CN1241636A - Nuclease plasmid resisting HPV16E6 and its application in treatment of human papilloma virus and tumor - Google Patents
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Abstract
本发明公开了抗HPV16E6核酶质粒及其在人乳头瘤病毒与肿瘤治疗中的应用。该质粒可溶于水或TE等缓冲液配成溶液,或用冷冻干燥法制成干粉。该质粒能在RNA水平阻断HPV16E6基因的表达,阻断HPV相关肿瘤的细胞周期进展,诱导肿瘤细胞凋亡,部分逆转宫颈癌等肿瘤的恶性表型,并使肿瘤细胞易于被免疫细胞所杀伤。可用于人乳头瘤病毒及其相关肿瘤,如宫颈癌、口腔癌、喉癌等肿瘤的治疗与研究。The invention discloses an anti-HPV16E6 ribozyme plasmid and its application in human papillomavirus and tumor treatment. The plasmid can be dissolved in water or buffer such as TE to make a solution, or it can be made into a dry powder by freeze-drying. The plasmid can block the expression of HPV16E6 gene at the RNA level, block the cell cycle progression of HPV-related tumors, induce tumor cell apoptosis, partially reverse the malignant phenotype of cervical cancer and other tumors, and make tumor cells susceptible to immune cells. kill. It can be used in the treatment and research of human papillomavirus and its related tumors, such as cervical cancer, oral cancer, laryngeal cancer and other tumors.
Description
本发明涉及含抗HPV16E6核酶基因的真核表达质粒，可用于人乳头瘤病毒及其相关肿瘤，如宫颈癌、口腔癌、喉癌等的治疗。The invention relates to a eukaryotic expression plasmid containing an anti-HPV16E6 ribozyme gene, which can be used for the treatment of human papillomavirus and related tumors, such as cervical cancer, oral cancer, laryngeal cancer and the like.
目前所知，人乳头瘤病毒(HPV，Human Papillomaviruses)是人类肿瘤病毒病因中最为重要的一类病毒。高危HPV易引起恶性肿瘤，与宫颈癌、阴茎癌、口腔癌、喉癌及皮肤癌等发病密切相关，其中最常见的类型是HPV16和HPV18。核酶(Ribozyme)是指有催化活性的RNA分子，它可以有效地、序列特异地切割靶RNA。由于Ribozyme可以序列特异性地切割靶RNA，从而可以在mRNA水平阻断靶基因的表达。研究已表明，HPV的致癌性主要与其E6、E7基因有关。因此本研究设计、合成了抗HPV16E6核酶，其靶位点是HPV16E6基因上的第170位点，并将其克隆于真核表达质粒PcDNA3中。目前国内外尚未见有关抗HPV16E6核酶的研究报道。As far known, human papillomavirus (HPV, Human Papillomaviruses) is the most important virus in the etiology of human tumor viruses. High-risk HPV can easily cause malignant tumors and is closely related to cervical cancer, penile cancer, oral cancer, laryngeal cancer and skin cancer. The most common types are HPV 16 and HPV 18 . Ribozyme refers to a catalytically active RNA molecule that can efficiently and sequence-specifically cleave target RNA. Since Ribozyme can sequence-specifically cut target RNA, it can block the expression of target genes at the mRNA level. Studies have shown that the carcinogenicity of HPV is mainly related to its E 6 and E 7 genes. Therefore, this study designed and synthesized an anti-HPV16E6 ribozyme whose target site is the 170th site on the HPV16E6 gene, and cloned it into the eukaryotic expression plasmid PcDNA3. At present, there is no research report on anti-HPV16E6 ribozyme at home and abroad.
本发明的目的是提供一种可用来治疗和研究人乳头瘤病毒及其相关肿瘤的含抗HPV16E6核酶基因的真核表达质粒。The purpose of the present invention is to provide a eukaryotic expression plasmid containing anti-HPV16E6 ribozyme gene which can be used for treating and studying human papillomavirus and related tumors.
