摘要
目的:为增强肿瘤细胞表面MHCⅠ类分子表达,并调动免疫系统更有效地抑制和杀伤肿瘤细胞,构建在HLA-B7启动子调控下,IRES连接的人TNFα和IFNβ基因的重组逆转录病毒载体.方法与结果:将TNFαcDNA克隆人pBIuescript sk(+)中,改换酶切位点后定向克隆人含HLA-B7启动子的质粒中,构建成pGL3B7TNF.IFNβcDNA经定向插入pGEM-7Zf(+)改换酶切位点切下,定向克隆人含内部核糖体进入位点(IRES)的质粒中,构建成pIRE-SIFN.B7pro-TNFα和IRES-IFN β基因片段分别于各自载体酶切取出,再次插入中间载体,改换酶切位点后,先后定向插入pBlucscript sk(+)中,构建成pBLB7TNIRIF.用SacI+SalI+PvuI酶切回收B7pro-TNFα-IRES-IFNβDNA片段,补平后平端连入GINa逆转录病毒载体的BamH I补平位点,构建成重组逆转录病毒载体GINNaB7TNIRIF(+),结论:该载体的构建为调控各种肿瘤细胞MHCⅠ类分子增强表达,杀伤和抑制肿瘤研究奠定了基础.
Aim: In order to increase major histocompatibility complex class I molecule expression on the tumor cells and prime an efficient antitumor immunity, A retroviral Vector containing the IRES linked HuTNFα and IFN β genes under the control of human HLA-B7 inducible promoter was constructed. Methods and Results: Human TNFa cDNA sequence was isolated from pE4 and cloned to pBluescript sk (+) , then released with Sac II /Hind III and ligated to the plasmid containing HLA-B7 promoter to generate pGL3B7TNF.Human IFN β cDNA sequence was isolated from pSPGNHIFNB10 and inserted into pGEM-7Zf(+), then released with Xba I /BamH I and ligated to the plasmid containing an internal ribosome entry site (IRES) to generate pIRESIFN. IRES-IFN β DNA fragment was isolated from plRE-SIFN and inserted into pBluescript sk(+)to generate pBLIRIF. B7pro-TNFa DNA fragment was isolated from pGL3B7TNF and insert to Sac I/EcoR I site of pBIIRIF to generate pBLB7TNIRIF. The Sac I/ Sal I /Pvu I fragment of B7pro-TNFα-IRES-IFN β was isolated, blunted and ligated to BamH I site of retroviral vector G1Na to generate GlNaB7TNIRIF(+) . Conclusion: The constructed vector fund to basis for cancer immune genetherapy.
出处
《胃肠病学和肝病学杂志》
CAS
1998年第4期308-313,共6页
Chinese Journal of Gastroenterology and Hepatology
基金
国家自然科学基金资助项目
基金号:39600172
关键词
干扰素
HLA-B7
逆转录病毒
载体
基因疗法
肿瘤
Interferon
tumor necrosis factor
gene expression
HLA-B7 promoter
retroviral vector
gene therapy
