WO2017036302A1 - 减毒鼠伤寒沙门氏菌基因工程菌在制备治疗肝癌药物上的应用 - Google Patents

减毒鼠伤寒沙门氏菌基因工程菌在制备治疗肝癌药物上的应用 Download PDF

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WO2017036302A1
WO2017036302A1 PCT/CN2016/095629 CN2016095629W WO2017036302A1 WO 2017036302 A1 WO2017036302 A1 WO 2017036302A1 CN 2016095629 W CN2016095629 W CN 2016095629W WO 2017036302 A1 WO2017036302 A1 WO 2017036302A1
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plasmid
methioninase
vnp20009
salmonella typhimurium
attenuated salmonella
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French (fr)
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赵子建
周素瑾
林艳
赵正刚
李芳红
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南京华贞生物医药科技有限公司
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Priority to US15/755,537 priority patent/US20180339032A1/en
Publication of WO2017036302A1 publication Critical patent/WO2017036302A1/zh
Priority to US17/067,636 priority patent/US11318172B2/en

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  • the invention relates to the technical field of medicine, in particular to the application of genetically engineered bacteria of attenuated Salmonella typhimurium in the preparation of a medicament for treating liver cancer.
  • Liver cancer is one of the most common malignant tumors in the world. China is the country with the highest incidence of liver cancer in the world, ranking second in common tumors after lung cancer. The incidence of liver cancer in China accounts for 45% of the world's total, and the mortality rate is the third highest among malignant tumors.
  • Treatment of hepatocellular carcinoma includes surgery, radiation therapy, and drug chemotherapy. Because liver cancer is less sensitive to radiotherapy, the treatment of conventional chemotherapy drugs (such as doxorubicin, fluorouracil, cisplatin) not only has serious side effects, but also can not significantly alleviate the disease. Therefore, the current treatment of liver cancer is still mainly surgical resection.
  • conventional chemotherapy drugs such as doxorubicin, fluorouracil, cisplatin
  • liver cancer is insidious, the early symptoms are lacking, the tumor cells grow rapidly, the progress is fast, the degree of malignancy is high, less than 30% of patients can undergo surgery, and the recurrence rate is high even after surgery, making the prognosis of liver cancer patients extremely poor.
  • the treatment of liver cancer is far from meeting the clinical needs, and it is urgent to develop new and effective therapeutic drugs.
  • VNP20009 is an attenuated Salmonella typhimurium strain with msbB and pur I gene deletion, which is genetically stable and sensitive to antibiotics.
  • the msbB gene is required for lipid acylation to endotoxin, and its deletion prevents the lipid-like A terminal from being acylated, which reduces toxicity; the pur I gene is involved in purine metabolism, and its deletion requires exogenous adenine for bacterial reproduction.
  • VNP20009 also reduces the tumor necrosis factor (TNF) produced by the body itself, thereby reducing the inflammatory response.
  • TNF tumor necrosis factor
  • VNP20009 has been widely used in cancer research, and it can be applied to a variety of mouse solid tumor models, including melanoma, lung cancer, colon cancer, breast cancer, and kidney cancer.
  • One of the main advantages of VNP20009 as a tumor gene therapy vector is its ability to highly target aggregation at tumor sites.
  • the researchers found that the number of VNP20009 in the tumor was 200 to 1,000 times higher than that in the main organs such as the liver. VNP20009 can preferentially aggregate and multiply in the hypoxic necrosis area of tumor tissue.
  • the amplification of bacteria in tumor tissue is significantly higher than that in normal tissues, making attenuated Salmonella a new antitumor agent and a carrier for tumor targeted therapy.
  • Salmonella causes tumor growth to slow down include: the nutrients required for tumor growth are consumed by bacteria, enzymes produced by bacteria, such as asparaginase, can deplete essential amino acids for tumor growth; bacteria are secreted into the extracellular microenvironment. Local toxins or tumor necrosis factor alpha produced may affect tumor angiogenesis; In addition, non-specific inflammatory responses at the site of bacterial growth can potentially activate anti-tumor T cells.
  • Tumor cells need sufficient nutrients in order to maintain their high proliferation rate.
