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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.
OBJECTIVE: To enhance the expression of MHC class I molecules on the surface of tumor cells and mobilize the immune system to more effectively inhibit and kill tumor cells, and to construct a recombinant retroviral vector containing the IRES-linked human TNFα and IFNβ genes under the control of the HLA-B7 promoter. METHODS AND RESULTS: The TNFα cDNA was cloned into pBIuescript sk(+) and the target site was cloned and cloned into a plasmid containing the HLA-B7 promoter. The pGL3B7TNF.IFNβ cDNA was then inserted into the pGEM-7Zf(+) exchangeable enzyme. The excision site was excised, and the cloned human plasmid containing the internal ribosome entry site (IRES) was constructed, and the pIRE-SIFN.B7pro-TNFα and IRES-IFNβ gene fragments were respectively cut into their respective vectors and inserted again. The vector was changed into restriction sites and inserted into pBlucscript sk(+) to construct pBLB7TNIRIF. The B7pro-TNFα-IRES-IFNβ DNA fragment was recovered by digesting with SacI+SalI+PvuI, and bluntly inserted into the GINa reverse transcriptase after filling. The BamH I filling site of the viral vector was constructed into a recombinant retroviral vector GINNaB7TNIRIF(+). Conclusion: The construction of this vector lays the foundation for the regulation of enhanced expression of MHC class I molecules in various tumor cells, killing and inhibiting tumor research.