山羊β防御素124定位及过表达附睾头上皮细胞系的建立

Localization of β-Defensin 124 and Construction of OverexpressedEpithelial Cell Strain of Epididymal Caput of Goats

为研究山羊β防御素124(BD124)的生物学功能,进行附睾头上皮细胞BD124的定位研究,并建立BD124过表达附睾头上皮细胞系,应用细胞免疫荧光法对山羊附睾头上皮细胞BD124进行定位;将山羊BD124基因编码区序列连接到LV5穿梭质粒后,测序验证LV5-BD124质粒构建情况,然后将其与包装质粒共转染293T细胞进行包装生产并重组LV5-BD124慢病毒,并采用有限稀释法测定病毒滴度;之后采用实时荧光定量PCR和Western blot检测确认LV5-BD124慢病毒侵染96、120 h后山羊附睾头上皮细胞中BD124 mRNA和蛋白的过表达情况;最后用嘌呤霉素进行2次筛选后获得BD124过表达稳转附睾头上皮细胞系。定位结果显示,BD124阳性绿色荧光信号在细胞核周围高度聚集,且在细胞质和细胞核内也有分布。测序结果显示,BD124序列插入LV5慢病毒载体后,表达的LV5-BD124慢病毒载体滴度为3.0×108 TU/mL。荧光定量PCR和Western blot结果显示,LV5-BD124附睾头上皮BD124 mRNA和蛋白表达量显著高于对照组;LV5-BD124侵染后的附睾头上皮细胞经2次嘌呤霉素筛选后,阳性细胞比例增加,传2代后细胞内荧光信号保持稳定。

To study the biological function ofβ-Defensin 124(BD124)of goats,conduct stduy on localization of BD124 of epididymal caput epithelial cell,construct the BD124 overexpressed epithelial cell strain of epididymal caput,in this study,the localization of BD124 of epididymal caput epithelial cell of goats was carried out with cell immunofluorescence.The complete coding sequence of the caprine BD124 was firstly ligated into the LV5 shuttle plasmid,verifying construction of LV5-BD124 plasmid by sequencing.Then,the recombinant LV5-BD124 shuttle plasmid and packaging plasmid were co-transfected into 293T cell to produce recombinant LV5-BD124 lentivirus,the titer of the lentivirus was determined by limiting dilution assay.Subsequently,the overexpression of BD124 mRNA and protein was determined by qRT-PCR and Western blot,after caprine epididymal caput epithelial cells were infected with recombinant LV5-BD124 lentivirus for 96 h and 120 h,respectively.The epididymal caput epithelial cell strain with the stable overexpression of BD124 was obtained by two times of screenings with puromycin.The results of localization showed that positive green fluorescence(FITC)signals of BD124 were highly assembled around the nucleus,as well as distributed in the cytoplasm and nucleus.The sequencing results indicated that after BD124 sequence was inserted into the LV5 lentivirus vector,and the virus titer of the expressed LV5-BD124 lentivirus vector was 3.0×108 TU/mL.qRT-PCR and Western blot results demonstrated that expression of BD124 mRNA and protein in epididymal caput epithelial cells of the LV5-BD124 was significantly higher than that of the control group.After the epididymal caput epithelial cells infected by LV5-BD124 were screened two times with puromycin,proportion of positive cells increased and the intracellular fluorescence signal remained stable after 2 generations of passages.