我们以HPV16E6基因为靶基因，设计了能特异切割它的核酶，并进行了克隆、表达与活性鉴定；证实该抗HPV16E6核酶能有效地、序列特异地切割HPV16E6mRNA。质粒可应用于人乳头瘤病毒及其相关疾病，如宫颈癌、喉癌、口腔癌等，的治疗与研究。细胞和动物实验证实，抗HPV16E6核酶能在RNA水平阻断HPV16E6基因的表达，阻断HPV相关肿瘤的细胞周期进展，诱导肿瘤细胞凋亡，部分逆转宫颈癌等肿瘤的恶性表型，因此可用于宫颈癌、口腔癌、喉癌等肿瘤的治疗。另一方面以核酶为研究工具，通过阻断癌基因的表达，可研究细胞癌基因、抑癌基因、信号转导机制的变化，从而深入探讨HPV的致癌机制。Taking the HPV16E 6 gene as the target gene, we designed a ribozyme that can specifically cut it, and carried out cloning, expression and activity identification; it was confirmed that the anti-HPV16E 6 ribozyme can effectively and sequence-specifically cut HPV16E 6 mRNA. Plasmids can be used in the treatment and research of human papillomavirus and related diseases, such as cervical cancer, laryngeal cancer, oral cancer, etc. Cell and animal experiments have confirmed that anti- HPV16E6 ribozyme can block the expression of HPV16E6 gene at the RNA level, block the cell cycle progression of HPV-related tumors, induce tumor cell apoptosis, and partially reverse the malignant phenotype of cervical cancer and other tumors. Therefore, it can be used in the treatment of cervical cancer, oral cancer, laryngeal cancer and other tumors. On the other hand, using ribozyme as a research tool, by blocking the expression of oncogenes, the changes of cellular oncogenes, tumor suppressor genes, and signal transduction mechanisms can be studied, so as to deeply explore the carcinogenic mechanism of HPV.
抗HPV16E6核酶质粒的有效浓度0.1ug/ul-100ug/ul，最佳浓度0.1ug/ul-10ug/ul.The effective concentration of anti-HPV16E 6 ribozyme plasmid is 0.1ug/ul-100ug/ul, and the optimal concentration is 0.1ug/ul-10ug/ul.
本发明的来源：Source of this invention:
(1)设计：以HPV16基因序列为分析序列，采用计算机软件针对HPV16E6基因设计特异性锤头状核酶。(1) Design: The HPV 16 gene sequence was used as the analysis sequence, and computer software was used to design a specific hammerhead ribozyme for the HPV16E6 gene.
(2)人工合成了抗16HRz ribozyme基因(各两条链)，并引入酶切位点，以便于克隆组装。A.5’CTAGATATCATGTACTGATGAGTCCGTGAGGACGAAAGTTGTTTGGGTAC3’(2) The anti-16HRz ribozyme gene (two strands each) was artificially synthesized, and enzyme cutting sites were introduced to facilitate cloning and assembly. A.5'CTAGATATCATGTACTGATGAGTCCGTGAGGACGAAAGTTGTTTGGGTAC3'
Xba I Kpn IB.5’CCAAACAACTTTCGTCCTCACGGACTCATCAGTACATGATAT3’Xba I Kpn IB.5'CCAAACAACTTTCGTCCTCACGGACTCATCAGTACATGATAT3'
(3)将抗16HRz ribozyme基因装入原核表达质粒中，进行核酶的体外切割实验，而后又将核酶基因装入真核表达质粒(PcDNA3)中，构建成抗HPV16E6核酶质粒。该质粒借助于脂质体或病毒载体等的携带，用于人乳头瘤病毒及其相关肿瘤，如宫颈癌、喉癌、口腔癌等，的治疗与研究。(3) The anti-16HRz ribozyme gene was loaded into the prokaryotic expression plasmid, and the ribozyme cutting experiment was carried out in vitro, and then the ribozyme gene was loaded into the eukaryotic expression plasmid (PcDNA3) to construct the anti- HPV16E6 ribozyme plasmid. The plasmid is carried by means of liposomes or virus vectors, etc., and is used for the treatment and research of human papillomavirus and related tumors, such as cervical cancer, laryngeal cancer, oral cancer, etc.
