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目的探讨慢病毒介导LOC100132354过表达载体的构建及鉴定。方法化学合成LOC100132354全基因序列,与GV303载体进行连接反应获得重组质粒;进一步制备感受态细胞,并行转化实验,PCR鉴定进行转化子,对阳性转化子进行测序;构建好的LOC100132354过表达载体转染至293T细胞包装病毒,采用荧光显微镜观察荧光情况,用q PCR检测LOC100132354的表达情况。结果酶切结果与预期一致,阳性转化子克隆测序结果与LOC100132354的RNA序列完全一致,293T细胞转染后荧光表达结果较强,q PCR结果显示293T细胞转染过表达载体后LOC100132354表达水平为对照组的555 427.148倍,差异有统计学意义(P<0.001)。结论成功构建了慢病毒介导LOC100132354过表达载体,为后续进一步研究提供了基础。
Objective To investigate the construction and identification of lentivirus-mediated LOC100132354 overexpression vector. Methods LOC100132354 gene was chemically synthesized and ligated with GV303 vector to obtain a recombinant plasmid. Competent cells were further prepared and transformed in vitro. Transformants were identified by PCR and the positive transformants were sequenced. The constructed LOC100132354 overexpression vector was transfected To 293T cell packaging virus, the fluorescence observed by fluorescence microscopy, qPCR detection of LOC100132354 expression. Results The results of digestion were consistent with the expected results. The results of positive clones were exactly the same as those of LOC100132354. The results of qPCR showed that the expression of LOC100132354 in transfected 293T cells was higher than that in control Group 555 427.148 times, the difference was statistically significant (P <0.001). Conclusion The lentivirus-mediated LOC100132354 overexpression vector was successfully constructed, which provided the basis for further research.