牛新孢子虫NcSRS2基因腺病毒穿梭载体的构建及在293细胞中的表达

Construction of bovine Neospora caninum NcSRS2 adenovirus shuttle vector and expression of NcSRS2 gene in 293 cells

应用PCR技术扩增牛新孢子虫NcSRS2基因,纯化PCR产物后与克隆载体pMD18-T Simple Vector连接,将PCR、酶切鉴定及测序分析正确的pMD-18T-NcSRS2重组质粒进行EcoRⅠ和XbaⅠ双酶切,克隆至相同酶切回收后的腺病毒穿梭载体pCR259中,再将PCR、酶切鉴定正确的pCR259-NcSRS2重组质粒转染293细胞,应用IF-AT和Western-blotting技术检测重组质粒在293细胞中的表达情况。结果显示,扩增的牛新孢子虫NcSRS2基因长度为1 227bp,与GenBank中发表的NcSRS2(AF061249)核苷酸序列同源性为99%,构建的pCR259-NcSRS2重组质粒在293细胞中得到瞬时表达,表达蛋白的相对分子质量为43 000,具有较好的反应原性。本试验为新孢子虫病腺病毒载体疫苗的构建奠定了基础。

In this research,bovine Neospora caninum NcSRS2 gene was amplified by PCR.The purified PCR products were ligated with pMD18-T simple vector.After enzyme digestion and sequence analysis,the correct pMD-18T-NcSRS2 gene was digested by EcoRⅠand XbaⅠ,and cloned into the adenovirus shuttle vector pCR259 which was digested by the same enzymes.The correct pCR259-NcSRS2 recombinant plasmid was transfected into 293 cells.The expression of recombinant plasmid in 293 cells was detected by IFAT and Western-blotting.The result shows that the length of bovine Neospora caninum NcSRS2 gene is 1 227 bp.which has 99% homology with sequence and sequence in GenBank.The pCR259-NcSRS2 recombinant plasmid could transiently express in 293 cells.The molecular weight of the expressed protein which has good reactionogenicity is 43 000.This research laid the foundation for the construction of Neospora caninum vector-adenovirus vaccine