml solution with PDS and placed in a 1 ml ampule. When used, each person is injected subcutaneously or intramuscularly with lral.

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Description

运载重组质粒的减毒沙门氏菌及其在抗肿瘤中的应用 技术领域
本发明属于生物技术领域， 具体为一种携带 siRNA-Stat3, siRNA-Survivin及共表达 siRNA-Stat3及 GRIM-19质粒的减毒沙门氏菌及其制备方法及其在治疗肿瘤的中的应用。 背景技术
肿瘤基因治疗的主要障碍之一是如何把抗肿瘤因子或其他治疗药物传递到肿瘤组织 内， 特别是深部肿瘤和转移瘤， 而不伤害正常组织。 沙门氏菌为兼性厌氧菌， 具有嗜肿瘤 活性， 在低氧或缺氧条件下的肿瘤深部可繁殖良好并产生溶瘤效应， 许多报告指出沙门氏 菌在肿瘤组织中繁殖的特异性与正常组织相比可超过 1000倍以上， 因此以减毒沙门氏菌 用于肿瘤治疗具有十分看好的应用前景。
癌基因 Stat3 是信号转导子与转录激活子 （Signal Transducer and Activator of Transcription, STAT)家族的重要成员， Stat3信号转导通路与细胞的增殖、 分化及凋亡密 切相关， 该通路持续激活可导致细胞异常增殖和恶性转化。 研究表明人前列腺癌组织及前 列腺癌细胞中 Stat3以及其下游基因， 如 Survivin、 cyclin DK c-Myc、 Bcl-2、 Bcl-xL以及 Mcl-1和 VEGF等， 常显示异常表达或活性增强， 与前列腺癌发生发展密切相关。 而 Stat3 '的活化是这种调控异常的关键一环。 阻断肿瘤细胞中癌基因 Stat3 信号转导通路可能起到 治疗肿瘤的作用。
RNA干涉 (RNA interference) 是一种转录后的基因沉默， 小干涉 RNA (small or short inference RNA /siR A) 能触发某种转录后监控程序， 识别有同源序列的 mRNA, 对其产 生特异性切割， 从而阻断其翻译功能。 但是在哺乳动物细胞中， RNAi 并不能完全阻断基 因的表达， 尤其是异常高表达的基因。 所以仍然需要寻求更好的治疗方案以加强疗效。
细胞死亡调节因子 GRIM-19 是 Grim ( gene associated with retinoid-IFN- induced mortality) 家族成员之一， 其过度表达会导致细胞凋亡。 GRIM-19是 Stat3的负调节因子， 能与 StaG的转录激活区 （Transactivation Domain/TAD) 结合， 抑制其转录活性， 因为下调 Stat3下游抗凋亡基因的转录可能会进一步损伤线粒体，使位于线粒体复合物 I的 GRIM-19 大量释放入胞浆， 从而加大凋亡的反应进程。
我们的前期工作已经成功建立了一种用于治疗前列腺癌及黑色素瘤的重组质粒， 并申 报了专利， 专利申请号： CN200510017056.9; 公幵号： CN1730658, 目前以减毒沙门氏菌 携带 siR A-Stat3， siR A-Survivi 及共表达 siRNA-Stat3及 GRIM-19的 pGRIM-19-Si-Stat3 重组质粒进行多种肿瘤治疗， 在国内外均未见报道。
发明内容
本发明提供减毒鼠沙门氏菌作为运载体携带 Stat3及 Survivin的特异性 siRNA, 联合 对 Stat3呈现拮抗作用的 GRIM-19基因共表达系统及其制备方法， 以及其在进行基因治疗 中的应用， 在对多种肿瘤的治疗过程取得了显著效果， 如前列腺癌、 肝癌、 肺癌、 胃癌、 喉癌、 乳腺癌及黑色素瘤等， 同时探讨了以减毒沙门氏菌为共表达质粒运载体进行深部肿 瘤的综合靶向治疗， 同时对转移癌的发生表现出显著的预防和抑制作用。
本发明釆取的技术方案是：
一、构建共表达 siRNA-Stat3和 GRIM-19基因的 pGRIM-19-Si-Stat3质粒。
1.1 pSHlSi-Stat3载体的构建
1.1.1 Stat3 siRNA模板寡核苷酸的设计
• 根据 genebank(NM 31500)人 Stat3基因 mRNA的已知序列， 确定合适的靶位点 认本 (2143-2162), 寡核苷酸链序列为 GCAGCAGCTGAACAACATG, 合成编码 siRNA的 DNA模 板： '
正义链： 5 ' GATCCGCAGCAGCTGAACAACATGTTCAAGAGACATG
TTGTTCAGCTGCTGCTTTTTTGGAAA 3'
反义链： 5， AGCTTTTCCAAAAAAGCAGCAGCTGAACAACATGTCT
CTTGAACATGTTGTTCAGCTGCTGCG 3'
稀释寡核苷酸至终浓度 Ι μ^μΐ;
1.1.2 Stat3 siRNA模板寡核苷酸退火
1.1.3连接
连接退火的 Stat3 siRNA模板寡核苷酸到线性化的 p&7e"cerTMneo 3.1-H1 siRNA表达载 体。 将连接产物转化入大肠杆菌。 筛选阳性重组克隆。
1.1.4重组质粒的鉴定
将 p&7em?erTMneo 3.1-Hl-Stat3 siRNA质粒 (以下简称 p5HlSi-Stat3)用限制性内切酶 (BamH I、 Hind III)进行双酶切， 反应条件如下： 质粒 8μ1; BamH I Ι μΐ; Hind III Ι μΐ; ΙΟχΗ Buffer 2μ1; ddH20 8μ1; 混合后 37°C水浴 2h。 取 5μ1酶切产物在 2%琼脂糖凝胶中电 泳， 释放出目的片段的质粒为阳性重组质粒。
阳性克隆经上海生工生物工程技术服务有限公司自动测序仪进行 cDNA序列测定。
1.2 pcDNA3.1- GRIM-19表达载体的构建
1.2.1 引物设计
根据 Genebank AF286697号编码 GRIM19全长序列由软件设计引物：
P1 : 5， -GAGAATTCATGGCGGCGTCAAAGG-3 ' (EcoR I )
P2: 5 ' -GAAAGCTT CAGGGCCTACGTGTACCACAT-3 ' (Hind III).
