抗肿瘤治疗性疫苗DNA的构建及纯化工艺研究

发布时间：2018-05-08 01:33

本文选题：VP3 + HN　；参考：《吉林大学》2007年硕士论文

【摘要】： 本研究利用分子生物学、分子免疫学、分子病毒学、生物化学等技术和手段进行了抗肿瘤治疗性重组核酸疫苗的构建及纯化工艺研究。首先将三个抑瘤机制不同的抗肿瘤基因,即鸡贫血病毒VP3基因、新城疫病毒HN基因及IL-18基因,克隆到真核表达载体pVAX1中,构建含有三种基因的真核重组表达质粒pVVP3IL-18HN。将重组表达质粒pVVP3IL-18HN转染人肝癌SMMC7221细胞,通过RT-PCR、western-blot及间接免疫荧光进行分析,结果表明,外源基因VP3基因及IL-18HN嵌合基因在SMMC7221细胞中获得了正确的表达。将已构建的抗肿瘤重组质粒(pVVP3IL-18HN),用发酵罐优化发酵工艺,通过阴离子交换层析方法(Q Sepharose XL)进行纯化,将得到的质粒DNA通过Source 15Q进行进一步的纯化及脱盐,浓缩。质量鉴定结果表明,通过本研究建立的制备工艺纯化获得的质粒DNA,外观澄清透明,无絮状颗粒;pH值为7.2±0.2;超螺旋质粒比例为87.5%;转化效率为6.20×10~5(cfu/μg质粒);纯度(OD 260/280)为1.86;未检测到宿主蛋白质、宿主基因组、RNA。本实验证明了所建立制备工艺的可行,为DNA疫苗的大规模生产提供了可借鉴的技术数据。
[Abstract]:In this study, molecular biology, molecular immunology, molecular virology, biochemistry and other techniques were used to construct and purify the recombinant nucleic acid vaccine of anti tumor therapeutic recombinant nucleic acid.
First, three antitumor genes, chicken anemia virus VP3 gene, Newcastle disease virus HN gene and IL-18 gene, were cloned into eukaryotic expression vector pVAX1, and the recombinant expression plasmid pVVP3IL-18HN. containing three genes was constructed to transfect the recombinant expression plasmid pVVP3IL-18HN into human hepatoma SMMC7221 cells by RT-PCR, weste Rn-blot and indirect immunofluorescence analysis showed that the foreign gene VP3 and IL-18HN chimeric genes were correctly expressed in SMMC7221 cells.
The constructed anti tumor recombinant plasmid (pVVP3IL-18HN) was optimized by the fermentation tank and purified by the anion exchange chromatography (Q Sepharose XL). The obtained plasmid DNA was further purified and desalted and concentrated by Source 15Q. The quality identification results showed that the preparation process was obtained through the preparation process of this study. Plasmid DNA was clear and transparent with no floc particles, pH value was 7.2 + 0.2, the ratio of super helix plasmid was 87.5%, the conversion efficiency was 6.20 x 10~5 (cfu/ g plasmid), and the purity (OD 260/280) was 1.86. The host protein and host genome were not detected, and RNA. experiment proved that the preparation process was feasible, which provided the large scale production of DNA vaccine. Technical data for reference.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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