  • methionine Metionine, Met
  • glutamine glutamine
  • arginine glutamine
  • Met dependence is a common feature of most tumor cells, such as breast cancer, liver cancer, lung cancer, colon cancer, kidney cancer, bladder cancer, melanoma, glioma, etc., while normal cells do not have Met dependence.
  • Some in vitro and in vivo experiments have confirmed that direct consumption of methionine-deficient diet can delay the proliferation of tumor cells.
  • the long-term lack or deficiency of Met in the diet will cause malnutrition and metabolic disorders, the cancer may be exacerbated by the long-term hypomethylation of DNA.
  • the technical problem to be solved by the present invention is to provide a genetic engineering bacteria for preparing a biopharmaceutical for treating liver cancer.
  • the invention provides a genetic engineering bacteria for preparing a medicament for treating liver cancer, wherein the genetic engineering bacteria is attenuated Salmonella typhimurium VNP20009 carrying a plasmid, and wherein the plasmid has an L-methioninase gene cloned thereon.
  • the plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3.1 plasmid, a pBR322 plasmid or a pET23a plasmid.
  • the genetic engineering bacteria is constructed by subcloning the L-methioninase gene into a plasmid to obtain an L-methioninase expression plasmid, and electroporating the L-methioninase expression plasmid into the attenuated Salmonella typhimurium VNP20009.
  • the electrical conversion conditions are a voltage of 2400 V, a resistance of 400 ⁇ , a capacitance of 25 ⁇ F, and a discharge time of 4 ms.
  • the L-methioninase gene is subcloned into the plasmid by Kpn I and Hind III cleavage sites to obtain L-methioninase.
  • the plasmid was expressed, and then the L-methioninase expression plasmid was electrotransformed into the attenuated Salmonella typhimurium VNP20009 to obtain a genetically engineered strain.
  • the above-mentioned attenuated Salmonella typhimurium and its genetically engineered bacteria are administered intravenously or by intervention.
  • the biopharmaceutical for treating liver cancer of the present invention is a safe, non-toxic and anti-tumor activity novel biopharmaceutical, using attenuated Salmonella typhimurium VNP20009 as a carrier and utilizing genetic recombination technology High expression of methioninase, strong anti-tumor activity, can meet the needs of use.
  • the preparation method is simple and easy to operate; and has a good application prospect.
  • Figure 1 is a 1% agarose gel electrophoresis map of plasmid pSVSPORT-L-methioninase.
  • Figure 2 is a graph showing the results of methioninase expression by Western blot.
  • Figure 3 is a graph showing the results of detecting methioninase activity in Salmonella.
  • Figure 4 is a graph showing tumor volume changes after administration of Salmonella.
  • Figure 5 is a photograph of the size of the tumor after 4 weeks of Salmonella injection, and the tumor in the black box.
  • Figure 6 is a graph showing the results of tumor size after 4 weeks of Salmonella.
  • Figure 7 is a graph showing the weight of tumors after 4 weeks of Salmonella.
  • Figure 8 is a graph showing changes in tumor volume by administration of L-methioninase.
  • Example 1 Construction of genetically engineered bacteria.
  • the L-methioninase (GenBank: L43133.1) gene was firstly subcloned into the pUC57 plasmid (Kinseri), and then subcloned into the pSVSPORT plasmid (invitrogen) by Kpn I and Hind III cleavage sites to obtain pSVSPORT-L- Methioninase expression plasmid.
  • the specific construction process is as follows:
  • the pSVSPORT plasmid was digested with Kpn I and Hind III, and the digestion system was: 2 ⁇ g of plasmid DNA, 3 ⁇ L of 10 ⁇ buffer, 1.5 ⁇ L of Kpn I enzyme, 1.5 ⁇ L of Hind III enzyme, and ddH 2 O was added to make up the volume to 30 ⁇ L, 37 ° C. Warm bath for 3h. Then, the digestion system was separated by electrophoresis on a 1% agarose gel, and a DNA band of 4.1 kb was cut out, and the DNA was purified using a gel recovery purification kit.
  • the DNA fragment of the L-methioninase coding region was subcloned into the pUC57 plasmid (Kinseri) by whole-genome synthesis, and digested with Kpn I and Hind III.
