稳定表达促红细胞生或素的重组腺病毒的构建及其对大鼠的促红细胞生成作用

用EPO基因组基因构建了腺病毒质粒型载体psp1B/hEPO,该质粒含有以RSV-LTR为启动子的完整的EPO基因表达盒.单独转染CHO细胞,经暂态表达检测到EPO的表达。用psp1B/hEPO与腺病毒拯救型载体pBHG11共转染293细胞,获得了表达EPO的重组腺病毒AdhEPO.经Southern杂交证实AdhEPO中有EPO表达盒,ELISA检测到了EPO阳性表达.用5×108pfu的AdhEPO给大鼠作一次性肌肉注射,观察到了其促进大鼠红细胞生成的短期效应。在注射后第1,3,5,7,10d分别检测了大鼠的红细胞压积、血红蛋白含量和红细胞计数等指标,发现大鼠的红细胞数量显著提高。在第10d红细胞压积从46±4%上升至65±6%。证实了重组腺病毒AdhEPO具有潜在的临床应用价值,可用于贫血症的基因治疗。

Construction of EPO Stable Expressing Recombinant Adenovirus and Its Erythropoiesis Effect on Rats

Erythropoietin expressing plasmid type adenovirus vector psp1B/hEPO was constructed.A completed EPO expression cassette promoted by RSV LTR was inserted into the Eco R Ⅴ site of the adenovirus vector pΔE1SP1B.The transient EPO expressing capability was detected by transfecting the plasmid of psp1B/hEPO into the CHO cell.By co transfecting psp1B/hEPO with the rescue type adenovirus vector—pBHG11,the recombinant adenovirus AdhEPO was obtained.EPO expressing cassette was found in AdhEPO by Southern blot and positive EPO expression was detected using ELISA method.5×10 8 pfu of AdhEPO was administrated to the SD rat by a single intramuscular injection to investigate the EPO expression and its effect of erythropoiesis.EPO expression and polycythemia were found in a period of 10 days.The hematocrites,hematogloblin and the number of RBC(Red blood cell)of rats were detected after injections of 1,3,5,7 and 10 days.The significant increase of RBC was found.The hematocrites increased from 46±4% to 65±6% after 10 days of injection.It was found that the potential value of clinical application of recombinant adenovirus—AdhEPO could be used for gene therapy of anemia.