以下所述的实例详细说明了本发明实施例1实验材料：1.pc16Rz、pcDNA3质粒的制备：pRSV-Rz523为抗HPV16E6核酶的真核表达质粒，pcDNA3为无目的基因的真核表达质粒，将其转化JM105受体菌，大量制备并纯化。2.CaSKi细胞、HL60细胞、K562细胞的培养：CaSKi细胞为HPV16阳性的人宫颈癌细胞株：HL60细胞为人粒细胞白血病细胞株，系NK抵抗细胞；K562细胞为人红白血病细胞株，系NK敏感细胞。以上三种细胞均由本室传代培养。3.动物：4周龄裸鼠，雌性，体重20-26克，由第一军医大学动物中心提供。4.主要试剂：G418，MTT，HPV16E6单抗，FITC标记的bcl-2、c-myc、fas、p-53单抗。实验方法：The example described below has described the embodiment of the present invention 1 experimental material in detail: 1. The preparation of pc16Rz, pcDNA3 plasmid: pRSV-Rz523 is the eukaryotic expression plasmid of anti-HPV16E6 ribozyme, and pcDNA3 is the eukaryotic expression plasmid of gene without purpose, It was transformed into JM105 recipient bacteria, prepared and purified in large quantities. 2.Cultivation of CaSKi cells, HL60 cells, and K562 cells: CaSKi cells are HPV16-positive human cervical cancer cell lines: HL60 cells are human myeloid leukemia cell lines, which are NK-resistant cells; K562 cells are human erythroleukemia cell lines, which are NK-sensitive cell. The above three types of cells were all subcultured in our laboratory. 3. Animals: 4-week-old nude mice, female, weighing 20-26 grams, provided by the Animal Center of First Military Medical University. 4. Main reagents: G418, MTT, HPV16E6 monoclonal antibody, FITC-labeled bcl-2, c-myc, fas, p-53 monoclonal antibody. experimental method:
1.转染CaSKi细胞：以脂质体法将浓度为10ug/ul的pc16Rz、pcDNA3分别转染CaSKi细胞，通过G418(400ug/ml)抗性筛选，将阳性克隆细胞扩增并保存，分别命名为CaSKi-R、CaSKi-P细胞。以RNA点杂交法检测抗HPV16E6核酶在CaSKi-R、CaSKi-P细胞中的表达，结果证实抗CaSKi-R细胞中能稳定表达。1. Transfect CaSKi cells: Transfect CaSKi cells with pc16Rz and pcDNA3 at a concentration of 10ug/ul by liposome method, and pass G418 (400ug/ml) resistance screening to amplify and save positive cloned cells, and name them respectively For CaSKi-R, CaSKi-P cells. The expression of anti-HPV16E6 ribozyme in CaSKi-R and CaSKi-P cells was detected by RNA dot blot method, and the results confirmed that it could be stably expressed in anti-CaSKi-R cells.
2.转染的CaSKi细胞生物学特性观察：于倒置显微镜下观察细胞的形态和生长状态，测定细胞生长曲线，测定细胞软琼脂克隆形成率，并通过免疫组化SABC法和流式细胞术检测转染的CaSKi细胞中HPV16E6蛋白的表达。CaSKi、CaSKi-R、CaSKi-P细胞生长速率相近。与CaSKi细胞相比，CaSKi-R细胞表达HPV16E6蛋白明显减少，软琼脂克隆形成率显著降低，而CaSKi-P细胞无此改变。2. Observation of the biological characteristics of the transfected CaSKi cells: observe the morphology and growth state of the cells under an inverted microscope, measure the cell growth curve, determine the formation rate of the soft agar colony of the cells, and detect it by immunohistochemical SABC method and flow cytometry Expression of HPV16E6 protein in transfected CaSKi cells. CaSKi, CaSKi-R, and CaSKi-P cells had similar growth rates. Compared with CaSKi cells, the expression of HPV16E6 protein in CaSKi-R cells was significantly reduced, and the colony formation rate in soft agar was significantly reduced, but there was no such change in CaSKi-P cells.