  • the digestion system was: 3 ⁇ g plasmid DNA, 3 ⁇ L 10 ⁇ buffer, 1.5 ⁇ L. Kpn I enzyme, 1.5 ⁇ L Hind III enzyme, add ddH 2 O to make up the volume to 30 ⁇ L, and warm at 37 ° C for 3 h. Then, the digestion system was separated by electrophoresis on a 1% agarose gel, and a 1.2 kb DNA band was excised, and the DNA was purified using a gel recovery purification kit.
  • pSVSPORT Kpn I/Hind III
  • L-methioninase coding region DNA fragment Kpn I/Hind III
  • the ligation product was transformed into competent cells of E. coli DH5 ⁇ (Takara). Take a tube of 50 ⁇ L of DH5 ⁇ competent cells and place them on ice. After they are thawed, add 5 ⁇ L of the above-mentioned ligation product, mix gently, then incubate on ice for 30 min; heat at 42 °C for 60 s, then let stand on ice. 2 min; add 500 ⁇ L of non-resistant LB liquid medium, incubate for 1 h at 37 ° C; centrifuge for 5 min at 4000 rpm, aspirate, retain about 100 ⁇ L of medium, pipette the bacterial pellet evenly and apply it to ampicillin-resistant Sexual LB medium on the plate. The plates were then incubated for 16 h in a 37 ° C incubator.
  • the monoclonal colonies were picked into 3 mL of LB-O medium containing ampicillin, and cultured at 37 ° C for 16 h.
  • the plasmid DNA was extracted and identified by Kpn I and Hind III digestion.
  • the positive clones were 4.1 kb. Two DNA bands of 1.2 kb are shown in Figure 1. The sequence of the positive clone was further confirmed by sequencing to be completely correct.
  • VNP20009-V and VNP20009-M were each electroporated into VNP20009 strain (YS1646, ATCC No. 202165), designated as VNP20009-V and VNP20009-M, respectively.
  • the specific construction process is as follows:
  • the competent bacteria VNP20009 was placed on ice, and after it was melted, it was transferred to a pre-cooled electric rotor, 2 ⁇ L of the plasmid was added thereto, and the mixture was gently incubated and incubated on ice for 1 min. Put the electric rotor into the electro-rotation instrument, the condition is set to voltage 2400V, resistance 400 ⁇ , capacitance 25 ⁇ F, discharge time 4ms. Immediately after the electric shock, add 1 mL of SOC medium and mix gently.
  • L-methionine and pyridoxal were mixed with VNP20009, VNP20009-V and VNP20009-M, respectively, and incubated at 37 ° C for 10 min, then terminated with 50% trichloroacetic acid, and the supernatant was centrifuged to obtain 3-methyl- 2-Benzothiazolinone hydrochloride hydrate (MBTH) was thoroughly mixed, and after incubation at 50 ° C for 30 min, the absorbance at 320 nm was measured, and the amount of enzyme that catalyzes the conversion of 1 ⁇ mol of ⁇ -ketobutyrate per minute was defined as one enzyme. Active unit. The results showed (Fig. 3) that the methioninase activity of Salmonella VNP20009-M was 10-fold higher than that of VNP20009 and VNP20009-V.
  • Example 2 Antitumor effect of VNP20009-L-methioninase strain.
  • High-metastatic hepatoma cells HCCLM3 were cultured in DMEM medium containing 10% fetal bovine serum, subcutaneously inoculated into the right axilla of nude mice with 2 ⁇ 10 6 cells, and the tumor-bearing nude mice were randomly divided into groups: PBS control. Group, VNP20009-V group and VNP20009-M group.
  • the operation method was the same as above.
  • the tumor-bearing nude mice were divided into two groups.
  • the results of the tumor volume change curve of nude mice were plotted. As shown in Fig. 8, there was no significant difference in tumor between the two groups.
  • the dose of L-methioninase 1 ng/only is equivalent to 2 ⁇ 10 6 CFU of L-methioninase contained in VNP20009-M.
  • the simple administration of 100-fold dose of L-methioninase had no significant anti-tumor effect.
  • the present invention shows that the genetically engineered bacteria have a significant inhibitory effect on liver cancer cells, and the attenuated Salmonella typhimurium strain carrying the plasmid with the L-methioninase gene can continuously express L-methioninase in the tumor tissue, and consumes a large amount of methionine and other nutrients.
