摘要
目的构建含编码牙龈卟啉单胞菌FimA蛋白和IL-15基因的真核共表达质粒,并检测其在哺乳动物细胞中的表达。方法应用基因重组技术,构建真核表达载体pIRES-fimA和真核共表达载体pIRES-fimA:IL15,通过酶切、PCR及DNA序列测定鉴定获得的质粒,用Lipofectamine2000介导转染CHO细胞,用Westernblot检测重组质粒在哺乳动物中的表达,酶联免疫吸附试验检测培养上清中的蛋白表达。结果PCR扩增获得的fimA和IL-15目的基因与预计相同,定向插入真核表达质粒pIRES中,插入位相正确,未改变阅读框架。转染的CHO细胞能够检测到目的基因的表达,在培养上清中也可以检测到蛋白质的表达。结论本实验成功构建了牙龈卟啉单胞菌FimA蛋白和IL-15的真核共表达质粒pIRES-fimA:IL15,为研制增强免疫应答的抗牙龈卟啉单胞菌DNA疫苗奠定了基础。
Objective To construct a eukaryotic co-expression plasmid plRES-fimA：IL15, which can be used as an immunoreaction-enhancing DNA vaccine against Porphyromonas gingivalis FimA, and investigate its expression in mammalian cells. Methods The eukaryotic co-expression plasmid pIRES-fimA：IL15 was constructed by molecular cloning methods and characterized by restricted endonuclease mapping, PCR and DNA sequencing. The plasmid was transfected into mammalian cell CHO using Lipofectamine 2000. Expression of fimA gene was detected by Western blot and the protein secretion in cultural medium was analyzed by ELISA. Results Endonuclease mapping showed that the target genes fimA and IL-15 obtained by PCR had the same molecular size as predicted. The DNA sequencing data also indicated that inserted fimA gene and IL-15 gene had correct DNA sequence and orientation. The recombined plasmid could express FimA in mammalian cell CHO transfeeted. FimA and 1L-15 could be secreted into cultural supematant detected by ELISA. Conclusion A new eukaryotic co-expression plasmid pIRES-fimA： ILlS was constructed and it could be applied for further immunization in animal as an effective anti-Porphyromonas gingivalis vaccine.
出处
《华西口腔医学杂志》
CAS
CSCD
北大核心
2006年第3期265-268,共4页
West China Journal of Stomatology
基金
国家自然科学基金资助项目(30371323)
山东省自然科学基金资助项目(21350005200337)
