抗肝癌基因疫苗的构建及纯化工艺研究
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摘要
研究背景：肿瘤基因疫苗(即DNA疫苗)是近年来国内外研究的热点,它主要通过激活患者自身免疫系统,以达到清除或控制肿瘤的目的。
研究目的：本实验将HBV X基因,MAGE-1基因与载体pVAX-1相连,构建针对肝癌的治疗性重组基因疫苗pVAX-aHCC并通过高效液相的方法将其纯化。旨在建立重组DNA疫苗的实验室制备工艺,并对该纯化重组DNA疫苗进行初步的质量控制及检定,为基因疫苗的药理学研究及规模化生产奠定基础。
研究方法：(1)利用真核表达载体质粒pVAX-1,将HBV X基因,MAGE-1基因与其相连,构建针对肝癌的治疗性重组基因疫苗pVAX-aHCC。(2)将已构建好的治疗性肝癌重组DNA疫苗(pVAX-aHCC)转化大肠杆菌JM109。制备LB种子液,摇瓶培养,收集菌体,以碱裂解、回收等方法处理后得到质粒DNA的粗制品。(3)利用色谱原理,通过Sepharose 6FF, Plasmid Select, DEAE Sepharose FF层析系统对其进行纯化,并按照FDA推荐的临床用DNA的质量监控标准,对纯化重组DNA疫苗进行质量控制及检定。
研究结果：(1)本试验成功构建了真核重组质粒pVAX-aHCC。(2)每1L发酵菌可获得12mg质粒,纯度OD260/280为1.79,超螺旋质粒比例为91.7%。经检测,纯化的质粒DNA疫苗中无RNA、菌体蛋白质、菌体基因组DNA、细菌内毒素等残留量；热稳定性良好,可以耐受一定范围的高温；并从转化效率、酶切效果等方面进行质量鉴定,结果显示,该制品符合FDA推荐的临床用质粒DNA的质量标准。
研究结论：本实验,成功地利用真核表达载体质粒pVAX-1,构建了新型肝癌治疗性重组DNA疫苗(pVAX-aHCC),并对重组的基因疫苗进行了纯化,经Sepharose 6FF, Plasmid Select, DEAE Sepharose FF层析系统纯化的质粒DNA纯度高,符合FDA推荐的临床用质粒DNA的质量标准。
Background:Anti-cancer (DNA vaccines) is a research hotspot in recent years at home and abroad. It is mainly by activating the patient's own immune system in order to achi eve the purpose of tumor removal or control.
Objective:To study the strategy of cooperative therapy of anti-cancer gene, two anti-tum-or gene, HBV X gene, MAGE-1 gene, were inserted into eukaryotic expressive plasmid vector to construct recombinant (pVAX-aHCC) and design experiment to study the anti-t umor effects of pVAXl-aHCC in vivo and ex vitro. The study established a solid founda-tion of cooperative cancer gene therapy, and has considerable significance in developing safe and/or effective anti-tumor medicine.
Methods:The recombinant expression plasmid of pVAX-aHCC was constructed by inserti-ng HBV X gene and MAGE-1 gene into the downstream of prom-oter (CMV). The con s-tructed recombinant anti-tumor DNA was amplified in JM109 strain of Escherichia coli. After fermated in complex medium, the fermentation technology of pVAX-aHCC was e-st ablished. The Bacterium were collected through centrifugalization and then the plasmid w as prepurified by alkaline lysis, calcium chloride precipitation, PEG precipitation and soo n. Then the plasmid was applied to a Sepharose 6FF, Plasmid Select, DEAE Sepharose FF column, and crude product was successfully isolated. When it comes to plasmid quail-ty and safety, several specifications were assessed by methods recommended by FDA, in-eluding supercoiled form percentage, transformation efficiency, identity, overall yield, puri-ty (OD260/280). Residual impurities levels such as RNA, host protein, host genomic DNA in final product were also detected.
Results:(1) In this study, a new type of liver cancer therapeutic recombinant DNA vacci-ne (pVAX-aHCC) was successfully constructed. (2) The result is as follows, pH 7.2±0. 2, supercoiled form percentage 91.7%, trasfection effiency 6.20×105 (cfu/μg plasmid),OD 260/280 1.79. Host protein, RNA and genomic DNA were undetectable. And the purified pl asmid was stable and can bear a certain high temperature. The result shows that the p-ur ification technology is feasible and it lays a basis for large-scale production of anti tu-m or therapeutic DNA vaccine as well as the others.
Conclusion:In this study, a new type of liver cancer therapeutic recombinant DNA vacci-ne (pVAX-aHCC) was successfully constructed through the use of eukaryotic expressionp lasmid pVAX-1. And recombinant genetic vaccines werepurified by Sepharose 6FF, Plas- mid Select, DEAE Sepharose FF chromato graphy system. The purified plasmid DNA of high purity, consistent with FDA recommended using plasmid DNA of clinical quality sta-ndards.