  • the substance makes the tumor cells lack nutrients and grows slowly, so it can be used to prepare drugs for treating liver cancer.
  • the above plasmid is not limited to the pSVSPORT plasmid, and the pTrc99A plasmid, the pcDNA3.1 plasmid, the pBR322 plasmid or the pET23a plasmid, and the above plasmid in which the L-methioninase gene was cloned have similar effects.

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Abstract

提供了一种减毒鼠伤寒沙门氏菌基因工程菌在制备治疗肝癌药物上的应用，该菌为携带克隆有甲硫氨酸酶基因的质粒的减毒鼠伤寒沙门氏菌VNP20009。还提供了该菌的构建方法。

Description

减毒鼠伤寒沙门氏菌基因工程菌在制备治疗肝癌药物上的应用 技术领域
本发明涉及医药技术领域，具体涉及减毒鼠伤寒沙门氏菌的基因工程菌在制备治疗肝癌药物上的应用。
背景技术
肝癌是世界上常见的恶性肿瘤之一。中国是全球肝癌发病率最高的国家，在常见肿瘤中仅次于肺癌，居第2位。中国肝癌发病率占全球的45％，死亡率高居恶性肿瘤的第三位。肝细胞癌的治疗包括手术治疗、放疗和药物化疗。由于肝癌对放射性治疗敏感性差，而常规化疗药物(如阿霉素、氟尿嘧啶、顺铂)的治疗不仅毒副作用严重，而且也不能明显缓解疾病，因此目前肝癌的治疗仍以手术切除为主。但是肝癌起病隐匿，早期缺乏典型症状，肿瘤细胞生长迅速，进展快，恶性程度高，能进行手术的患者不到30％，而且即使手术以后复发率也很高，使得肝癌患者的预后极差。据统计，95％以上的肝癌患者治疗失败。目前，肝癌治疗药物远未满足临床需求，亟待开发新的有效的治疗药物。
沙门菌属是一群在人和动物肠道内寄生的革兰氏阴性、侵袭性细胞内兼性厌氧菌。VNP20009为msbB、pur I基因缺失的减毒鼠伤寒沙门菌株，遗传稳定，对抗生素敏感。msbB基因为脂质酰化为内毒素所必需，其缺失使类脂质A末端不能酰化，降低了毒性；pur I基因参与嘌呤代谢，其缺失使细菌的繁殖需要外源性腺嘌呤。VNP20009还降低了自身诱导机体产生的肿瘤坏死因子(tumor necrosis factor，TNF)，从而降低炎性反应。因此，它的低致病性提高了用于临床治疗的安全性。VNP20009已被广泛用于癌症研究，它可作用于多种小鼠实体瘤模型，包括黑色素瘤、肺癌、结肠癌、乳腺癌、肾癌。VNP20009作为肿瘤基因治疗载体的一个主要优点是它能够高度靶向聚集于肿瘤部位。研究人员在多种实体肿瘤的小鼠模型中发现，VNP20009在肿瘤内的数量较肝脏等主要器官内高200～l000倍。VNP20009能在肿瘤组织缺氧坏死区优先聚集并繁殖，相同时间内肿瘤组织内细菌的扩增代次显著高于正常组织，使得减毒沙门菌成为新型抗瘤制剂和肿瘤靶向治疗的载体成为可能。沙门菌导致肿瘤生长减慢可能机制包括：肿瘤生长所需要的营养物质为细菌所消耗，细菌所产生的酶，如天冬酰胺酶，可耗竭肿瘤生长必需氨基酸；细菌向胞外微环境分泌的局部毒素或者产生的肿瘤坏死因子α，都可影响肿瘤血管形成； 此外，在细菌生长部位的非特异性炎症反应可潜在激活抗肿瘤T细胞。
