供转导PDGF-A基因的高滴度逆转录病毒的制备

目的制备高滴度供转导血小板衍生生长因子Ａ链（ＰＤＧＦ－Ａ）基因的重组逆转录病毒，为体内、外深入研究该基因表达产物自分泌和旁分泌的生物学功能奠定基础。方法将人ＰＤＧＦ－ＡｃＤＮＡ片段插入含巨细胞病毒（ＣＭＶ）启动子序列的逆转录病毒ＧＩＮａ载体的多克隆位点中，将构建的重组质粒导入病毒包装细胞ＰＡ３１７中，经Ｇ４１８筛选并扩增，以病毒液感染ＮＩＨ３Ｔ３细胞，测病毒滴度，选择出抗性克隆，进行免疫组织化学检测，表达产物经ＳＤＳ－ＰＡＧＥ电泳、Ｗｅｓｔｅｒｎｂｌｏｔ分析和３Ｈ－ＴｄＲ掺入法测生物学活性。结果病毒滴度最高为１．４×１０５ＣＦＵ／ｍｌ；免疫荧光染色法和ＳＤＳ－ＰＡＧＥ电泳、Ｗｅｓｔｅｒｎｂｌｏｔ证实ＰＤＧＦ－ＡＡ的表达；抗性细胞条件培养基的细胞活性高达５１．２Ｕ／（１０６·ｄ）。结论供转导ＰＤＧＦ－Ａ基因的高滴度逆转录病毒得以制备，该基因获得有效表达，表达产物具有明显的致丝裂活性。

Preparation of the high-titer retroviral viruses for transducting PDGF-A gene

OBJECTIVE: To prepare high-titer recombinant retrovirus for transducting platelet-derived growth factor A chain(PDGF-A) gene, which will pave the way for future research in vitro and in vivo on biological function of autocrine and paracrine of PDGF-A gene expression product. METHODS: The cDNA fragment of human PDGF-A was inserted into the polyclonal sites of retroviral vector GINa containing cytomegalovirus (CMV) promotor. The constructed recombinant plasmid was transformed into PA317 packaging cell line. The positive clones were obtained by selection in G418-medium and amplified. The supernatant virus was used to infect NIH3T3 cells and titrated. After drug resistance selection, NIH3T3 cell clones were amplified, and a histochemical analysis was performed. SDS-PAGE electrophoresis and Western blot as well as 3H-TdR incorporation methods were adopted to assay the presence and the mitogenic activity of the PDGF in the conditioned medium. RESULTS: The highest titer of the virus was 1.4 x 10(5) CFU/ml. The PDGF-A expression was verified with SDS-PAGE electrophoresis and Western blot and immunofluorescent staining. The cell mitogenic activitiy in the media was up to 51.2 U/(10(6).d). CONCLUSIONS: We had made high-titer recombinant retrovirus for transducting human PDGF-A gene. The gene was expressed with high efficiency. The expressed product has significant mitogenic effect on NIH3T3 cell line.