水牛miR-302s真核表达载体的构建及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2019.09.001

摘要/Abstract

摘要： 试验旨在克隆并构建水牛miR-302s慢病毒真核表达载体（bbu-miR-302s），对其进行生物信息学分析，并尝试将该载体应用于水牛体细胞重编程中。以水牛基因组DNA为模板扩增得到bbu-miR-302s前体序列，测序正确后将其连入pLVX-IRES-ZsGreen1构建重组慢病毒真核表达载体。重组的慢病毒真核表达载体经过酶切鉴定正确后，采用脂质体转染方法包装慢病毒颗粒，通过感染HEK-293T细胞及猪和水牛体细胞，检测重组慢病毒载体的有效性。bbu-miR-302s有效感染水牛胎儿成纤维细胞（BFF）后，经诱导培养，检测能否产生水牛诱导多能干细胞（iPSC）。采用CoGeMiR数据库查询法和SnapGene Viewer软件进行miR-302s家族在基因组中的定位分析；利用ClustalX 1.83软件分析miR-302s序列保守性；利用TargetScan和miRWalk软件预测bbu-miR-302s主要的靶基因，并运用DAVID程序对靶基因进行信号通路富集。测序结果显示，扩增获得的序列是水牛miR-302s家族，包装的重组慢病毒滴度为7.2×10⁶TU/mL，并可以有效感染3个物种的体细胞。bbu-miR-302s病毒感染BFF后，细胞经历形态变化并发生聚集形成克隆样，碱性磷酸酶检测阳性，但多能基因及表面抗原检测结果均为阴性，说明单因子miR-302s不足以完全重编程BFF为水牛iPSC，但促进了重编程进程。CoGeMiR数据库检索发现，miR-302s家族主要位于LARP7基因的内含子区；SnapGene Viewer软件进一步分析发现，bbu-miR-302s位于水牛7号染色体LARP7基因的内含子区。序列同源性分析表明，miR-302s家族成员间及miR-302s簇成员在不同物种间均高度保守。水牛miR-302s主要靶基因共255个，这些靶基因主要集中在33个信号通路中，其中PGK信号通路与胰高血糖素信号转导及调控干细胞信号通路最为显著。本研究结果为后续开展miR-302s簇在体细胞重编程中的作用奠定基础。

关键词: bbu-miR-302s; 慢病毒真核载体; 生物信息学分析

Abstract: The aim of this study was to clone and construct a buffalo miR-302s (bbu-miR-302s) lentivirus expression vector,analyze its bioinformatics,and try to apply the vector to buffalo somatic cell reprogramming.The bbu-miR-302s precursor sequence was amplified using buffalo genomic DNA as template,after the TA clone was sequenced,it was ligated into pLVX-IRES-ZsGreen1 lentiviral expression vector.After the vector was identified by enzyme digestion,it was transferred into HEK-293T cells by lipofection to package the lentivirus,and bbu-miR-302s lontivirus infect the HEK-293T and fibroblast cells of pig and buffalo.After buffalo fetal fibroblasts (BFFs) were infected by bbu-miR-302s lentivirus,the production of buffalo induced pluripotent stem cells (iPSC) was detected by induction culture.The location analysis of the miR-302s family in the genome was performed using the CoGeMiR database and SnapGene Viewer,its conservative was analyzed using ClustalX 1.83 software;TargetScan and miRWalk were used to predict the major target genes,and DAVID program was used to enrich the signal pathway of target genes.The sequencing results showed that the amplified sequence was buffalo miR-302s family,the titer of the packaged lentivirus was 7.2×10⁶ TU/mL,which could effectively infect the somatic cells of three species.After BFFs were infected by bbu-miR-302s lentivirus,the cells changed morphologically and gathered to form clones.Alkaline phosphatase was positive,but the results of pluripotency gene and surface antigen were negative,which indicated that single factor miR-302s was not enough to completely reprogram BFF into iPSC,it promoted the reprogramming process.The CoGeMiR database showed that the miR-302s family was mainly located in the intron region of LARP7 gene,SnapGene Viewer software analysis result showed that bbu-miR-302s was located in the intron region of LARP7 gene in buffalo chromosome 7.The miR-302s clusters of different species and members of miR-302s family were highly conserved.A total of 255 major target genes of buffalo miR-302s were predicted,and these target genes were mainly concentrated in 33 pathways,among which PGK signal pathway,glucagon signal transduction and regulation of stem cell signal pathway were the most significant.The results laid a foundation for further research on the role of the miR-302s cluster in somatic cell reprogramming.

Key words: bbu-miR-302s; lentivirus eukaryotic vector; bioinformatics analysis

中图分类号:

乔树叶, 黄时海, 邓彦飞, 邓海莹, 罗婵, 石德顺, 李湘萍. 水牛miR-302s真核表达载体的构建及生物信息学分析[J]. 中国畜牧兽医, 2019, 46(9): 2497-2506.

QIAO Shuye, HUANG Shihai, DENG Yanfei, DENG Haiying, LUO Chan, SHI Deshun, LI Xiangping. Construction of Eukaryotic Expression Vector of Buffalo miR-302s and Its Bioinformatics Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2497-2506.

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