肿瘤细胞为了维持其高增殖率，需要足够的营养。除糖之外，对甲硫氨酸(Methionine,Met)、谷胺酰氨、精氨酸等需要量尤其大。研究证实Met依赖性是绝大多数肿瘤细胞的共同特征，如乳腺癌、肝癌、肺癌、结肠癌、肾癌、膀胱癌、黑色素瘤、神经胶质瘤等，而正常细胞不存在Met依赖性。一些体内体外实验相继证实，直接食用甲硫氨酸缺乏的膳食可以延缓肿瘤细胞的增殖。但是若膳食中Met长期缺乏或不足，将会引起机体营养不良、代谢障碍，还可因DNA长期处于低甲基化状态加剧癌变。那么，通过甲硫氨酸酶(L-methioninase)特异性地将Met分解，从而降低体内甲硫氨酸水平，则能更加有效地抑制肿瘤细胞生长或使之消退。动物实验已证明经腹膜内注射甲硫氨酸酶可以抑制裸鼠的吉田肉瘤和肺肿瘤的增长。临床试验选四个分别患有乳腺癌、肺癌、肾癌和淋巴瘤的患者每24h静脉注射一次甲硫氨酸酶，发现甲硫氨酸酶可以明显减少血浆中甲硫氨酸含量。但是，由于哺乳动物本身不表达甲硫氨酸酶，所以外源性给与的方式有一定的副作用，往往引起机体的免疫反应。
发明内容
本发明所要解决的技术问题在于提供一种基因工程菌在制备治疗肝癌的生物药物上的应用。
为解决上述技术问题，本发明采用的技术方案如下：
本发明提出一种基因工程菌在制备治疗肝癌的药物上的应用，所述基因工程菌为携带质粒的减毒鼠伤寒沙门氏菌VNP20009，且其中所述质粒上克隆有L-methioninase基因。
其中，所述的质粒为pSVSPORT质粒、pTrc99A质粒、pcDNA3.1质粒、pBR322质粒或pET23a质粒。
所述基因工程菌的构建方法为：将L-methioninase基因亚克隆至质粒中，得到L-methioninase表达质粒，将L-methioninase表达质粒电转化至减毒鼠伤寒沙门氏菌VNP20009，即得。所述的电转化条件为电压2400V，电阻400Ω，电容25μF，放电时间4ms。
最为优选，在构建基因工程菌的过程中，当选用pSVSPORT质粒时，将L-methioninase基因通过Kpn I和Hind III酶切位点亚克隆至质粒中，得到L-methioninase 表达质粒，然后将L-methioninase表达质粒电转化至减毒鼠伤寒沙门氏菌VNP20009中得到基因工程菌。
上述减毒鼠伤寒沙门氏菌及其基因工程菌的给药方式为静脉或介入注射。
有益效果：与现有技术相比，本发明的用于治疗肝癌的生物药物，是一种安全无毒具备抗肿瘤活性的新型生物药物，以减毒鼠伤寒沙门氏菌VNP20009为载体，利用基因重组技术高表达甲硫氨酸酶，抗肿瘤活性强，能满足使用需求。制备方法简单，容易操作；具有很好的应用前景。
附图说明
图1是质粒pSVSPORT-L-methioninase酶切鉴定1％琼脂糖凝胶电泳图。
图2是Western blot鉴定methioninase表达结果图。
图3是检测沙门氏菌中methioninase活性结果图。
图4是给与沙门菌后肿瘤体积变化曲线图。
图5是给沙门菌4周后将小鼠麻醉，肿瘤的大小拍照结果图，黑色框中为肿瘤。
图6是给沙门菌4周后肿瘤的大小结果图。
图7是给沙门菌4周后肿瘤的重量结果图。
图8是给予L-methioninase肿瘤体积变化曲线图。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：基因工程菌的构建。
(1)构建表达L-methioninase基因的质粒。
先合成L-methioninase(GenBank：L43133.1)基因亚克隆至pUC57质粒(金斯瑞公司)，接着通过Kpn I和Hind III酶切位点亚克隆至pSVSPORT质粒(invitrogen)，得到pSVSPORT-L-methioninase表达质粒。具体构建过程如下：
将pSVSPORT质粒用Kpn I和Hind III双酶切，酶切体系为：2μg质粒DNA，3μL 10× buffer，1.5μL Kpn I酶，1.5μL Hind III酶，加入ddH2O补足体积至30μL，37℃温浴3h。然后将酶切体系在1％的琼脂糖凝胶中通过电泳分离，切出4.1kb大小的DNA条带，用胶回收纯化试剂盒纯化DNA。
