禽传染性支气管炎病毒S_1基因玉米表达载体的构建

Maize Expression Vector Construction of S1gene of Avian Infectious Bronchitis Virus

禽传染性支气管炎病毒主要编码3种结构蛋白,其中S蛋白的S1蛋白是宿主的主要保护性抗原,能诱导机体产生中和抗体和血凝抑制抗体,并与病毒的组织嗜有关。本研究选取具有代表性的禽传染性支气管炎病毒分离株SAIB14和SAIB4,以其S1基因克隆质粒pUCSAIB14和pUCSAIB4为模板,用具有高保真度的pfu酶分别扩增,得到含先导序列的S1基因片段。把扩增产物分别通过ClaI和BamHI酶切纯化,替换中间载体pUGFPocs的GFPm1基因,构建中间表达载体pUSAIB14和pUSAIB4。用KpnI和HindIII双酶切重组质粒pUSAIB14和pUSAIB4,回收Ubi-S1-Tocs片段,定向插入到pCAMBIA1300载体的多克隆位点。经酶切和PCR鉴定,证明获得了S1基因的玉米表达载体pCUSAIB14和pCUSAIB4。外源基因导入受体植物能否有效表达,表达载体构建是关键因素,构建时选用表达载体的类型、启动子和终止子、目的基因插入位点等都有影响。本研究用BamHI和ClaI双酶切载体pUGFPocs,用S1基因扩增片段替换GFPm1基因,将S1基因置于Ubi启动子和T-ocs终止子的控制之下,有助于S1蛋白的高效表达,最后将Ubi-S1-Tocs单元HindIII和KpnI双酶切下来,插入到pCAMBIA1300的多克隆位点,得到可用于农杆菌介导的转化或直接转化的植物表达载体pCUSAIB4和pCUSAIB14。Ubi启动子来自玉米,属组成型启动子,?

The S 1 gene of avian infectious bronchitis virus(SAIB 4 and SAIB 14 )were amplified by PCR with high-fidelity poly-erase(pfu)and special primers with ClaI and BamHI,The purified S1PCR product and pUGFPocs were digested by ClaI and BamHI respectively,ligeat and transformed into E.coli.JM109strain.The result of PCR and enzyme digestion of plasmid proved that the recombination vector(named pUSAIB 4 and pUSAIB 14 )was obtained.The pUSAIB 4 and pUSAIB 14 was digested by KpnI and HindIII respectively.The Ubi-S 1 -ocs fragment was taken out,inserted into the multi-cloning site of pCAMBIA1300,The result of PCR and enzyme digestion of plasmid proved that recombinatin vector was obtained(named pCUSAIB 4 and pCUSAIB14).The successful construction of pCUSAIB 4 and pCUSAIB 14 vector is the key foundation of S 1 gene expressed in maize.