猪带绦虫六钩蚴TSOL18基因密码子优化真核载体的构建及其在体内、外表达的实验研究
发布时间：2018-11-04 16:30
【摘要】： 目的:构建猪带绦虫六钩蚴期密码子优化TSOL18基因的重组核酸疫苗优化PVAX1/TSOL18,观察其在体外CHO-K1仓鼠卵巢细胞亚株中的表达以及经肌肉接种后在小鼠骨骼肌内的表达,为猪带绦虫囊尾蚴病DNA疫苗的进一步研究奠定基础。方法:按照哺乳动物的密码子使用偏好,对猪带绦虫六钩蚴TSOL18基因进行优化,使用GeneOptimizer软件对TSOL18基因的氨基酸编码基因进行优化,但不改变氨基酸。根据优化后的基因序列,由南京金思特科技有限公司进行全基因合成。利用PCR扩增该基因序列,将其插入真核表达载体PVAX1,经酶切,测序鉴定。大量提取重组质粒,将重组质粒优化PVAX1/TSOL18经梭华-SofastTM介导,体外转染CHO-K1细胞,通过RT-PCR、细胞裂解物的SDS-PAGE电泳及Western-blotting分析检测其在细胞内是否有表达,再将密码子优化的PVAX1/TSOL18和PVAX1/TSOL18进行体内转染继以免疫组化法观察其在肌肉组织内的表达效果并作比较。结果:优化密码子的PVAX1/TSOL18重组质粒经酶切电泳和测序结果与预期设计的基因序列一致。真核细胞转染后通过RT-PCR结果显示目的条带约为414bp;真核细胞转染后通过SDS-PAGE电泳及Western-blotting检测表明能够在CHO-K1细胞中表达出约为18kDa的目的蛋白。免疫组化染色后在重组质粒注射部位肌肉组织的肌纤维内显示棕黄色阳性分泌颗粒,并且优化PVAX1/TSOL18组比PVAX1/TSOL18组棕黄色阳性分泌颗粒多,空载体组与空白对照组骨骼肌组织未见明显的棕黄色颗粒。结论:成功构建PVAX1/TSOL18密码子优化基因的真核表达质粒;并且初步揭示了优化PVAX1/TSOL18在体内和体外的表达,优化PVAX1/TSOL18比PVAX1/TSOL18在体内表达量更多,为进一步在体验证密码子优化PVAX1/TSOL18保护性和进行大规模现场研究奠定了基础。
[Abstract]:Objective: to construct a recombinant nucleic acid vaccine to optimize the TSOL18 gene of Taenia solium and to observe its expression in CHO-K1 hamster ovarian cell subline in vitro and in the skeletal muscle of mice after intramuscular inoculation. To lay a foundation for further research on DNA vaccine of cysticercosis solium. Methods: according to the preference of mammalian codon, the TSOL18 gene of Taenia solium was optimized, and the amino acid encoding gene of TSOL18 gene was optimized by GeneOptimizer software, but the amino acid was not changed. According to the optimized gene sequence, the whole gene synthesis was carried out by Nanjing Kinster Technology Co., Ltd. The gene was amplified by PCR and inserted into eukaryotic expression vector PVAX1,. A large number of recombinant plasmids were extracted, and the optimized PVAX1/TSOL18 was transfected into CHO-K1 cells by Sova-SofastTM in vitro. The expression of RT-PCR, cell lysates was detected by SDS-PAGE electrophoresis and Western-blotting analysis. Then the codon optimized PVAX1/TSOL18 and PVAX1/TSOL18 were transfected in vivo and then the expression of codon optimized PVAX1/TSOL18 and PVAX1/TSOL18 in muscle tissue was observed by immunohistochemical method and compared. Results: the PVAX1/TSOL18 recombinant plasmid with optimized codon was sequenced by restriction endonuclease electrophoresis. The result of RT-PCR showed that the target band of eukaryotic cells was about 414bp.After the transfection of eukaryotic cells, SDS-PAGE electrophoresis and Western-blotting detection showed that the target protein was about 18kDa in CHO-K1 cells. After immunohistochemical staining, brown positive secretory granules were found in muscle fibers of muscle tissue at the injection site of recombinant plasmid, and there were more brown positive secretory granules in optimized PVAX1/TSOL18 group than in PVAX1/TSOL18 group. There were no obvious brown granules in skeletal muscle of empty carrier group and blank control group. Conclusion: the eukaryotic expression plasmid of PVAX1/TSOL18 codon optimization gene was successfully constructed. It was revealed that the expression of PVAX1/TSOL18 in vivo and in vitro was optimized, and the expression of PVAX1/TSOL18 was more than that of PVAX1/TSOL18 in vivo, which laid a foundation for further codon optimization of PVAX1/TSOL18 protection and large-scale field research.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
本文编号：2310450
[Abstract]:Objective: to construct a recombinant nucleic acid vaccine to optimize the TSOL18 gene of Taenia solium and to observe its expression in CHO-K1 hamster ovarian cell subline in vitro and in the skeletal muscle of mice after intramuscular inoculation. To lay a foundation for further research on DNA vaccine of cysticercosis solium. Methods: according to the preference of mammalian codon, the TSOL18 gene of Taenia solium was optimized, and the amino acid encoding gene of TSOL18 gene was optimized by GeneOptimizer software, but the amino acid was not changed. According to the optimized gene sequence, the whole gene synthesis was carried out by Nanjing Kinster Technology Co., Ltd. The gene was amplified by PCR and inserted into eukaryotic expression vector PVAX1,. A large number of recombinant plasmids were extracted, and the optimized PVAX1/TSOL18 was transfected into CHO-K1 cells by Sova-SofastTM in vitro. The expression of RT-PCR, cell lysates was detected by SDS-PAGE electrophoresis and Western-blotting analysis. Then the codon optimized PVAX1/TSOL18 and PVAX1/TSOL18 were transfected in vivo and then the expression of codon optimized PVAX1/TSOL18 and PVAX1/TSOL18 in muscle tissue was observed by immunohistochemical method and compared. Results: the PVAX1/TSOL18 recombinant plasmid with optimized codon was sequenced by restriction endonuclease electrophoresis. The result of RT-PCR showed that the target band of eukaryotic cells was about 414bp.After the transfection of eukaryotic cells, SDS-PAGE electrophoresis and Western-blotting detection showed that the target protein was about 18kDa in CHO-K1 cells. After immunohistochemical staining, brown positive secretory granules were found in muscle fibers of muscle tissue at the injection site of recombinant plasmid, and there were more brown positive secretory granules in optimized PVAX1/TSOL18 group than in PVAX1/TSOL18 group. There were no obvious brown granules in skeletal muscle of empty carrier group and blank control group. Conclusion: the eukaryotic expression plasmid of PVAX1/TSOL18 codon optimization gene was successfully constructed. It was revealed that the expression of PVAX1/TSOL18 in vivo and in vitro was optimized, and the expression of PVAX1/TSOL18 was more than that of PVAX1/TSOL18 in vivo, which laid a foundation for further codon optimization of PVAX1/TSOL18 protection and large-scale field research.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
【引证文献】
相关硕士学位论文 前1条
1 刘立营;基于全基因合成技术的南极假丝酵母脂肪酶B基因的密码子优化研究[D];华中科技大学;2011年
，本文编号：2310450
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