通过全基因合成得到L-methioninase编码区域DNA片段亚克隆至pUC57质粒(金斯瑞公司)，用Kpn I和Hind III双酶切，酶切体系为：3μg质粒DNA，3μL 10×buffer，1.5μL Kpn I酶，1.5μL Hind III酶，加入ddH2O补足体积至30μL，37℃温浴3h。然后将酶切体系在1％的琼脂糖凝胶中通过电泳分离，切出1.2kb大小的DNA条带，用胶回收纯化试剂盒纯化DNA。
将pSVSPORT(Kpn I/Hind III)和L-methioninase编码区域DNA片段(Kpn I/Hind III)连接，连接反应中加入2μL载体、6μL插入片段、1μL T4DNA连接酶，16℃温浴16h。
将连接产物转化到E.coli DH5α(Takara)的感受态细胞中。取一管50μL的DH5α感受态细胞置于冰上，待其融化后，向其中加入5μL上述连接产物，轻弹混匀，后于冰上孵育30min；42℃热击60s，后冰上静置2min；加入500μL无抗性的LB液体培养基，37℃震荡培养1h；4000rpm离心5min，吸走，保留100μL左右的培养基，用移液器将细菌沉淀吹打均匀后涂布在含氨苄青霉素抗性的LB培养基平板上。然后将平板置于37℃温箱培养16h。
克隆生长出来后，挑单克隆菌落至3mL含氨苄青霉素的LB-O培养液中，37℃震荡培养16h，提取质粒DNA，用Kpn I和Hind III酶切鉴定，阳性克隆中可得到4.1kb、1.2kb的两条DNA条带，如图1所示。再通过测序进一步确定阳性克隆的序列完全正确。
(2)构建携带质粒的VNP20009菌和携带克隆有L-methioninase的基因的质粒的VNP20009菌。
将pSVSPORT和pSVSPORT-L-methioninase表达质粒分别电转化至VNP20009菌株(YS1646，ATCC号202165)，分别命名为VNP20009-V和VNP20009-M。具体构建过程如下：
将感受态细菌VNP20009置于冰上，待其融化后移入预冷电转杯，向其中加入2μL质粒，轻弹混匀，于冰上孵育1min。将电转杯放入电转仪，条件设置为电压2400V，电阻400Ω，电容25μF，放电时间4ms。电击完立刻加入1mL SOC培养基，轻轻混匀。37℃震荡培养1h；4000rpm离心5min，吸走，保留100μL左右的培养基，用移液器将 细菌沉淀吹打均匀后涂布在含氨苄青霉素抗性的LB-O培养基平板上。然后将平板置于37℃温箱培养16h。VNP20009-V和VNP20009-M用LB-O培养后，提取质粒，酶切鉴定正确。
取1×108沙门菌用蛋白裂解液提取蛋白，进行10％SDS-PAGE电泳，再稳压冰浴电转至PVDF膜，BSA室温封闭1h后，TBST漂洗3×5min，加入兔抗L-methioninase抗体(1：2000)，4℃孵育过夜。TBST漂洗3次，每次5min再加HRP标记的抗兔二抗(1：5000)，室温孵育1h，TBST漂洗3次，每次5min，ECL化学发光法显影。结果如图2所示，在分子量约43kD处有特异性条带，说明VNP20009-M与VNP20009、VNP20009-V相比，L-methioninase表达量显著提高。
将L-甲硫氨酸和吡哆醛分别与VNP20009、VNP20009-V和VNP20009-M菌体混合，37℃孵育10min后用50％三氯乙酸终止，离心取上清，与3-甲基-2-苯并噻唑啉酮腙盐酸盐水合物(MBTH)充分混匀，50℃孵育30min后，测定320nm处的吸光值，以每分钟催化转化1μmolα-酮丁酸的酶量定义为1个酶活性单位。结果显示(图3)，沙门菌VNP20009-M的甲硫氨酸酶活性比VNP20009和VNP20009-V高10倍。
实施例2：VNP20009-L-methioninase菌株的抗肿瘤效果。
1、用含有10％胎牛血清的DMEM培养基培养高转移性肝癌细胞HCCLM3，以细胞数2×106个皮下接种于裸鼠右侧腋下，并将荷瘤裸鼠随机分组：PBS对照组、VNP20009-V组和VNP20009-M组。