1.2.2扩增人 GRIM-19全长序列
以正常人胎盘组织为模板进行 PCR， 扩增人 GRIM-19全长序列：
反应循环条件为： 94°C预变性 5min; 94 °C 30 Sec, 62°C45Sec, 72 °C lmin， 30个 循环； 72°C延伸 10min。
PGR产物电泳， 用 QIAquick Gel Extration Kit进行回收。
1.2.3 MD18-T-GRIM-19重组质粒制备
于 0.5ml Ep管中， 加入下述试剂： 回收 DNA片段 4.5μ1; pMD18-T vector 0.5μ1 (片段 量： 载体量 =3〜8： 1)； solution I 5μ1; 将其混匀后， 于 16°C水浴过夜。 转化， 鉴定并测 序。
1.2.4连接反应
用 Kpnl和 EcoRI分别酶切 pMD18-T- GRIM-19重组质粒及 pcDNA3.1载体， 进行连 接， 构建 pcDNA3.1- GRIM-19重组质粒（以下略称为 pGRIM-19)。 将连接产物转化大肠 杆菌 JM109, 用含有 Amp的培养板筛选阳性克隆。 '
1.2.5重组质粒的鉴定
从培养板中挑取单一菌落， 提取质粒。 用 Kpnl和 EcoRI进行双酶切鉴定。
1.3共表达 siRNA-Stat3及 GRIM-19基因 p GRIM-19-Si-Stat3真核重组质粒的构建
1.3.1 引物设计- 依据引物设计原则，参照 pcDNA3.1图谱设计引物 P3和 P4，并在其上下游分别引入 Bgl II 和 Nm l酶切位点： 长度为 213bp。
P3： 5'CGAGATCTGAATTCATATTTGCATGTCGCTATG3 ' P4: 5' TCGCGAAGGAAACAGCTATGACCATGATTAC 3'
1.3.2扩增 HI启动子及 siRNA-Stat3序列
以 pSHlSi-Stat3载体为模板进行 PCR， 扩增 HI启动子及 siRNA-Stat3序列：
反应循环条件为： 94 °C预变性 5min; 94 °C 30 Sec, 55。C45Sec， 72 °C lmin, 30个循 环； 72 °C延伸 10min。 PCR产物电泳， 特异带 QIAquick Gel Extration Kit回收。 与 pMD18-T 载体进行连接， 转化并测序。
1.3.3连接反应
用 Bgl II Si Nru I 分别酶切 pMD18-T- HI Si-Stat3 重组质粒， pcDNA3.1 质粒及 pcDNA3.1- GRIM-19重组质粒， 并进行连接， 构建 pcDNA3.1- HI Si-Stat3重组质粒（以下 略称为 pHlSi-Stat3 ) 及 pcDNA3.1- HI- Stat3-G IM-19 (以下略称为 p GRIM-19-Si-Stat3 ) 重组质粒。 将连接产物转化大肠杆菌 JM109, 用含有 Amp的培养板筛选阳性克隆。
1.3.4鉴定
从培养板中挑取单一菌落， 提取质粒。
①用 Bgl II和 Nru I进行双酶切鉴定。
②用 Kpn I和 EcoR I进行双酶切鉴定。
二将该重组质粒电转化入减毒沙门氏菌
2.1 电转化感受态的制备
用枪头挑取单克隆减毒沙门氏菌菌落，投入盛有 5ml LB液体培养基的 50ml离心管中。 (同时做培养基和枪头的空白对照） 37°C， 220rpm， 培养 14-16个小时。 第二天， 以 1 : 100的比例将这 5ml菌液倒入 500ml LB液体培养基中， 37°C， 220rpm, 振摇 2-3h， 每半 小时测一次 OD值， 当 OD值达到 0.3-0.4时， 停止培养。 将菌液在冰上预冷 30分钟， 随 后将菌液分装到 100ml预冷的离心管中， 4°C， 4200rpm离心 10min。 弃上清， 离心管中加 人 lmmol/L冰预冷的 HEPES (pH7.0) lml,使沉淀重悬后，再加入 lmmol/L冰预冷的 HEPES (pH7.0) 49ml, 4°C， 4200rpm离心 10min。 重复 2次。 弃上清， 往离心杯中加入少量 10% 甘油 (灭菌， 预冷)， 重悬菌体， 再加满 10%甘油， 4°C， 4200rpm, 离心 10min。 弃上清， 每个离心管中加入 500W 10%的甘油,使沉淀悬浮后，将菌液在冰上以 300μ1/管分装于 1.5ml 的离心管中， 投入液氮 lmin, -80°C保存。
2.2 电转化步骤
取 1 μΐ 纯化后的重组质粒于 1.5ml的离心管中，将其和 0.1CM的电极杯一起置于冰上 预冷。将 lOOul解冻的感受态细胞转移至此 1.5ml 的离心管中，小心混匀，冰上放置 10min。 打开电转仪， 调至 Manual, 调节电压为 2. 5 kV， 25 F， 200Ω。 将此混合物转移至已预冷 的电极杯中， 轻轻敲击电极杯使混合物均匀进入电极杯的底部； 将电极杯推入电转化仪， 按一下 pulse键， 听到蜂鸣声后， 向电击杯中迅速加入 ΙΟΟΟμΙ的 LB液体培养基， 重悬细 胞后， 转移到 1.5ml的离心管中。 37°C， 250rpm复苏 lh。 取 20ul转化产物加 160 μΐ LB 涂板， 7°C过夜培养， 次日查看转化结果。 其余菌液加 1 : 1的 30%的甘油后混匀一 80°C保 存。
本发明技术方案主要创新点：
1,本研究成功构建 pHlSi-Stat3， pGRIM-19及可同时表达 GRIM-19和 siR A-Stat3的 共表达的 pGRIM-19-Si-Stat3重组质粒。
2. 体内实验证明 GRIM-19和 siR A-Stat3的共表达可以起到很好的协同作用抑制肿瘤 的生长及转移。
3. 减毒沙门氏菌具有噬肿瘤特性，在肿瘤组织中繁殖的特异性与正常组织相比可超过 1000倍以上， 1以减毒沙门氏菌携带可表达 siRNA-Stat3, GRIM19可运载效应质粒到达深 部肿瘤并可起到显著抑制肿瘤生长和转移的作用， 并且能够明显延长小鼠的生存'时间。
本发明优点及有益效果是
癌基因 Stat3是转录信号转导子与激活子家族的重要成员，在很多人及鼠的恶性肿瘤中 如头颈部鳞状细胞癌、 多发性黑色素瘤、 乳腺癌、 前列腺癌以及肺癌等有 Stat3的过度激活 及表达， 活化的 Stat3对肿瘤细胞的形成、 生长、 凋亡抑制等过程起着重要的调控作用， 提 示其调控异常与肿瘤发生发展密切相关。 因此阻断肿瘤细胞中癌基因 Stat3信号转导通路可 能起到治疗肿瘤的作用。
我们的前期工作表明， 虽然 siR A-Stat3可以显著下调 Stat3基因的表达， 通过诱导细胞 凋亡促进对肿瘤细胞的抑制效果，但是在哺乳动物细胞中， R Ai并不能完全阻断基因的表 达， 尤其是异常高表达的基因。 为进一步寻找最佳肿瘤治疗模式， 我们选择联合基因治疗 手段， 即在对 Stat3进行 RNA沉默的基础上， 增加了 GRIM-19基因的联合表达。
' GRIM- 19基因（ gene associated with retinoid-IFN-induced mortality 19 )是 Grim家族成员 之一，它是一种由 IFN-e联合 RA (Retinoic acid/维甲酸)诱导表达的新的细胞死亡调节因子。 GRIM-19是一种 16-kDa的蛋白， 分布在细胞核和胞桨中， 广泛表达于大多数组织， Zhang 等采用酵母双杂交文库筛选法证实了 GRIM-19可结合于原癌基因 Stat3并阻抑依赖 Stat3的 基因表达， 推测 Stat3的 TAD可能是 GRIM-19结合的直接位点， 与 Stat3绑定， 形成点状密集 结构共定位于核周， 从而抑制 Stat3的转录活性， 因此 GRIM-19是 Stat3的活性抑制物， 有人 将 GRIM-19列入为抗癌基因系列。 GRIM-19与 Stat3结合，并下调这些抗凋亡蛋白与其它前 凋亡调节子的结合可能会进一步损伤线粒体， 导致氧化磷酸化的中断， 从而加大凋亡的反 应进程。 研究证实在某些肿瘤组织中 GRIM-19表达抑制或表达缺失， 而在相邻的正常组织 中 GRIM-19表达却正常。显示 GRIM-19可能为维持正常组织或抑制癌症发生所必须。本研 究应用共表达 si-Stat3及 GRIM-19基因真核重组质粒， 既以 RNAi下调 Stat3表达， 同时提供外 源性 GRIM-19, 以期增强抑制前列腺癌的治疗目的。
