CAJ | 학술논문

[目的]在体外分离培养牛乳腺上皮原代细胞，并构建牛白细胞介素-17（ bIL-17）基因真核表达质粒，观察重组质粒在牛乳腺上皮细胞的表达．[方法]提取牛脾脏细胞总RNA，通过RT-PCR扩增bIL-17，将bIL-17连入pMD19-T进行测序，测序成功后将bIL-17插入含有增强型绿色荧光蛋白报告基因的真核表达质粒pEGFP-N3上，构建真核表达质粒pEGFP-N3-bIL-17．用脂质体法将pEGFP-N3-bIL-17质粒转染于牛乳腺上皮细胞，荧光显微镜观察绿色荧光蛋白在细胞中的表达，RT-PCR检测bIL-17基因在细胞内的转录，定量ELISA检测bIL-17蛋白在细胞内的表达情况．[结果和结论]成功构建具有绿色荧光和新霉素抗性的双选择标记的牛白细胞介素-17真核表达载体，并可在牛乳腺上皮细胞成功表达．
[목적]재체외분리배양우유선상피원대세포，병구건우백세포개소-17（ bIL-17）기인진핵표체질립，관찰중조질립재우유선상피세포적표체．[방법]제취우비장세포총RNA，통과RT-PCR확증bIL-17，장bIL-17련입pMD19-T진행측서，측서성공후장bIL-17삽입함유증강형록색형광단백보고기인적진핵표체질립pEGFP-N3상，구건진핵표체질립pEGFP-N3-bIL-17．용지질체법장pEGFP-N3-bIL-17질립전염우우유선상피세포，형광현미경관찰록색형광단백재세포중적표체，RT-PCR검측bIL-17기인재세포내적전록，정량ELISA검측bIL-17단백재세포내적표체정황．[결과화결론]성공구건구유록색형광화신매소항성적쌍선택표기적우백세포개소-17진핵표체재체，병가재우유선상피세포성공표체．
[Objective] To construct bovine interleukin-17 ( bIL-17 ) gene eukaryotic expression vector and detect its expression in bovine mammarily epithelial cells which were primarily cultured in vitro.[Method] Bovine interleukin-17 gene (bIL-17) was amplified from the spleen tissue of cow by RT-PCR, which was inserted into pMD19-T vector and then sequenced .The bIL-17 was inserted into an expression vector pEGFP-N3 carrying the enhanced green fluorescent protein to generate a new plasmid pEGFP-N3-bIL-17.The pEGFP-N3-bIL-17 eukaryotic expression vector was transfected into bovine primary mamma-ry epithelial cells with Lipofectamine TM2000 liposome.After the transfection, the green fluorescent pro-tein was observed under fluorescence microscopy , and bIL-17 transcription was examined by RT-PCR and bIL-17 protein expression in cells detected by ELISA methods .[Result and conclusion] The recombinant vector pEGFP-N3-bIL-17 with a green fluorescence and neomycin resistance was constructed and bIL-17 protein could be normally expressed in mammary epithelial cells .