摘要
为探索研制丙型肝炎疫苗的新途径,以期获得防治丙型肝炎的重组腺病毒减毒活疫苗,我们构建了表达丙型肝炎病毒(Hepatitis C virus ,HCV)非结构蛋白3(non structural protein 3,NS3)抗原的重组腺病毒RAd NS3,并检测其在体外表达。应用PCR从真核表达质粒pRC/NS3 中扩增编码HCV NS3 蛋白(329-935aa)的基因片段,定向克隆到重组腺病毒AdEasy-1系统的穿梭质粒pAdTrack CMV上,采用细菌内同源重组"两步转化法"构建携带HCV NS3基因的重组腺病毒基因组质粒pAd HCV NS3,转染293 细胞,成功包装出重组腺病毒RAd NS3,利用它有效地感染人肝癌细胞株HepG2,经RT PCR及免疫印迹等不同方法检测表明,被感染细胞能表达HCVNS3蛋白,为后续进行重组腺病毒在动物体内诱导抗HCV免疫应答能力的研究奠定了基础。
To develop an attenuated recombinant adenoviral vaccine against Hepatitis C virus infection , we construct the recombinant adenoviral vector expressing non-structural protein 3(NS3) antigen of HCV(RAd-NS3). The HCV NS3 gene enconding for 329-935aa was amplified from the plasmid pRC/NS3 by PCR and cloned into the adenoviral shuttle plasmid pAdTrack-CMV. A two-step transformation protocol was employed for the construction of recombinant adenoviral plasmid pAdEasy-HCV NS3. The identified recombinant DNA was transfected into 293 cells to package adenovirus. From cell lysis , the presence of recombinant adenovirus was continued by PCR, and RT-PCR demonstrated the transcription of NS3 in HepG2 cells infected with RAd-NS3, and Western blot revealed that NS3 protein can express in infected HepG2 cells. These results indicated that tumor cells infected with recombinant adenovirus vector RAd-NS3 can express NS3 antigen ,which will be potentially used as a candidate vaccine for the prophylaxis and treatment of HCV infection.
出处
《中国病毒学》
CAS
CSCD
2005年第2期101-104,共4页
Virologica Sinica
基金
国家863高技术计划项目资助(No.2002AA214161)