3.将CaSKi、CaSKi-R、CaSKi-P细胞按常规方法收集，上流式细胞仪检测，每例样品测5千个细胞核，检测结果输至计算机作数据处理，专用软件作细胞周期分析。结果发现CaSKi-R细胞凋亡率明显高于CaSKi和CaSKi-P。利用流式细胞仪测定CaSKi、CaSKi-R、CaSKi-P三种细胞bcl-2、c-myc、fas、p-53等基因表达的变化情况，发现上述基因的表达均有改变。与CaSKi细胞相比，CaSKi-R细胞bcl-2表达降低，c-myc表达增强，fas表达增强，p-53表达增强。而CaSKi-P与CaSKi相比，上述基因表达无明显改变。研究结果证实，转染核酶可上调c-myc、fas、p-53的表达，降低bcl-2的表达，从而启动凋亡机制，促进肿瘤细胞凋亡。3. Collect CaSKi, CaSKi-R, and CaSKi-P cells according to conventional methods, and use flow cytometry to detect 5,000 nuclei in each sample. The test results are sent to the computer for data processing, and special software is used for cell cycle analysis. The results showed that the apoptosis rate of CaSKi-R cells was significantly higher than that of CaSKi and CaSKi-P. Flow cytometry was used to measure the changes in the expression of genes such as bcl-2, c-myc, fas, and p-53 in CaSKi, CaSKi-R, and CaSKi-P cells, and it was found that the expressions of the above genes were all changed. Compared with CaSKi cells, CaSKi-R cells showed decreased expression of bcl-2, enhanced expression of c-myc, enhanced expression of fas, and enhanced expression of p-53. Compared with CaSKi-P, the expression of the above genes had no obvious change. The results of the study confirmed that transfection of ribozymes can up-regulate the expression of c-myc, fas, p-53, and reduce the expression of bcl-2, thereby initiating the apoptosis mechanism and promoting the apoptosis of tumor cells.
4.免疫活性细胞的杀伤活性检测：从健康人血中诱导和制备免疫活性NK、LAK、CD3AK细胞，并分别诱导CaSKi、CaSKi-R细胞与rIL-2共激活杀伤细胞(称CASKI和CASKI-R细胞)，将它们作为效应细胞(简称E)。以CaSKi-R、CaSKi-P、CaSKi、K562和HL60细胞为靶细胞(简称T)，取效靶比分别为20∶1、10∶1、5∶1、2.5∶1、1∶1，于96孔板内混合培养24小时后掺入MTT(1mg/ml)，以单独的效应细胞和靶细胞为对照，每个实验均设三个复孔。MTT作用4小时后加入异丙醇振荡，测570nmOD值，取每个实验OD570值均数，计算效应细胞杀伤百分率，比较杀伤活性。NK、LAK、CD3AK细胞对CaSKi-R细胞杀伤率显著高于CaSKi细胞，CaSKi、CaSKi-R分别与rIL-2共激活杀伤细胞的杀伤活性无明显差别，对CaSKi-R的杀伤活性高于CaSKi细胞。4. Detection of killing activity of immunocompetent cells: Induce and prepare immunocompetent NK, LAK, and CD3AK cells from healthy human blood, and induce CaSKi, CaSKi-R cells and rIL-2 to co-activate killer cells (called CASKI and CASKI- R cells), and they are used as effector cells (referred to as E). CaSKi-R, CaSKi-P, CaSKi, K562 and HL60 cells were used as target cells (T for short), and the effect-to-target ratios were 20:1, 10:1, 5:1, 2.5:1, and 1:1, respectively. MTT (1 mg/ml) was added to the 96-well plate after 24 hours of mixed culture, and the individual effector cells and target cells were used as controls, and three replicate wells were set up for each experiment. After 4 hours of MTT action, add isopropanol to shake, measure 570nm OD value, take the average value of OD570 in each experiment, calculate the killing percentage of effector cells, and compare the killing activity. The killing rate of NK, LAK, and CD3AK cells to CaSKi-R cells was significantly higher than that of CaSKi cells, and there was no significant difference in the killing activity of CaSKi, CaSKi-R and rIL-2 respectively, and the killing activity of CaSKi-R was higher than that of CaSKi cell.
5.裸鼠成瘤性实验：取一定数量、指数期生长、活力在95％以上的CaSKi、CaSKi-R、CaSKi-P细胞，依次接种于裸鼠右后肢大腿外侧皮内，约1×106个瘤细胞；肿瘤移植后对成瘤情况每周观察2次，共观察6周，记录肿瘤的长，宽。结果发现，CaSKi-R细胞的成瘤能力显著低于CaSKi细胞，而CaSKi-P与CaSKi细胞成瘤能力无明显差别。5. Tumorability experiment in nude mice: Take a certain number of CaSKi, CaSKi-R, and CaSKi-P cells with exponential growth and viability above 95%, and inoculate them sequentially in the outer thigh of the right hind limb of nude mice, about 1×10 6 tumor cells; after tumor transplantation, the tumor formation was observed twice a week for a total of 6 weeks, and the length and width of the tumor were recorded. The results showed that the tumorigenic ability of CaSKi-R cells was significantly lower than that of CaSKi cells, while there was no significant difference between CaSKi-P and CaSKi cells.