. 目前， 肿瘤基因治疗的主要障碍之一是如何把抗肿瘤因子或其他治疗药物传递到肿瘤 组织内， 特别是深部肿瘤和转移瘤， 而不伤害正常组织。 许多报告指出沙门氏菌能在肿瘤 组织中繁殖的特异性与正常组织相比可超过 1000-10 000倍以上， 因此以减毒沙门氏菌用 于肿瘤治疗具有十分看好的应用前景。 以减毒沙门氏菌直接用于肿瘤治疗， 以其为运载体 运载抗肿瘤药物或肿瘤拮抗因子的研究不断增多， 但至今没有以沙门氏菌运载特异性 siR A表达载体， 并以肿瘤为靶向进行 R A沉默的研究报导。 我们首次以减毒鼠伤寒沙 门氏菌为 siR A-Stat3运载体， 并联合 Stat3拮抗基 因 GRIM-19, 以期实现特异性肿瘤的 综合靶向治疗。
本发明通过前列腺癌原位组织块移植模型等多种肿瘤模型， 模拟了从原发灶产生， 到 转移灶形成的完整的前列腺癌转移的病理生理过程， 并且釆用了减毒鼠沙门氏菌作为运载 体进行基因治疗， 对前列腺癌的治疗取得了显著效果。 经组织细菌克隆形成试验和荧光显 微镜观察， 减毒鼠沙门氏菌表现出优先在肿瘤组织中聚集和复制的特性。 以下一些因素可 能对减毒沙门氏菌优先在肿瘤组织中复制给予解释：
• (1)低氧环境不仅允许兼性厌氧菌生长，而且可以侵入并最后杀伤巨噬细胞和中性粒细 胞， 同时进入到巨噬细胞内的伤寒菌又更容易随巨噬细胞侵入到肿瘤组织内繁殖； （2)肿瘤 细胞内营养丰富， 快速生长的肿瘤组织内容易形成低氧区和肿瘤坏死区， 使肿瘤组织内环 境与正常组织不同； （3)最近研究发现在肿瘤细胞表面存在补体抑制因子， 此外， 不规则的 血管分布和肿瘤内的压力阻止抗体和裂解沙门氏菌的补体渗透， 肿瘤组织中很少发现有中 性粒细胞，这主要是因为肿瘤细胞和基质细胞分泌肿瘤坏死因子 β (TOF e )或其他免疫抑制 因子， 从而使中性粒细胞的激活和浸润受到抑制， 因此沙门氏菌在肿瘤组织中找到一个安全 的定居所； （4) 由于沙门氏菌的兼性厌氧特性使其既能在有一定氧含量的小转移性瘤细胞内 定居，也能在较大瘤组织内深度缺氧的中心进行定居，并呈现出溶解与杀伤肿瘤组织的功能；
(5)有报告指出， 敲除沙门氏菌基因组中的致病岛 2(SPI2)可使细 菌丧失抗肿瘤的活性， 已 知 SPI2是沙门氏菌在宿主内生长、在巨噬细胞和上皮细胞内存活所必需； (6)有报告指出当 沙门氏菌侵袭巨噬细胞以后本身亦能诱导细胞凋亡，这可能是使所携带外源基因释放的机制 之一。
本发明首次应用减毒沙门氏菌携带 Stat3或 Survivin特异性 siR A, 联合对 Stat3呈现 拮抗作用的 GRIM-19基因共表达系统，进行了抗多种肿瘤的体内外研究。以肿瘤局部注射 或小鼠尾静脉注射 Stat3序列特异性 siRNA与 GRIM-19联合表达载体系统有明显的协调治 疗作用。 结果表明减毒沙门氏菌具有噬肿瘤特性， 在肿瘤组织中繁殖的特异性与正常组织 相比可超过 1000倍以上， 15天后在正常组织中表达量明显降低， 并且该系统对实验性肿 瘤具有显著的治疗效果， 可以延长小鼠的生存时间。
附图说明
' 图 1 : 共表达质粒 pGRIM-19-Si-Stat3构建过程；
图 2 (A) pSHlSi-Stat3表达质粒酶切鉴定结果；
图 2 (B) 以pCXN2mycAGRIM-19表达质粒为模板扩增GRIM-19全长； ' 图 2 (C) Kpnl和 EcoRI双酶切鉴定 pMD18-T-GRIM-19质粒；
图 2 (D) Kpnl和 EcoRI双酶切鉴定 pGRIM-19重组质粒；
图 2 (E) 以 pHlSi-Stat3质粒为模板扩增 HI启动子和 Si-Stat3片断；
图 2 (F) Bgl ll和 Nrul双酶切鉴定 pMD18-T- HlSi-Stat3质粒；
图 2 (G) J)HlSi-Stat3重组质粒及 pGRIM-19-Si-Stat3重组质粒的鉴定
图 3 (A)细胞免疫化学染色显示: PGRIM-19-Si-Stat3组 GRIM-19表达增强；
图 3 (B)细胞免疫化学染色显示 Stat3表达减弱。
图 4 MTT实验检测各组质粒对肿瘤细胞增殖的抑制
图 5 Annexin V-CY3凋亡试剂盒检测共表达 pGRIM-19-Si-Stat3质粒可以诱导肿瘤细 胞凋亡；
图 6细菌在小鼠体内的分布分析
(A) Amp平板计数， 肿瘤组织细菌分布与其余脏器相比具有统计学意义 .
(B) 荧光显微镜下观察冰冻切片， 肿瘤组织内有大量的绿色荧光颗粒； 其余脏器仅见极 少的绿色荧光
图 7. 共表达质粒对裸鼠前列腺癌肿瘤具有生长抑制作用
图 8共表达质粒对裸鼠喉癌肿瘤具有生长抑制作用
图 9共表达质粒对裸鼠乳腺癌肿瘤具有生长抑制作用
图 10共^达质粒对裸鼠肺癌肿瘤具有生长抑制作用
图 11. 裸鼠前列腺癌肿瘤生长曲线
图 12减毒沙门氏菌携带重组质粒对小鼠原位肿瘤具有生长抑制作用
图 13 减毒沙门氏菌携带重组质粒对小鼠前列腺癌原位肿瘤具有生长抑制作用 图 14 前列腺癌原位肿瘤转移部位
' 图 15 酶谱分析表明共表达质粒组 MMP-2的表达量降低
图 16 转染后肿瘤组织 Stat3和 GRIM-19基因表达分析 图 17转染后肿瘤组织 Stat3和 GRIM-19蛋白表达分析
图 18 转染后肿瘤组织 Stat3下游基因转录表达分析
图 19 TUNEL试剂盒检测肿瘤组织凋亡
具体实施方式
一、 材料
1. 主要试剂
T4 DNA连接酶购自美国 Promega公司； BamH I, Hind III, Nru I, Kpn I等内切酶 购自大连宝生物工程公司； DNA purification system Wizard plus SV Minipreps购自美国 Promega公司； 胰化蛋白胨及酵母提取物购自 OXOID公司； 琼脂糖及琼脂粉购自大连宝 生物工程公司； DNA凝胶回收试剂盒购自大连宝生物工程公司； DNA Marker DL2000， 1Kb Ladder DNA marker均为 Takara公司产品。引物合成及测序由上海生工生物工程技术 服务公司完成。 溴化乙锭、 琼脂糖、 SDS、 TEMED、 丙烯酰胺、 N, N—二甲基双丙烯酰 胺、 MTT、 DTT、 DMSO、 PI、 PMSF购自美国 Sigma公司； 氨苄青霉素、 卡那霉素购自 北京鼎国公司； DEPC购自德国 Merk公司；高保真 Taq DNA聚合酶、 DAB、 Triton X- 100、 dNTP、 MMV逆转录酶及质粒提取和纯化试剂盒购自 Promega公司； R A酶及蛋白酶 K 购于美国 Ambion公司。Transwell细胞培养小室及 matrigel为美国 BD生物科学公司产品； Lipofetion2000及 Trizol为美国 Invitrogen公司产品；胰酶、 IMDM培养基为美国 Hyclone 公司产品； 新生牛血清购自杭州四季青公司； Annexin V-CY3凋亡试剂盒购自 SIGMA公 • 司； 其它常规化学试剂均为分析纯产品。
2. 主要仪器