研究目的：本实验将HBV X基因,MAGE-1基因与载体pVAX-1相连,构建针对肝癌的治疗性重组基因疫苗pVAX-aHCC并通过高效液相的方法将其纯化。旨在建立重组DNA疫苗的实验室制备工艺,并对该纯化重组DNA疫苗进行初步的质量控制及检定,为基因疫苗的药理学研究及规模化生产奠定基础。
研究方法：(1)利用真核表达载体质粒pVAX-1,将HBV X基因,MAGE-1基因与其相连,构建针对肝癌的治疗性重组基因疫苗pVAX-aHCC。(2)将已构建好的治疗性肝癌重组DNA疫苗(pVAX-aHCC)转化大肠杆菌JM109。制备LB种子液,摇瓶培养,收集菌体,以碱裂解、回收等方法处理后得到质粒DNA的粗制品。(3)利用色谱原理,通过Sepharose 6FF, Plasmid Select, DEAE Sepharose FF层析系统对其进行纯化,并按照FDA推荐的临床用DNA的质量监控标准,对纯化重组DNA疫苗进行质量控制及检定。
研究结果：(1)本试验成功构建了真核重组质粒pVAX-aHCC。(2)每1L发酵菌可获得12mg质粒,纯度OD260/280为1.79,超螺旋质粒比例为91.7%。经检测,纯化的质粒DNA疫苗中无RNA、菌体蛋白质、菌体基因组DNA、细菌内毒素等残留量；热稳定性良好,可以耐受一定范围的高温；并从转化效率、酶切效果等方面进行质量鉴定,结果显示,该制品符合FDA推荐的临床用质粒DNA的质量标准。
研究结论：本实验,成功地利用真核表达载体质粒pVAX-1,构建了新型肝癌治疗性重组DNA疫苗(pVAX-aHCC),并对重组的基因疫苗进行了纯化,经Sepharose 6FF, Plasmid Select, DEAE Sepharose FF层析系统纯化的质粒DNA纯度高,符合FDA推荐的临床用质粒DNA的质量标准。
Background:Anti-cancer (DNA vaccines) is a research hotspot in recent years at home and abroad. It is mainly by activating the patient's own immune system in order to achi eve the purpose of tumor removal or control.
Objective:To study the strategy of cooperative therapy of anti-cancer gene, two anti-tum-or gene, HBV X gene, MAGE-1 gene, were inserted into eukaryotic expressive plasmid vector to construct recombinant (pVAX-aHCC) and design experiment to study the anti-t umor effects of pVAXl-aHCC in vivo and ex vitro. The study established a solid founda-tion of cooperative cancer gene therapy, and has considerable significance in developing safe and/or effective anti-tumor medicine.
Methods:The recombinant expression plasmid of pVAX-aHCC was constructed by inserti-ng HBV X gene and MAGE-1 gene into the downstream of prom-oter (CMV). The con s-tructed recombinant anti-tumor DNA was amplified in JM109 strain of Escherichia coli. After fermated in complex medium, the fermentation technology of pVAX-aHCC was e-st ablished. The Bacterium were collected through centrifugalization and then the plasmid w as prepurified by alkaline lysis, calcium chloride precipitation, PEG precipitation and soo n. Then the plasmid was applied to a Sepharose 6FF, Plasmid Select, DEAE Sepharose FF column, and crude product was successfully isolated. When it comes to plasmid quail-ty and safety, several specifications were assessed by methods recommended by FDA, in-eluding supercoiled form percentage, transformation efficiency, identity, overall yield, puri-ty (OD260/280). Residual impurities levels such as RNA, host protein, host genomic DNA in final product were also detected.
Results:(1) In this study, a new type of liver cancer therapeutic recombinant DNA vacci-ne (pVAX-aHCC) was successfully constructed. (2) The result is as follows, pH 7.2±0. 2, supercoiled form percentage 91.7%, trasfection effiency 6.20×105 (cfu/μg plasmid),OD 260/280 1.79. Host protein, RNA and genomic DNA were undetectable. And the purified pl asmid was stable and can bear a certain high temperature. The result shows that the p-ur ification technology is feasible and it lays a basis for large-scale production of anti tu-m or therapeutic DNA vaccine as well as the others.
Conclusion:In this study, a new type of liver cancer therapeutic recombinant DNA vacci-ne (pVAX-aHCC) was successfully constructed through the use of eukaryotic expressionp lasmid pVAX-1. And recombinant genetic vaccines werepurified by Sepharose 6FF, Plas- mid Select, DEAE Sepharose FF chromato graphy system. The purified plasmid DNA of high purity, consistent with FDA recommended using plasmid DNA of clinical quality sta-ndards.
引文
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[2].Ribas A, Butterfield LH, Economous JS. Genetic immunotherapy for cancer[J]. Oneologist. 2000,5(2):87-98.
[3].Tan PH, Chan CL, George AJ. Strategies to improve non-viral vectors potential applications in cli-nical transplantation[J]. Expert OpinBiol Therapy.2006,6(6):619-630.
[4].Madden CR. Slagle BL. Stimulation of cellular proliferation by hepatitis B virus X protein[J]. DisMarkers.2001,17(3):153-157.
[5].Chen YB, Yan ML. Gong JP, et al. Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2 mdrl[J]. Chin Med.2007,120(8):703-707.
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