摘要
为了构建隐孢子虫鼠基因型CP15基因的重组真核表达质粒,检测其重组质粒在Hela细胞中的表达。采用PCR方法,从pMD18-T-CP15菌液中扩增了CP15及含寡聚脱氧核苷酸(CpG-ODN)的CP15基因,酶切测序鉴定;将该基因亚克隆至pVAX1载体中,用脂质体介导的方法转染Hela细胞,RT-PCR法、间接免疫荧光法和Western-blot法检测表达蛋白的生物学活性。结果显示,扩增了约360 bp和380 bp的隐孢子虫鼠基因型CP15和含CpG-ODN的CP15基因,酶切测序鉴定成功构建了pVAX1-CP15和含有CpG-ODN的重组真核表达质粒pVAX1-C-CP15。转染Hela细胞后,用RT-PCR法检测到外源基因有效的转录,用间接免疫荧光、SDS-PAGE和Western-blot法检测到外源基因的有效表达。结果表明,构建的重组真核表达质粒能够在Hela细胞中成功转录和表达,并且表达产物具有良好的免疫活性。这为下一步深入研制具有高保护性的隐孢子虫核酸疫苗奠定了基础。
In order to construct recombinant expression plasmids with Cryptosporidium mouse genotype CP15 gene and express them in Hela cell,CPl5 gene and CP15 gene containing CpG-ODN（C CP15） were amplified from pMD18-T CP15 by PCR. The CP15 gene and C-CP15 gene were identified by enzyme digestion and sequencing. Then the CP15 gene and C-CP15 gene were cloned into the pVAX 1 vector. Then Hela cells were transfected with recombinant expression plasmids by Lipofectarnine and assayed by RT- PCR,indirect immunofluorescence,SDS-PAGE and Western blot assay. In results,the CP15 gene was about 360 bp and the C CP15 gene was about 380 bp in size. The recombinant plasmids pVAX1-CP15 and pVAX1 C-CP15 were constructed successfully, and confirmed by enzyme digestion and sequencing. RT-PCR,indiret imnmnofluorescence, SDS PAGE and Western-blot assay showed that the recombinant plasmids were effectively transcripted and expressed in Hela cell,and the expressed product had good immunity activity. The results provide the basis for further research and development of higher protective vaccines against cryptosporidiosis.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2011年第5期519-525,共7页
Chinese Veterinary Science
基金
国家"十一五"高技术研究发展计划(863)项目(2006AA10A207)
国家"十一五"科技支撑计划项目(2007BAD40B05)
上海市科技兴农重点攻关项目[沪农科攻字(2005)第3-4号]
中央级公益性科研院所基本科研业务费专项资金项目(2010JB12)