PCR扩增仪 （GeneAmp, 美国）； 超净工作台 （YZ-875苏州净化设备厂）； .自动高压 蒸气消毒器（SONY, 日本）； 低温冰箱（-80°C SANYO, 日本）； 恒温水浴箱（江苏常州 国华仪器厂）； 高速低温离心机 （TOMY GRX-220, 日本）； 电热鼓风干燥箱 （上海实验仪 器总厂）； 电子天平 （OHAUS , 美国）； 电泳仪 （Bio-rad， 美国）； 凝胶成像系统 （上海天 能科技有限公司）;相差显微镜 (OLYMPUS,日本);全自动显微镜数码摄像系统 (OLYMPUS , 日本)； 酶标仪 (TAKARA, 日本)； 荧光显微镜 (OLYMPUS , 日本)； 流式细胞仪 (CC^LTER, 美国)； 去离子水装置 （日本)； PCR扩增仪（GeneAmp, 美国）； 可见、 紫外分光光度计及 分析工作站 （岛津 Shimadazi, 日本）； 加样器 （JECONS , 芬兰）； 自动 C02恒温培养箱 ( SANYO, 日本）； 蛋白质电泳装置及转移系统 （Bio-rad， 美国）。
3. 质粒和菌株
pSilencer™nGO 3.1-H1 siRNA表达载体购自 Ambion公司。 带有 U6启动子及 GFP的质粒 pGCsilencer™ U6/Neo/GFP购自上海吉凯化学公司。减毒沙门氏菌购自美国 Berna生物公司。 PMD-18T vector购自宝泰克公司。 pcDNA3.1表达载体及大肠杆菌 JM109购自美国 Invitrogen 公司。 '
4. 细胞株
人激素非依赖性高转移前列腺癌 PC-3M细胞株，人乳腺癌 MCF-7细胞株，人喉癌 HEP-2 细胞株， 人肺癌 A549细胞株， 人黑色素瘤 A375细胞株， 人肝癌 Bel-7402细胞株， 购自美国 ATCC公司。 小鼠前列腺癌 RM-1细胞株， 购自上海细胞所。 各细胞株用含 10 %新生牛血清 的 IMDM培养液在 37 °C、 5 % C02 的孵箱内培养， 以 0.25 %胰酶消化传代。
5. 实验动物
实验动物均购自中国医学科学院实验动物研究所。 BALB/C nu/nu雄性裸鼠， 150只， 4〜 6周龄， 体重 18〜20g， 饲养于恒定温度 （22-25 °C )、 恒定湿度 （40%-50% ) 的 SPF层流室 中， 经高压灭菌的标准饲料和水供动物自由食用。 C57BL6雄性近交系小鼠 50只， 鼠龄 8周, 体重 18〜20g ,词养环境恒温、 恒湿、 清洁、 无特殊病原体， 定期更换垫料， 清洁饮用水及 伺料供小鼠自由摄入。
二、方 法
―、 构建共表达 siRNA-Stat3和 GRIM-19基因的 pGRIM-19-Si-Stat3质粒。
1.1 pSHlSi-Stat3载体的构建
1.1.1 Stat3 siRNA模板寡核苷酸的设计
根据 genebank(NM 31500)人81&13基因^11^八的已知序列， 根据本研究室前期工作， 确 合适的靶位点 (2143-2162)， 寡核苷酸链序列为 GCAGCAGCTGAACAACATG, 合成编码 siRNA的 DNA模板：
正义链： 5， GATCCGCAGCAGCTGAACAACATGTTCAAGAGACATG
TTGTTCAGCTGCTGCTTTTTTGGAAA 3'
反义链： 5， AGCTTTTCCAAAAAAGCAGCAGCTGAACAACATGTCT
CTTGAACATGTTGTTCAGCTGCTGCG 3'
稀释寡核苷酸至终浓度 1 μ£/μ1。
1.1.2 Stat3 siRNA模板寡核苷酸退火
2μ1正义 siRNA模板寡核苷酸， 2 μΐ反义 siRNA模板寡核苷酸， 46μ1 l xDNA退火溶液； 加热混合物至 90Ό3分钟， 冷却至 37°C， 孵化 1小时。
1.1.3连接
连接退火的 Stat3 siRNA模板寡核苷酸到线性化的 p¾/encerTMneo 3.1-H1 siRNA表达载 体。 用 45μ1去核酸酶水稀释 5μ1退火的 Stat3 siRNA模板寡核苷酸， 终浓度为 8 μ§/μ1; 建立 10 μΐ连接反应体系： 设 1个阴性对照， 4°C过夜。 反应体系如下： 稀释退火的 siRNA模板寡核 苷酸 Ι μΐ; 去核酸酶水 6μ1; 10xT4 DNA连接酶缓冲液 Ι μΐ; p&'/eMCerTMneo 3.1-H1 siRNA载体 Ι μΐ; Τ4 DNA连接酶 （5υ/ μ1) 1 μ1。
1.1.4连接产物转化大肠杆菌
1.1.4.1 大肠杆菌 JM109感受态细胞的制备
将宿主菌 JM109接种于 LB固体培养基上， 培养过夜。 次日， 从 LB 平板上挑取单菌 落， 接种于 3ml LB 液体培养基中， 37°C下振荡培养 12 小时左右， 直至对数生长后期。 将该菌悬液以 1 : 50 的比例接种于 100ml LB 液体培养基中， 37°C振荡培养 2-3 小时至 OD600达 0.45」0.55。 冰浴菌液 10分钟， 4000g离心 lOmin收集 50ml菌体。 10ml冰预冷 0.1M CaCl2重悬。 2ml冰预冷 0.1M CaCI2重悬沉淀， 置于 4°C， 16h后取 ΙΟΟμΙ用于转化 试验， 或加入甘油至终浓度 10%， -70°C保存备用。
1.1.4.2重组质粒的转化
ΙΟΟμΙ感受态细胞加入连接反应物 1 μ1， 混匀后冰浴 30min。 42°C水浴 90s， 然后迅速 移入冰浴中放置 2min。 向管中加入 LB液体培养基 800μ1， 37°C温和震荡培养 lh。 用无菌 弯头玻璃铺菌器将 200μ1菌液铺于含 Amp的 LB平板 （5(^g/mL) 表面， 待表面液体吸收 后， 倒置平皿 37Ό培养 16-20h。 .
1.1.5 阳性重组克隆的筛选 (碱裂解法）
挑取生长于选择培养板的单菌落， 加到含氨苄青霉素 （lOO g/mL) 的 5ml LB液体培 养基中， 37°C振荡培养过夜。碱裂解法小量制备质粒， 过程如下： 将 1.5ml培养物倒入 Ep 管中， 7 000g离心数秒， 吸去培养液， 重复前一步骤， 使细菌沉淀尽可能干燥。 将细菌沉 淀重悬于 100μl用冰预冷的溶液solution I (50mmol/L葡萄糖、 25mmol/L Tris.Cl pH 8.0、 10mmol/L EDTA pH8.0)中， 剧烈震荡， 室温放置 5min。 力 Β 200μ1 新配制的溶液 solution II (0.2mol/L NaOH, 1%SDS) , 盖紧管口， 快速颠倒离心管数次， 以混合 '内容物， 然后将离心管放置于冰上 5min。 加冰预冷的溶液 solutionlll (5mol/L乙酸钾， 11.5ml冰乙 酸， 28.5ml dd¾0)，盖紧管口，颠倒离心管 20sec，将管置于冰上 5min。 12 000g离心 10min， 将上清转移至另一 Ep管中，加等量酚：氯仿 (25 : 24)，振荡混匀， 4°C 12， 000g离心 2min， 将上清转移至另一 Ep管中。加两倍体积的乙醇，振荡混合，于室温放置 2min。离心 15min， 弃上清， 再加 70%乙醇 lml以洗涤双链 DNA， 离心 10min， 弃上清， 室温干燥沉淀， 然后 加含 RNA酶的 dd¾0 (20μβ/ιη1)30μ1重新溶解核酸沉淀。
1.1.6重组质粒的鉴定
将 p&7ewcerTMneo 3.1-Hl-Stat3 siRNA质粒 (以下简称 pSHlSi-Stat3)用限制性内切酶 (BamH I、 Hind III) 进行双酶切， 反应条件如下： 质粒 8μ1; BamH I Ι μΐ; Hind III Ι μΐ; ΙΟχΗ Buffer 2μ1; ddH20 8μ1; 混合后 37°C水浴 2h。 取 5μ1酶切产物在 2%琼脂糖凝胶中电 泳， 释放出目的片段的质粒为阳性重组质粒。
阳性克隆经上海生工生物工程技术服务有限公司自动测序仪进行 cDNA序列测定。
1.2 pcDNA3.1- GRIM-19表达载体的构建
1.2.1 引物设计
根据 Genebank AF286697号编码 GRIM19全长序列由软件设计引物：
P1 : 5 ' -GAGAATTCATGGCGGCGTCAAAGG-3 ' (EcoR I )
P2: 5 ' -GAAAGCTT CAGGGCCTACGTGTACCACAT-3 ' (Hind III) .
