表达人GM-CSF重组痘苗病毒抗肿瘤作用的实验研究

EXPERIMENTAL STUDY ON ANTI-TUMOR EFFECT MEDIATED BY GM-CSF EXPRESSED BY RECOMBINANT VACCINIA VIRUS

将ｈＧＭ－ＣＳＦｃＤＮＡ插入含有Ｐ１１ｋ及Ｐ２５ｋ双向启动子的痘苗病毒载体ｐＪ１２０的Ｐ１１ｋ启动子下游，而Ｐ２５ｋ下游则为ＬａｃＺ基因，构建成表达质粒ｐＪ１２０／ＧＭ－ＣＳＦ。以此质粒与痘苗病毒天坛株共转染ＴＫ－１４３细胞，在ＢＵｄＲ存在下，利用蓝斑筛选及纯化获得重组病毒ＲＶＪ１２０／ＧＭ－ＣＳＦ，Ｓｏｕｔｈｅｒｎｂｌｏｔ和ＰＣＲ－Ｓｏｕｔｈｅｒｎｂｌｏｔ结果均证实，重组病毒ＤＮＡ中ＧＭ－ＣＳＦ基因整合，ＴＦ１测活显示重组病毒可表达有活性的ＧＭ－ＣＳＦ，且分泌到上清中。将表达ＧＭ－ＣＳＦ的重组痘苗病毒体外感染肿瘤细胞制备的裂解物，接种于荷瘤大鼠，能明显地抑制大鼠Ｗａｌｋｅｒ实体瘤的生长。

hGM-CSF were inserted into the down stream of P11K promoter of pJ120,which contains LacZ gene under P25K promoter and the flanking vaccinia virus TK gene sequence.The constructed plasmid pJ120/GM-CSF was cotransfected with VV into TK -143cells.Through homologous recombination,RVJ120/GM-CSF was obtained by BUdR and X-gal.It was proved that GM-CSF had been inserted into the recombinants by PCR-Southern blot,and could be detected in the supernatant of cells infected with RVJ120/GM-CSF by MTT assay.The effect of GM-CSF expressed by RVJ120/GM-CSF on the growth of tumor in rats was also studied.It was shown that the lysate prepared with RVJ120/GM-CSF infected cells can evidently inhibit the growth of Walker tumor in rats.