2、用LB-O培养VNP20009-V和VNP20009-M，当OD≈0.6时，收集菌体，然后用PBS重悬。在种瘤后第3天，以1×104CFU/g(约为2×105CFU/只)的剂量，采用尾静脉注射方式给药，对照组注射同等体积PBS。给药后每隔2～3天，观察小鼠状态，用游标卡尺测量肿瘤大小(体积＝0.52×长×宽2)，绘制裸鼠肿瘤体积变化曲线(图4)。给药后第30天，对照组和实验组各选取3只小鼠，麻醉小鼠拍照(图5)，并随机选取对照组和实验组各2只，剥离裸鼠肿瘤，称重，拍照(图6、7)。结果如图4、5所示，造模后，PBS以及空菌对照组小鼠的肿瘤都正常生长并增长迅速，而给予沙门菌VNP20009-M治疗后，部分小鼠肿瘤减小甚至完全消失。VNP20009-M组的大部分小鼠的肿瘤生长停滞，肿瘤体积和重量(图6、7)约为VNP20009-V组的1/2，PBS对照组的1/5。结果说明沙门菌VNP20009-M对于该肝癌肿瘤具有显著的抑制效应。
3、操作方法同上述1，将荷瘤裸鼠分为两组，同样采取静脉注射的方式，分别给予PBS、L-methioninase 100ng/只，用游标卡尺测量肿瘤大小(体积＝0.52×长×宽2)，绘制裸鼠肿瘤体积变化曲线结果，如图8所示，两组之间肿瘤无显著性差异。L-methioninase1ng/只的剂量等同于2×106CFU的VNP20009-M所含L-methioninase。由此可见，单纯给予100倍剂量的L-methioninase无明显的抗肿瘤效应。这说明随着L-methioninase的耗竭或降解，单次给药没有发挥作用，而以VNP20009为载体持续高表达L-methioninase则弥补了这个缺陷，表现出显著的抗肿瘤效应。
本发明表明基因工程菌对肝癌细胞具有显著的抑制效应，带有克隆有L-methioninase基因的质粒的减毒鼠伤寒沙门氏菌可以在肿瘤组织持续表达L-methioninase，大量消耗甲硫氨酸及其它营养物质，使得肿瘤细胞缺乏营养，生长缓慢，因此可以用于制备治疗肝癌的药物。上述质粒并不局限于pSVSPORT质粒，pTrc99A质粒、pcDNA3.1质粒、pBR322质粒或pET23a质粒以及克隆有L-methioninase基因的上述质粒同样具有类似效果。

Claims (6)

  1. 一种基因工程菌在制备治疗肝癌药物上的应用，所述基因工程菌为携带质粒的减毒鼠伤寒沙门氏菌VNP20009，且其中所述质粒上克隆有L-methioninase基因。
  2. 根据权利要求1所述的应用，其特征在于，所述的质粒为pSVSPORT质粒、pTrc99A质粒、pcDNA3.1质粒、pBR322质粒或pET23a质粒。
  3. 根据权利要求1或2所述的应用，其特征在于，所述基因工程菌的构建方法为：将L-methioninase基因亚克隆至质粒中，得到L-methioninase表达质粒，将L-methioninase表达质粒电转化至减毒鼠伤寒沙门氏菌VNP20009，即得。
  4. 根据权利要求3所述的应用，其特征在于，所述的电转化条件为电压2400V，电阻400Ω，电容25μF，放电时间4ms。
  5. 根据权利要求3所述的应用，其特征在于，在构建基因工程菌的过程中，当选用pSVSPORT质粒时，将L-methioninase基因通过Kpn I和Hind III酶切位点亚克隆至质粒中，得到L-methioninase表达质粒，然后将L-methioninase表达质粒电转化至减毒鼠伤寒沙门氏菌VNP20009中得到基因工程菌。
  6. 根据权利要求1所述的应用，其特征在于，给药方式为静脉或介入注射。
PCT/CN2016/095629 2015-08-31 2016-08-17 减毒鼠伤寒沙门氏菌基因工程菌在制备治疗肝癌药物上的应用 WO2017036302A1 (zh)

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