1.2.2扩增人 GRIM-19全长序列
以正常人胎盘组织为模板进行 PCR， 扩增人 GRIM-19全长序列：
反应循环条件为： 94°C预变性 5min; 94 °C 30 Sec, 62 °C 45 Sec, 72°C lmin， 30个 循环； 72°C延伸 10min。
PCR产物电泳， 用 QIAquick Gel Extration Kit进行回收： 用手术刀片从琼脂糖凝胶中 切下含 DNA片段的凝胶， 放入 Ep管中， 将其捣碎； 向管内加入相当于凝胶体积三倍的溶 胶液， 50°C水浴 10min， 其间轻弹 Ep管壁数次， 使凝胶完全溶化； 将融化了的胶加入回 收柱中， 13 OOOrpm离心， lmin; 向柱中再次加入 750μ1溶胶液， 13 OOOrpm离心. lmin， 弃 溶胶液； 向柱中加入 500μ1 ΡΕ溶液， 13 OOOrpm离心 lmin， 弃去 PE; 再次 13 OOOrpm离心 lmin, 弃去残余的 PE, 室温干燥 lOmin; 向柱中加入 30μ1无菌水， 室温放置 lOmin, 充分 溶解 DNA; 13 OOOrpm离心 lmin, 得到 30μ1 DNA溶液； 取一部分回收的 DNA在 1.5%琼 脂糖凝胶中电泳， 以验证回收效率。
1.2.3 pMD18-T-GRIM-19重组质粒制备
于 0.5ml Ep管中， 加入下述试剂： 回收 DNA片段 4.5μ1; pMD18-T vector 0.5μ1 (片段 量： 载体量 =3〜8： 1)； solution I 5μ1; 将其混匀后， 于 16°C 水浴过夜。 转化， 鉴定并测 序。
1.2.4连接反应
用 Kpnl和 EcoRI分别酶切 pMD18-T- GRIM-19重组质粒及 pcDNA3.1载体， 进行连 接， 构建 pcDNA3.1- GRIM-19重组质粒 （以下略称为 pGRIM-19)。 将连接产物转化大肠 杆菌 JM109, 用含有 Amp的培养板筛选阳性克隆。
1.2.5重组质粒的鉴定
从培养板中挑取单一菌落， 提取质粒。 用 Kpnl和 EcoRI进行双酶切鉴定。
1.3共表达 siRNA-Stat3及 GRIM-19基因真核重组质粒的构建 1.3.1 引物设计：
依据引物设计原则，参照 pcDNA3.1图谱设计引物 P3和 P4，并在其上下游分别引入 Bgl II 和 Nru I酶切位点： 长度为 213bp。
P3： 5'CGAGATCTGAATTCATATTTGCATGTCGCTATG3 '
P4: 5' TCGCGAAGGAAACAGCTATGACCATGATTAC 3'
1.3.2扩增 HI启动子及 siR A-Stat3序列
以 pHlSi-Stat3载体为模板进行 PCR, 扩增 HI启动子及 siRNA-Stat3序列：
反应循环条件为： 94 °C预变性 5min; 94 °C 30 Sec, 55 °C45Sec, 72 °C lmin, 30个循 环； 72 °C延伸 10min。 PCR产物电泳， 特异带 QIAquick Gel Extration Kit回收。 与 pMD18-T 载体迸行连接，.转化并测序。
1.3.3连接反应
用 Bgl II和 Nru I分别酶切 pMD18-T- HI Si-Stat3 重组质粒， pcDNA3.1 质粒及 pcDNA3.1- GRIM-19重组质粒， 并进行连接， 构建 pcDNA3.1- HI Si-Stat3重组质粒（以下 略称为 pHlSi-Stat3 )及 pcDNA3.1- HI- Stat3-GRIM-19 (以下略称为 p GRIM-19-Si-Stat3 ) 重组质粒。 将连接产物转化大肠杆菌 JM109, 用含有 Amp的培养板筛选阳性克隆。
1.3.4鉴定
从培养板中 ¾ ^取单一菌落， 提取质粒。
①用 Bgl II和 Nm I进行双酶切鉴定。
②用 Kpn I和 EcoR I进行双酶切鉴定。
二.将重组质粒电转化入减毒沙门氏菌
2.1 电转化感受态的制备
用枪头挑取单克隆减毒沙门氏菌菌落，投入盛有 5ml LB液体培养基的 50ml离心管中。 (同时做培养基和枪头的空白对照） 37°C， 220rpm, 培养 14-16个小时。 第二天， 以 1 : 100的比例将这 5ml菌液倒入 500ml LB液体培养基中， 37°C， 220rpm, 振摇 2-3h， 每半 小时测一次 OD值， 当 OD值达到 0.3-0.4时， 停止培养。 将菌液在冰上预冷 30分钟， 随 后将菌液分装到 100ml预冷的离心管中， 4°C， 4200rpm离心 10min。弃上清， 离心管中加 入 lmmol/L冰预冷的 HEPES (pH7.0) lml,使沉淀重悬后，再加入 lmmol/L冰预冷的 HEPES (pH7.0) 49ml, 4°C， 4200rpm离心 10min。 重复 2次。 弃上清， 往离心杯中加入少量 10% 甘油 (灭菌， 预冷)， 重悬菌体， 再加满 10%甘油， 4°C， 4200rpm, 离心 10min。 弃上清， 每个离心管中加入 500μΐ 10%的甘油，使沉淀悬浮后，将菌液在冰上以 300W/管分華于 1.5ml 的离心管中， 投入液氮 lmin, -80°C保存。
2.2 电转化步骤
取 1 μΐ纯化后的重组质粒于 1.5ml的离心管中，将其和 0.1CM的电极杯一起置于冰上预 冷。 将 lOOul解冻的感受态细胞转移至此 1.5ml 的离心管中， 小心混匀， 冰上放置 10min。 打开电转仪， 调至 Manual, 调节电压为 2. 5 kV， 25μΡ, 200Ω。将此混合物转移至已预冷的 电极杯中， 轻轻敲击电极杯使混合物均勾进入电极杯的底部； 将电极杯推入电转化仪， 按 一下 pulse键， 听到蜂鸣声后， 向电击杯中迅速加入 ΙΟΟΟμΙ的 LB液体培养基， 重悬细胞后， 转移到 1.5ml的离心管中。 37°C， 250rpm复苏 lh。 取 20ul转化产物加 160 μΐ LB涂板， 7°C过 夜培养， 次日査看转化结果。 其余菌液加 1 : 1的 30%的甘油后混匀一 80°C保存。
3. 体外研究
应用真核细胞转染技术转染 PC-3M， RM-1细胞株， MCF-7细胞株， HEP-2细胞株， A549 细胞株， Bel-7402细胞株， A375细胞株；应用半定量 RT-PCR, 免疫细胞化学染色和 Western blot检测重组质粒及相关基因的基因及蛋白水平的表达； 应用流式细胞术检测细胞周期及 凋亡峰； 应用 Annexin V-CY3凋亡试剂盒检测细胞早期凋亡； 建立稳定转染 pGC-Si-Stat3质 粒的细胞系， 应用酶谱分析检测细胞 MMP-2的表达； 应用 Transwell小室侵袭实验检测细胞 侵袭能力。
4. 体内实验
复制裸鼠移植前列腺癌模型， 乳腺癌模型， 喉癌模型， 肝癌模型， 肺癌模型及黑色素 瘤模型并应用显微外科技术建立小鼠前列腺癌原位组织块移植模型， 应用减毒沙门氏菌局 部注射或尾静脉注射将重组质粒带入瘤体内观察抑瘤作用；应用 TUNEL试剂盒检测肿瘤细 胞凋亡； 应用 Northen blot和 Western blot检测相关基因的基因与蛋白水平的表达； 应用免 疫组织化学技术检测 Stat3 GRIM-19及 Ki-67的表达； 应用酶谱分析检测组织 MMP-2的表 达。
4.1裸鼠肿瘤移植瘤模型的建立
将肿瘤细胞用胰蛋白酶消化成细胞悬液， 经离心， 将细胞团块用 IMDM培养液吹散， 经台盼蓝实验 i 实细胞活力≥95%，最终细胞浓度为 2χ107个 /ml，取瘤细胞 2χ10δ个（0.1ml) 接种于裸鼠左侧背部近前肢皮下组织内。 隔天用游标卡尺进行测量， 待移植瘤长至直径 5mm时将裸鼠随机分组进行实验。
4.1.1 治疗过程
每组 5只裸鼠， 分为 5组， 分组如下： mock组， pHlSi-Sc ble组， pHlSi-Stat3组， pGRIM-19组， pGRIM-19-Si-Stat3组。 每组肿瘤局部注射携带相应组质粒的减毒沙门氏菌， 分 2点注射， 每点 50 μ1， 细菌浓度为 10 ίΙι/100μ1
4.1.2治疗效果观察指标
4.1.2.1全身 '膚况
观察实验动物的活动、 进食和体重。
4.1.2.2肿瘤体积的变化
治疗后隔天一次用精密卡尺测量肿瘤最大长径和短径， 计算肿瘤体积。 体积 = 0.52χ 长径 X短径 2。 .
