犬新孢子虫吉林株SAG1基因真核表达载体的构建

根据GenBank中已发表的犬新孢子虫SAG1基因序列(AF132217),设计了一对含有Kozak序列、起始密码子、终止密码子、BamHⅠ和EcoRⅠ酶切位点的引物。以含有SAG1基因的重组质粒pMD-18T-SAG1为模板,经PCR扩增获得SAG1基因,利用BamHⅠ和EcoRⅠ酶切该片段,回收含有BamHⅠ和EcoRⅠ酶切位点粘性末端的SAG1基因片段,将此基因片段克隆至相同酶切回收后的pVAX1真核表达载体中,获得重组质粒pVAX1-SAG1,经PCR鉴定、酶切分析和重组质粒的序列测定、比较,证实了重组质粒的正确性。

Construction of an eukaryotic expression vector of SAG1 gene from Neospora caninum Jilin strain

Based on the SAG1 gene sequence of Neospora caninum in the GenBank(AF132217),a pair of primers containing Kozak sequence,initiation codon,termination codon,and BamHⅠ and EcoRⅠ enzyme digestion sites were designed.Using the recombinant plasmid pMD-18T-SAG1 containing SAG1 gene as template,the SAG1 gene was amplified by PCR.The PCR product was digested with BamHⅠand EcoRⅠ,and ligated into the digested eukaryotic expression vector pVAX1 with the same restriction endonuclease.Finally the recombinant plasmid named pVAX1-SAG1 was obtained.Further verification was performed by enzyme digestion,PCR amplification and sequencing.