实施例2：Example 2:
将浓度为0.1ug/ul的pc16Rz、pcDNA3质粒分别转染CaSKi细胞，然后按照实施例1的方法进行(1)细胞生长曲线测定和形态学观察；(2)细胞软琼脂克隆率形成实验；(3)细胞凋亡率测定和凋亡基因分析；(4)免疫活性细胞杀伤实验；(5)裸鼠成瘤性实验。可以得出以下结论：0.1ug/ul的pc16Rz能降低肿瘤细胞软琼脂克隆形成率，提高肿瘤细胞凋亡率，启动凋亡基因，提高肿瘤细胞对免疫活性细胞杀伤的敏感性，降低肿瘤细胞在裸鼠体内的成瘤能力。The pc16Rz and pcDNA3 plasmids with a concentration of 0.1ug/ul were transfected into CaSKi cells respectively, and then carried out (1) cell growth curve determination and morphological observation according to the method of Example 1; (2) cell soft agar cloning rate formation experiment; ( 3) Cell apoptosis rate measurement and apoptosis gene analysis; (4) Immunocompetent cell killing test; (5) Nude mouse tumorigenicity test. The following conclusions can be drawn: 0.1ug/ul pc16Rz can reduce the soft agar colony formation rate of tumor cells, increase the apoptosis rate of tumor cells, activate apoptosis genes, improve the sensitivity of tumor cells to the killing of immune active cells, and reduce the tumor cell Tumor formation ability in nude mice.
实施例3：Example 3:
将浓度为100ug/ul的pc16Rz、pcDNA3质粒分别转染CaSKi细胞，然后按照实施例1的方法进行(1)细胞生长曲线测定和形态学观察；(2)细胞软琼脂克隆率形成实验；(3)细胞凋亡率测定和凋亡基因分析；(4)免疫活性细胞杀伤实验；(5)裸鼠成瘤性实验。可以得出以下结论：10ug/ul的pc16Rz能降低肿瘤细胞软琼脂克隆形成率，提高肿瘤细胞凋亡率，启动凋亡基因，提高肿瘤细胞对免疫活性细胞杀伤的敏感性，降低肿瘤细胞在裸鼠体内的成瘤能力。The pc16Rz, pcDNA3 plasmids that concentration is 100ug/ul transfect CaSKi cell respectively, then carry out (1) cell growth curve measurement and morphological observation according to the method for embodiment 1; (2) cell soft agar clone rate formation experiment; (3) ) Determination of cell apoptosis rate and analysis of apoptosis genes; (4) Immunocompetent cell killing experiment; (5) Nude mouse tumorigenicity experiment. The following conclusions can be drawn: 10ug/ul pc16Rz can reduce the soft agar colony formation rate of tumor cells, increase the apoptosis rate of tumor cells, activate apoptosis genes, improve the sensitivity of tumor cells to the killing of immune active cells, and reduce the tumor cells in the naked eye. Tumor formation ability in mice.
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| WO2002092796A1 (en) * | 2001-05-15 | 2002-11-21 | Liang Qiao | Papilloma pseud-virus and it's preparation |
| CN1293093C (en) * | 2002-08-30 | 2007-01-03 | 马润林 | Pronucleus preparation of nipple tumour virus capsid protein and application |
| CN104404076A (en) * | 2014-11-04 | 2015-03-11 | 珠海雅马生物工程有限公司 | Method for knockout of human papillomavirus E6E7 gene by zinc finger nucleases |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002092796A1 (en) * | 2001-05-15 | 2002-11-21 | Liang Qiao | Papilloma pseud-virus and it's preparation |
| US8129144B2 (en) | 2001-05-15 | 2012-03-06 | Loyola University Chicago | Papilloma pseudovirus and preparation |
| CN1293093C (en) * | 2002-08-30 | 2007-01-03 | 马润林 | Pronucleus preparation of nipple tumour virus capsid protein and application |
| CN104404076A (en) * | 2014-11-04 | 2015-03-11 | 珠海雅马生物工程有限公司 | Method for knockout of human papillomavirus E6E7 gene by zinc finger nucleases |
| CN104404076B (en) * | 2014-11-04 | 2017-10-03 | 珠海雅马生物工程有限公司 | The method that human papillomavirus E 6/E 7 gene is knocked out using Zinc finger nuclease |
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