4.1.2.3病理学检査
接种后当对照组肿瘤长至足够大时统一拉颈处死所有小鼠切除肿块称重， 分别行病理 学检査 （10%甲醛固定后， HE染色）。 Stat3 GRIM-19 Ki67免疫组织化学染色。 结果判 定： 以在肿瘤细胞胞浆或细胞间质内出现棕黄色颗粒， 且着色强度高于背景非特异性染色 者判定为阳性。
4.1.3肿瘤细胞凋亡检测 （TUNEL检测）
4.1.4肿瘤组织基因及蛋白质水平检测
提取肿瘤组织 RNA及蛋白， 半定量 PCR分析 Stat3 GRIM-19在基因水平'的表达， Western blotting分析 Stat3 GRIM- 19在蛋白水平的表达。
4.2原位移植瘤建立
4.2.1 小鼠背部皮下移植瘤模型的建立
C57BL6近交系小鼠 10只， 用强力碘消毒小鼠背部， 取鼠源性肿瘤细胞溶液接种于小 鼠背部皮下， 接种细胞浓度为 2χ107个 /ml， 每只接种 100μ1。 隔日观察肿瘤生长情况。
4.2.2小鼠前列腺癌原位移植瘤模型及肝癌原位移植瘤模型的建立
用于外科原位移植 （SOI) 的肿瘤组织来源于上述鼠背皮下肿瘤模型。 状态较好的肿 瘤组织靠近外周， 色白有光泽， 质韧有弹性， 不易破碎。 将取出的瘤块置于低温、 无菌的 0.9.生理盐水中， 在 10倍显微镜下切割成直径 1.5mm大小的肿瘤块。 C57BL6近交系小 鼠用戊巴比妥钠 （60mg/Kg) 麻醉， 取仰卧位， 强力碘常规消毒， 铺无菌洞巾。 下腹部正 中切口长约 1.5-2.0cm, 显露腹腔。将膀胱向上提起， 并用无菌棉签抵住膀胱腹恻壁， 暴露 腹侧前列腺。 镜下剥离前列腺腹侧筋膜， 用尖刀分离两腹侧叶， 将肿瘤块置于两腹侧叶间 形成的缝隙中，'用 9-0可吸收线缝合两腹侧叶及其表面筋膜， 使肿瘤块被严密包埋。 恢复 脏器原来的解剖位置， 用 5-0肠线全层连续缝合关腹， 待鼠苏醒后回笼。 术中操作要求轻 柔、 准确， 避免损伤正常组织和肿瘤组织。
4.2.3实验动物分组
每种肿瘤共有 40只 C57BL6小鼠进行原位移植。 分组同上。
4.2.4肿瘤生长和转移的评价
C57BL6小鼠濒死或处死时采用过量麻醉方法处死,手术显微镜下观察原位移植的肿瘤 生长情况和淋巴结转移情况,取肝、肺、脾、 肾、 椎体、 淋巴结和可疑的骨骼进行福尔马林 固定,石蜡包埋,切片 , HE染色后光镜下观察有无转移灶。
细胞数目。
结 果
1 . 经测序及酶切鉴定， 成功构建了共表达 siRNA-Stat3和 GRIM-19基因的 pGRIM-19-Si-Stat3质粒。
1.1 pSHlSi-Stat3载体的构建
用 BamH I和 Hind III双酶切 pSHlSi-Stat3表达质粒， 1.5%琼脂糖凝胶电泳显示分别出 现 66bp和 4.31cb的酶切片断， 结果见图 1。 DNA测序结果证实 Si-Stat3和 Si-Scramble片 段与
Figure imgf000013_0001
3.1-H1 siRNA表达载体连接反应正确， 结果见图 2 (A)。
1. 2 pGRIM-19表达载体的构建
' 如图 2 (B)， 以 pCXN2mycA GRIM-19质粒为模板作 PCR， 得到 GRIM-19的全长， 为 435bp。如图图 2 (C)所示， 将重组质粒用 Kpnl和 EcoRI作双酶切， 可得到两个片段， 大片段为载体片段， 大小为 2692bp; 小片段为 GRIM-19片段， 大小为 435bp。测'序结果与 Genebanl (NM_015965 )序列完全一致， 同时两端已成功地加入了限制性酶切位点。
将重组质粒用 Kpnl和 EcoRI作双酶切， 可得到两个片段， 大片段为载体片段， 大小为 5428bp; 小片段为 GRIM-19片段， 大小为 435bp， 结果见图 2 (D)。
1.3 PHlSi-Stat3表达载体的构建
以 pHlSi-Stat3质粒为模板扩增 HI启动子和 Si-Stat3片断，所得产物大小为 204bp,结 果见图 2 (E)。 将重组质粒用 Bgl ll和 Nral双酶切， 大片段为载体片段， 大小为 2692bp; 小片段为 HlSi-Stat3片段， 大小为 204bp,结果见图 2 (F)。 DNA测序结果证实 HlSi-Stat3 片段与 pcDNA3.1表达载体连接反应正确。
1.4 PHlSi-Stat3重组质粒及 PGRIM-19-Si-Stat3重组质粒的鉴定
如图 2 (G)所示， 将重组质粒用 Bgl ll和 Nm l双酶切， 大片段为载体片段， 大小分 别为 5428bp和 5860bp; 小片段为 HlSi-Stat3片段， 大小为 204bp。 将 pGRIM-19-Si-Stat3 重组质粒用 Kpnl和 EcoRI作双酶切，可得到两个片段，大片段为载体片段，大小为 5428bp; 小片段为 GRIM-19片段， 大小为 435bp。
2. 体外实验证明重组质粒对肿瘤细胞具有增殖抑制及促进凋亡的作用。
2. 1 PC-3M细胞免疫化学染色
细胞免疫化学染色结果如图 3显示， PGRIM-19-Si-Stat3组 GRIM-19表达增强， 细胞 浆、胞核中均可见颗粒样分布的深浅不一的棕黄色物质； PGRIM-19-Si-Stat3组 Stat3 mRNA 表达减弱。
2.2重组质粒转染对 PC-3M细胞的生长抑制作用
2.2.1 相差显微镜下观察
对照组细胞贴壁生长， 状况良好， 多为梭形， 大小适中， 核仁清晰， 可见核分裂相， 细胞折光性好，细胞增殖旺盛； pHlSi-Stat3组和 pGRIM-19组随转染时间的延长细胞生长缓 慢， 形态不规则， 细胞皱缩， 颗粒增多， 细胞碎片增加； pGRIM-19-Si-Stat3组可见细胞内 颗粒物质增加， 失去原有形态， 细胞核固缩， 裂解成质膜包绕的碎片， 细胞数目较对照组 明显减少， 部分细胞漂浮在培养液中。
2.2.2 MTT实验检测各组质粒对 PC-3M细胞增殖的抑制
如图 4所示， 三组重组质粒均能显著性的抑制 PC-3M细胞增殖， 抑制率分别为 51.7%， 42.3%和 20.9%, 与对照组相比，差异有显著性 (P<0.01);而脂质体对照组和 pHlSi-Scramble 对照组细胞体外增殖活性无明显差异 （P>0.05); 共表达 siRNA-Stat3及 GRIM-19基因真核 蕙组质粒与单用相比抑制作用明显增强 （ <0.05)。
2. 3重组质粒可诱导 PC-3M细胞凋亡
2.3.1应用流式细胞术检测 PC-3M细胞凋亡峰
重组质粒转染组 PC-3M 细胞于转染后 72h 较对照组出现了明显的细胞凋亡， (P<0.01)(表 1)， 在 G1期前出现的亚二倍峰即为凋亡峰。
表 1 共表达 PGRIM-19-Si-Stat3质粒诱导肿瘤细胞凋亡及对细胞周期的影响
(# P<0.05 versus pHlSi-Scram ble; *P<0.05 versus pHl Si-Scramble )
Apoptotic cells G0-G1 S
Group (η =·3)
(%； mean ± SD) (%； mean ± SD) (%； mean ± SD)
mock 0.3±0.95 40.2±2.02 57.1±7.11
pHl Si-Scramble 1.8±0.21 39.7±2.31 49.5±6.02
pGRIM-19 24.4±1.89* 55.4±2.95* 43.2±4.56
