用逆转录病毒载体表达嗜亲性病毒受体基因

Expression of Receptor Gene for Ecotropic MuLVs in Packaging Cells Using a Retroviral Vector

将含有嗜亲性鼠白血病病毒（ＥｃｏＭｕＬＶ）受体基因开放读码框架的ＤＮＡ片段（Ｗ１），克隆到逆转录病毒表达载体ｐＺｉｐ－ＮｅｏＳＶ（ｘ）的５＇－端长末端重复序列（ＬＴＲ）的ＢａｍＨＩ位点，构建成重组质粒ｐＺｉｐ－Ｗ１。用磷酸钙沉淀法将重组质粒转染到辅助包装细胞ＧＰ＋ｅｎｖＡｍ１２和ＧＰ＋Ｅ８６中，经Ｇ４１８筛选，２周后获得的抗性细胞克隆能有效地表达嗜亲性鼠白血病病毒的受体基因，该细胞培养２４ｈ后收集上清，分别做ＸＣ蚀斑和ＩＤ病灶实验，均证明该上清液内有毒力较强的ＥｃｏＭｕＬＶ。

An expression plasmid pZip W1 was constructed by insersion of a DNA fragment containing receptor gene for Eco MuLV open reading frames into BamHI site downstream 5' LTR of retroviral vector pZip Neo SV(x).The plasmid pZip W1 was transfected into Am12 and E86 packaging cells by calcium phosphate precipitation technique and cells resistant against G418 could efficiently express the receptor gene for Eco MuLVs after selecting with G418 for 2 weeks.After 24 hours culturing of the transfected cells,the supernatant was harvested then the supernatant was titrated by XC plaque assay and ID focus assay.The high titre of Eco MuLVs was found in the supernatant.