反义人血管生成素的真核表达载体的构建及其体外功能的初步研究

目的构建含反义人血管生成素的真核表达载体,并转染人肺癌细胞株A549,使之特异性地封闭A549细胞中血管生成素的表达,以达到抑制肿瘤生长的目的.方法用RT-PCR的方法合成血管生成素的cDNA,将其反向克隆入逆转录病毒质粒载体中,通过PA317细胞包装为假病毒颗粒,体外转染肺癌细胞株A549.结果构建出含反义血管生成素的逆转录病毒载体,感染A549细胞后,能有效抑制A549细胞血管生成素蛋白的表达.结论利用反义血管生成素的逆转录病毒载体能够有效抑制培养细胞血管生成素的表达,为今后的体内应用奠定了基础.

Construction of angiogenin antisense eukaryotic expression vector and its effect in vitro

Objective To construct recombinant retroviral vector expressing antisense ANG and transfect it into the human lung cancer cell line A549 to inhibit the expression of ANG.Methods The cDNA of ANG was amplified by using RT-PCR from A549 and cloned in the antisense orientation to pBabe,a retroviral vector.The pseudovirion was produced by PA317 packaging cell transfected by the recombinant retroviral vector and was used to infect A549 cell in vitro.Result After A549 was infected by recombinant retroviral vector expressing antisense ANG,ANG protein level decreased greatly.Conclusion Recombinant retroviral vector expressing antisense ANG can efficiently inhibit ANG expression in A549 cell in vitro,providing the basis for further study in vivo.