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目的：构建可用于肿瘤基因治疗研究的基因表达载体ｐＣＥＰｍＢ７，并研究该质粒转染小鼠宫颈癌Ｕ１４细胞后的体内致瘤性变化。方法：（１）用限制性核酸内切酶ＨｉｎｄⅢ和ＸｈｏⅠ分别对质粒ｐＣＥＰ４和ｐＬＸＳＮｍＢ７进行双酶切，分别回收１０．４ｋｂ的线性ｐＣＥＰ４ＤＮＡ片断和０．９ｋｂ的ｍＢ７ｃＤＮＡ片断，将上述两片断混合，在Ｔ４－ＤＮＡ连接酶的作用下连接，连接液转化受体菌ＴＧ－１，琼脂糖凝胶电泳鉴定连接是否正确；（２）用电穿孔法将ｐＣＥＰｍＢ７转染Ｕ１４细胞，转染后的细胞经ＨｙｇｒｏｍｙｃｉｎＢ（２００μｇ／ｍｌ）选择培养２周后得到选择阳性混合克隆细胞Ｕ１４／ｐＣＥＰｍＢ７，用流式细胞仪检测Ｕ１４／ｐＣＥＰｍＢ７细胞中Ｂ７表达阳性细胞的百分率；（３）将Ｕ１４和Ｕ１４／ｐＣＥＰｍＢ７细胞分别以不同的剂量皮下接种昆明鼠，找出两种细胞的最小成瘤剂量（ｍｉｎｉｍａｌｔｕｍｏｒｉｇｅｎｉｃｄｏｓｅｓ，ＭｉＴＤ）。结果：（１）连接后的新质粒经双酶切和单酶切后，琼脂糖凝胶电泳鉴定连接正确，该质粒即为ｐＣＥＰｍＢ７；（２）流式细胞仪检测结果显示，Ｕ１４细胞的Ｂ７表达阳性率为５．２４％，Ｕ１４／ｐＣＥＰｍＢ７细胞的Ｂ７表达阳性率为４?
OBJECTIVE: To construct a gene expression vector pCEPmB7 that can be used for tumor gene therapy research, and to study the in vivo tumorigenicity of this plasmid after transfection of murine cervical cancer U14 cells. Methods: (1) Plasmids pCEP4 and pLXSNmB7 were double-digested with restriction endonucleases HindIII and XhoI, respectively, and a 10.4 kb linear pCEP4 DNA fragment and a 0.9 kb mB7 cDNA fragment were recovered, and the two fragments were mixed together. Connected by T4-DNA ligase, the adaptor was transformed into TG-1, and the connection was confirmed by agarose gel electrophoresis; (2) pCEPmB7 was transfected into U14 cells by electroporation, and the transfected cells were Two weeks after selection, Hygromycin B (200 μg/ml) was used to obtain positive selection of mixed clonal cells U14/pCEPmB7. The percentage of B7 positive cells in U14/pCEPmB7 cells was detected by flow cytometry; (3) U14 and U14/pCEPmB7 cells were separated. Inoculate Kunming mice subcutaneously with different doses to find the minimal tumorigenicity of both cells (minimal tumorigenicity) ses, MiTD). RESULTS: (1) After the two ligated plasmids were digested by double enzyme and digested by single enzyme, agarose gel electrophoresis confirmed that the connection was correct, the plasmid was pCEPmB7; (2) flow cytometry results showed that U14 cells B7 The positive rate of expression was 5.24%. The positive rate of B7 expression in U14/pCEPmB7 cells was 4?