丙型肝炎病毒融合抗原基因NS3CE对番茄的遗传转化

Transformation of Hepatitis C Virus Chimeric NS3CE Genes into Tomato

丙型肝炎病毒是危害人类健康重要的病源之一 ,研制安全、经济、有效和易于推广使用的抗病毒疫苗是预防和控制 HCV的有效手段。本实验用 PCR方法克隆了 HCV中能够产生中和性抗体、具有很好免疫原性的非结构 N S3区基因片段及核心抗原 CE基因片段 ,并在两段基因之间加入连接肽 SPGS的密码子序列 ,构建成融合抗原基因 N S3- CE。将该融合基因连接到载体 p BI12 1上 ,构建植物表达载体 p BINS3CE。通过根癌农杆菌 EHA10 5介导获得转基因番茄。并进行了 PCR扩增、Southern杂交及 RT- PCR等分子检测 ,结果证明外源基因已整合到转基因番茄的基因组中 ,并在转录水平得到表达。

Hepatitis C virus(HCV) is one of the most widespread viral disease infected human health. Prevention and control of hepatitis C by using safe, economical and universal vaccination is highly effective. As the target genes NS3 and CE were obtained by PCR amplification. The 5' terminal of CE cDNA fragment was linked up with the 3' terminal of NS3 cDNA fragment by a oligonucleotide linker SPGS to form a chimeric gene NS3CE (2 0kb). The chimeric gene NS3CE was recombined with the expression vector pBI121, and constructed a plant expression vector pBINS3CE. Leaf disks were cut from sterilized leaves of ‘Zhongshu 5’ ( Lycopersicon esculentum ), inoculated with Agrobacterium tumefaciens EHA105 containing NS3CE . Plants were regenerated on selective media with Kanamysin. The presence of the NS3CE gene was confirmed by PCR and Southern blot in plants surviving sections. RTPCR results showed that NS3CE gene has been expressed at the transcription level.