牦牛lncRNAENSBGRT00000000387.1慢病毒载体构建及其对卵泡GCs凋亡的影响

doi:10.11843/j.issn.0366-6964.2023.03.019

摘要/Abstract

摘要： 旨在构建牦牛lncRNA ENSBGRT00000000387.1过表达慢病毒载体，并将其感染牦牛卵泡颗粒细胞(granulosa cells，GCs)，了解它对细胞凋亡的影响。本研究提取牦牛卵泡RNA，以RT-PCR技术扩增lncRNA ENSBGRT00000000387.1全长序列，而后构建其重组质粒，并以慢病毒包装系统(pVSVG:pMDL:pRev) 包装后利用qPCR法测定在293T细胞上的感染滴度，再感染分离培养的牦牛GCs，以qPCR检测GCs中lncRNA ENSBGRT00000000387.1的表达水平，并以流式细胞仪检测GCs的凋亡率，以Western blot和qPCR分别检测促凋亡基因CASPASE3、BAX和抑凋亡基因BCL-2的mRNA和蛋白表达水平。结果显示，由牦牛卵泡中成功扩增出lncRNA ENSBGRT00000000387.1的全长2 566 bp序列，将其与LV-EF1a-EGFP-2A载体同源区的片段同源重组，经酶切鉴定及测序验证表明成功构建了重组质粒，将之经慢病毒包装系统包装和浓缩后，在293T细胞的感染效率达(75.77±0.850)%，qPCR测定结果显示该慢病毒滴度为7.32×10⁸ TU·mL^-1，表明目的lncRNA过表达慢病毒载体成功构建；以该慢病毒感染牦牛卵泡GCs的感染效率达(52.93±1.168)%，qPCR结果显示感染GCs中目的lncRNA表达水平显著升高(P<0.01)；流式细胞仪检测发现慢病毒感染组细胞凋亡率显著下降(P<0.01)，促凋亡基因CASPASE3和BAX在mRNA和蛋白水平的表达量显著降低(P<0.01)，而抑凋亡基因BCL-2表达量显著升高(P<0.01)。本试验成功构建了lncRNA ENSBGRT00000000387.1慢病毒载体，成功感染牦牛卵泡GCs后发现其具有抑制细胞凋亡的作用，说明其可能在牦牛卵泡功能中发挥作用。

关键词: lncRNA, 慢病毒载体, 颗粒细胞, 凋亡, 牦牛

Abstract: The purpose of this study was to construct yak lncRNA ENSBGRT00000000387.1 overexpression lentivirus vector and infect yak follicular granulosa cells (GCs) to understand its effect on cell apoptosis. The yak follicular RNA was extracted, and the full-length sequence of lncRNA ENSBGRT00000000387.1 was amplified by RT-PCR, and then its recombinant plasmid was constructed. After it was packaged with lentivirus packaging system (pVSVG: pMDL: pRev), the infective titer on 293T cells was determined by qPCR, and then the isolated and cultured yak GCs were infected. The expression level of lncRNA ENSBGRT00000000387.1 in GCs was detected by qPCR, and the apoptosis rate of GCs was detected by flow cytometry. Western blot and qPCR were used to detect the mRNA and protein expression levels of pro-apoptotic gene CASPASE3, BAX and anti-apoptotic gene BCL-2, respectively. The full-length sequence of 2 566 bp of lncRNA ENSBGRT00000000387.1 was successfully amplified from yak follicles, and it was homologously recombined with the fragment of the homologous region of LV-EF1a-EGFP-2A vector. The restriction enzyme digestion identification and sequencing verification showed that the recombinant plasmid was successfully constructed. After it was packaged and concentrated in the lentivirus packaging system, the infection efficiency in 293T cells reached (75.77±0.850) %. The qPCR test results showed that the titer of the lentivirus was 7.32×10⁸ TU·mL^-1, indicating that the target lncRNA overexpression lentivirus vector was successfully constructed. The infection efficiency of yak follicular GCs infected with lentivirus was (52.93±1.168)%. The qPCR results showed that the expression level of target lncRNA in infected GCs was significantly increased (P<0.01). Flow cytometry showed that the apoptosis rate of cells in lentivirus infection group decreased significantly (P<0.01), the expression of pro-apoptotic genes CASPASE 3 and BAX at mRNA and protein levels decreased significantly (P<0.01), and the expression of anti-apoptotic gene BCL-2 increased significantly (P<0.01). In this experiment, lncRNA ENSBGRT00000000387.1 lentivirus vector was successfully constructed. After successfully infecting yak follicular GCs, it was found to have the effect of inhibiting apoptosis, indicating that it may play a role in yak follicular function.

Key words: lncRNA, lentivirus vector, granulosa cells, apoptosis, yak

中图分类号:

S823.85

孟朝轶, 王运路, 徐业芬, 牛家强, 索朗斯珠, 郭敏, 席广银. 牦牛lncRNAENSBGRT00000000387.1慢病毒载体构建及其对卵泡GCs凋亡的影响[J]. 畜牧兽医学报, 2023, 54(3): 1058-1070.

MENG Zhaoyi, WANG Yunlu, XU Yefen, NIU Jiaqiang, SUOLANG Sizhu, GUO Min, XI Guangyin. Construction of Yak lncRNA ENSBGRT00000000387.1 Lentivirus Vector and Its Effect on Apoptosis of Yak Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1058-1070.

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