pHlSi-Stat3 33.9±2.05* 63.1±2.56* 33.9±4.33#
■ pGRIM-19-Si-Stat3 45.5±1.73* 67.8±4.28* 35.7±3.29#
2.3.2 Annexin V-CY3凋亡试剂盒检测细胞凋亡
如图 5所示， 通过激光共聚焦显微镜可见脂质体对照组及 pHlSi-Scramble组 PC-3M 细胞呈均一绿色，表明转染重组质粒的 PC-3M细胞发生早期凋亡。 pGRIM-19，pHlSi-Stat3, pGRIM-19-Si-Stat3转染组呈不同程度的 annexin V阳性表达， 凋亡细胞被 AnnCy3与 6-CF 红绿两种荧光标记而呈现黄色。
3. 体外实验证明减毒沙门氏菌能够携带重组质粒进入深部肿瘤组织， 并对肿瘤细胞具有 增殖抑制及促迸凋亡的作用。
3.1细菌在小鼠体内的分布
无菌状态下取肿瘤、 脾脏、 肝脏和肺脏各 100mg， 研磨， 以冷的 PBS 5倍稀释后， 接 种到含 Amp 的 LB平皿， 37 °C过夜， 次日计算形成的单个克隆数 (图 6A)。 肿瘤组织形 成单个克隆数 1890±98.2个； 脾脏组织形成 10±3. 5个； 肝脏组织形成 8±4. 3个； 肺脏 组织形成 2±0. 3个。 肿瘤与其余脏器相比具有统计学意义 ( <0.01)。
' 取各脏器制成冰冻切片， 荧光显微镜下观察， 肿瘤组织内有大量的绿色荧光颗粒， 代 表细菌的分布； 其余脏器仅见极少的绿色荧光 （图 6Β)。
3.2重组质粒对裸鼠肿瘤及小鼠原位肿瘤生长的抑制 用 PC-3M细胞接种裸鼠， 第 12天全部出瘤， 平均体积为 97.5±11.36mm3， 分组治疗后， 裸鼠平均体重， 平均肿瘤重量和体积见表 2， 肿瘤生长抑制见图 7， 肿瘤生长曲线见图 11。 共表 达质粒组与对照组及单独应用组明显缩小， 具有统计学意义（ <0.01 )。 平均体重未见统计学差 O ).05)。 B侯癌 （图 8 ) ， 乳腺癌 （图 9) ， 肺癌 （图 10) 裸鼠皮下移植瘤模型均证明 PGRIM-19-Si-Stat3重组质粒具有协同作用， 与对照组相比平均肿瘤重量和体积均降低。
表 2各组裸鼠平均 M， 平均肿瘤 fig和 M¾比较
尸<0.05 versus pHlSi-Scramble; *P<0.05 versus pHlSi-Scramble
mean weight of mean weight of mean volume of the nude mice(g) tumor (g) tumor (mm3)
mock 26.52±3.06 2.72±0.53 1284.41±289.37
pHl Si-Scramble 25.36±2.58 2.18±0.64 1100.62±235.21 pGRIM-19 24.31±2.36 1.22±0.25 357.27±70.07* pHlSi-Stat3 25.12±2.77 0.94±0.3l" 271.46±65.11* pGRIM-19-Si-Stat3 27.17±2.92 0.26±0.11* 84.19±24.33*
成功建立小鼠前列腺癌原位组织块移植瘤模型及肝癌模型， 并用尾静脉注射减毒沙门 氏菌携带重组质粒到达深部肿瘤进行治疗 (图 12， 13 , 表 3)， 模型组可以发生各种脏器的转 移， 很好的模拟了晚期癌症的进展与转移 （图 14) 。 肿瘤细胞发生凋亡， 转移部位低于对 照组， 具有统计学差异 （表 4)。
表 3各组小鼠平均 £， 平均肿瘤 和 比较
(^P<0.01 versus pHlSi-Scramble)
mean weignt of mean weight mean volume
the nude mice(g) of tumor (g) of tumor (mm3)
mock 26.52±3.06 2.41±0.77 1673.3±488.61
pGC-Si-Scramble 25.36±2.58 1.98±0.58 1259.56±485.38
pGC-Si-Stat3 24.31±2.36 0.59±0.33* 345.78±183.17* 表 4各组小鼠肿瘤转移部位比较
Spleen Liver Kidney Lung Bladder lymphonode
(+) (+) (+) (+) (+) (+) mock 2 1 1 5 8 8
pGC-Si-Scramble 1 2 0 3 6 8
pGC-Si-Stat3 0 0 0 1 1 2
3.3 转染后肿瘤组织 MMP-2的表达分析
由酶谱分析表明 （图 15 )， 脂质体对照组和 pHlSi-Scramble对照组肿瘤组织 MMP-2 的活性明显高于 pGRIM-19, pHlSi-Stat3及 pGRIM-19- Si-Stat3转染组， P<0.05。 并且脂 质体对照组和 pHlSi-Scramble对照组病理学检查可见肌肉浸润及淋巴结转移，转染组未见 转移。
3.4转染后肿瘤组织 Stat3和 GRIM-19基因表达分析
RT-PCR结果显示， pGRIM-19组和 pGRIM-19-Si-Stat3组 GRIM-19 mRNA表达增强（图 •16)； PHlSi-Stat3组和 pGRIM-19-Si-Stat3组 Stat3 mRNA表达减弱 （图 17)。 3.5 转染后肿癉组织 Stat3和 GRIM-I9蛋白表达分析
图 26显示 Western blot分析结果， pGRIM-19组和 pGRIM-19-Si-Stat3组 GRIM-19蛋 白表达增强； pHlSi-Stat3组和 pGR!M-19-Si-Stat3组 Stat3 蛋白表达减弱。
3.6转染后肿瘤组织相关因子蛋白表达分析
Western blot 分析结果显示 （图 18 )， pGRIM-19 组， pGRIM-19-Si-Stat3 组和 pGRIM-19-Si-Stat3组 Bcl-2， cyclin Dl， c-Myc, VEGF蛋白表达减弱，其中 pGRIM-19-Si-Stat3 组减弱更明显。
3.7 转染后肿瘤组织细胞凋亡的检测
用苏木素 -伊红 （HE ) 染色普通光镜下观察： pGRIM-19， PHlSi-Stat3 及 PGRIM-19-Si-Stat3转染组肿瘤组织细胞核固缩碎裂、 呈蓝黑色、 胞浆呈淡红色（凋亡细 胞）， 正常细胞核呈均匀淡蓝色或蓝色， 坏死细胞核呈很淡的蓝色或蓝色消失。
原位细胞凋亡捡测可见 pGRIM-19， PHlSi-Stat3及 pGRIM-19- Si-Stat3转染组肿瘤组织细 胞核中有棕黄色颗粒， 提示有凋亡细胞（图 19) 。
实施例
将本发明与 PDS配制成 3 X 108/ml的溶液， 装入 lml安瓶中。 使用时， 每人皮下或肌 肉注射 lral。

Claims

权利要求书
1、 一种运载重组质粒的减毒沙门氏菌， 它是由下列步骤得到的- 一、构建共表达 siRNA-Stat3和 GRIM-19基因的 pGRIM-19-Si-Stat3质粒：
1.1 pSHlSi-Stat3载体的构建
1.1.1 Stat3 siRNA模板寡核苷酸的设计
根据 genebank(NM 31500)人 Stat3基因 mRNA的已知序列， 确定合适的靶位点 (2143-2162), 寡核苷酸链序列为 GCAGCAGCTGAACAACATG, 合成编码 siRNA的 DNA模 板- 正义链： 5， GATCCGCAGCAGCTGAACAACATGTTCAAGAGACATG
