人乳腺癌细胞cDNA文库的构建及鉴定

Construction and characterization of cDNA library from human breast cancer cell

目的 :应用前期制备的一株乳腺癌单克隆抗体 (M4G3) ,筛选出高表达其相关抗原的乳腺癌细胞系T47D ,并利用此细胞系构建了以λgt11为载体的cDNA文库 ,为完成此乳腺癌特异抗原基因的克隆与表达奠定基础。方法 :在4株乳腺癌细胞中应用M4G3单抗进行免疫组化和WesternBlot检测 ,以其中一株T47D细胞为材料构建以λgt11为载体的cDNA文库 ,并进行文库质量鉴定。结果 :文库含有9 97×105个重组子 ,重组效率达到96 7 % ,插入的cDNA片段平均长度为1 0kb。结论 :与M4G3单抗结合的乳腺癌抗原分子量约为48000dalton。构建的T47D细胞cDNA文库完整 ,具有较好的质量 ,为进一步筛选奠定了基础。

Objective:To clarify the specific antigen matched M4G3McAb of breast cancer prepared previously.Methods:Four strains of breast cancer cells had been cultured in vitro and screened by immunocytochemistry and Western blotting with M4G3ascites.And then one cDˉNA library of breast cancer cell(T47D)has been constructed withλgt11as vector.The quality of library has been identified subsequently.Results:The cDNA library consists of9.97×10 5 inˉdependent clones in which the percentage of recombinant clones is about96.7%.The average size of inserts is1.0kb.Conclusion:The breast cancer antigen specifically combined with M4G3McAb has48000dalton molecule weight.cDNA expression library constructed with good quality lays solid foundation for further screening.In addition,the library is also used for screening other breast cancer relevant genes.