TTGTTCAGCTGCTGCTTTTTTGGAAA 3'
反义链： 5 ' AGCTTTTCC AA AAAAGC AGC AGCTGAAC AAC ATGTCT
CTTGAACATGTTGTTCAGCTGCTGCG 3'
稀释寡核苷酸至终浓度 1 μ§/μ1;
1.1.2 Stat3 siRNA模板寡核苷酸退火
1.1.3连接
连接退火的 Stat3 siRNA模板寡核苷酸到线性化的 pS /ewcerTMneo 3.1-H1 siRNA表达载 体， 将连接产物转化入大肠杆菌， 筛选阳性重组克隆；
1.1.4重组质粒的鉴定
. 将 p5¾7e"cerTMneo 3.1-Hl-Stat3 siRNA质粒，简称 p5Hl Si-Stat3，用限制性内切酶 BamH I、 Hind III进行双酶切， 反应条件如下： 质粒 8μ1; BamH I Ι μΐ; Hind III Ι ΐ; ΙΟχΗ Buffer 2μ1; dd¾0 8μ1; 混合后 37°C水浴 2h。 取 5μ1酶切产物在 2%琼脂糖凝胶中电泳 ·， 释放出 目的片段的质粒为阳性重组质粒；
阳性克隆经自动测序仪进行 cDNA序列测定；
1.2 pcDNA3.1- GRIM-19表达载体的构建
1.2.1 引物设计
根据 Genebank AF286697号编码 GRIM19全长序列由软件设计引物：
P1 : 5 ' -GAGAATTCATGGCGGCGTCAAAGG-3 ' (EcoR I )
P2: 5 ' -GAAAGCTT CAGGGCCTACGTGTACCACAT-3 ' (Hind III)
1.2.2扩增人 GRIM-19全长序列
以正常人胎盘组织为模板进行 PCR, 扩增人 GRIM-19全长序列：
反应循环条件为： 94°C预变性 5min; 94 °C 30 Sec, 62°C45Sec， 72°C lmin， 30个 循环； 72°C延伸 lOmin;
PCR产物电泳， 用 QIAquick Gel Extration Kit进行回收；
1.2.3 pMD18-T-GRIM-19重组质粒制备
于 0.5ml Ep管中， 加入下述试剂： 回收 DNA片段 4·5μ1; pMD 18-T vector 0.5μ1 (片段 量： 载体量 =3〜8： 1)； solution I 5μ1; 将其混匀后， 于 16°C水浴过夜， 转化， 鉴定并测 序；
1.2.4连接反应
用 Kpnl和 EcoRI分别酶切 pMD18-T- GRIM-19重组质粒及 pcDNA3.1载体， 进行连 接， 构建 PCDNA3.1- GRIM-19重组质粒（以下略称为 pGRIM-19)， 将连接产物转化大肠 杆菌 JM109, 用含有 Amp的培养板筛选阳性克隆；
1.2.5重组质粒的鉴定
从培养板中挑取单一菌落， 提取质粒。 用 Kpnl和 EcoRI进行双酶切鉴定；
1.3共表达 siRNA-Stat3及 GRIM-19基因 p GRIM-19-Si-Stat3真核重组质粒的构建
1.3.1 引物设计：
依据引物设计原则，参照 pcDNA3.1图谱设计引物 P3和 P4,并在其上下游分别引入 Bgl II 和 Nru l酶切位点： 长度为 213bp;
P3： 5'CGAGATCTGAATTCATATTTGCATGTCGCTATG3 '
P4: 5' TCGCGAAGGAAACAGCTATGACCATGATTAC 3'
1.3.2扩增 HI启动子及 siRNA-Stat3序列
以 pSHlSi-Stat3载体为模板进行 PCR, 扩增 HI启动子及 siRNA-Stat3序列：
反应循环条件为： 94 °C预变性 5min; 94 °C 30 Sec, 55 °C45Sec, 72 °C lmin, 30个循 环； 72 °C延伸 10min。 PCR产物电泳， 特异带 QIAquick Gel Extration Kit回收， 与 pMD18-T 载体进行连接，，转化并测序；
1.3.3连接反应
用 Bgl II和 Nru I分别酶切 pMD18-T- HI Si-Stat3 重组质粒， pcDNA3.1 质粒及 pcDNA3.1- GRIM-19重组质粒， 并进行连接， 构建 pcDNA3.1- HI Si-Stat3重组质粒（以下 略称为 pHlSi-Stat3 )及 pcDNA3.1- HI- Stat3-GRIM-19 (以下略称为 p GRIM-19-Si-Stat3 ) 重组质粒。 将连接产物转化大肠杆菌 JM109, 用含有 Amp的培养板筛选阳性克隆；
1.3.4鉴定
从培养板中挑取单一菌落， 提取质粒；
①用 Bgl II和 Nra I进行双酶切鉴定；
②用 Kpn I和 EcoR I进行双酶切鉴定；
二.将重组质粒电转化入减毒沙门氏菌
2.1 电转化感受态的制备
用枪头挑取单克隆减毒沙门氏菌菌落，投入盛有 5ml LB液体培养基的 50ml离心管中； (同时做培养基和枪头的空白对照） 37°C， 220rpm, 培养 14-16个小时； 第二天， 以 1 : 100的比例将这 5ml菌液倒入 500ml LB液体培养基中， 37。C， 220rpm, 振摇 2-3h， 每半 小时测一次 OD值， 当 OD值达到 0.3-0.4时， 停止培养。 将菌液在冰上预冷 30分钟， 随 后将菌液分装到 100ml预冷的离心管中， 4°C， 4200rpm离心 10min。弃上清， 离心管中加 人 lmmol/L冰预冷的 HEPES (pH7.0) lml,使沉淀重悬后，再加入 lmmol/L冰预冷的 HEPES (pH7.0) 49ml, 4°C， 4200rpm离心 lOmin; 重复 2次； 弃上清， 往离心杯中加入^、量 10% 甘油 (灭菌， 预冷)， 重悬菌体， 再加满 10%甘油， 4°C， 4200rpm， 离心 10min。 弃上清， 每个离心管中加入 500W 10%的甘油，使沉淀悬浮后，将菌液在冰上以 300 /管分装于 1.5ml 的离心管中， 投入液氮 lmin, -80°C保存；
2.2 电转化步骤
' 取 1 μΐ 纯化后的重组质粒于 1.5ml的离心管中，将其和 0.1CM的电极杯一起置于冰上 预冷；将 lOOul解冻的感受态细胞转移至此 1.5ml 的离心管中，小心混匀，冰上放置 lOmin; 打开电转仪， 调至 Manual, 调节电压为 2. 5 kV， 25μΡ, 200Ω; 将此混合物转移'至已预冷 的电极杯中， 轻轻敲击电极杯使混合物均匀进入电极杯的底部； 将电极杯推入电转化仪， 按一下 pulse键， 听到蜂鸣声后， 向电击杯中迅速加入 ΙΟΟΟμΙ的 LB液体培养基， 重悬细 胞后， 转移到 1.5ml的离心管中。 37°C， 250rpm复苏 111; 取 20ul转化产物加 160 μΐ LB 涂板， 7°C过夜垮养， 次日査看转化结果； 其余菌液加 1 : 1的 30%的甘油后混匀一 80Ό保 存。
2、 如权利要求 1所述的运载重组质粒的减毒沙门氏菌的制备方法。
3、 如权利要求 1 所述的运载重组质粒的减毒沙门氏菌在制备治疗肿瘤的药物中的应 用。
4、 如权利要求 3 所述的运载重组质粒的减毒沙门氏菌， 治疗的肿瘤包括： 人前列腺 癌细胞、 肺癌、 胃癌、 喉癌、 乳腺癌、 肝癌、 黑色素瘤。
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