三角帆蚌IGF2基因的亚细胞定位及重组蛋白促体外细胞活性分析  

Subcellular localization of IGF2 gene and analysis of recombinant protein to promote in vitro cellular activity in Hyriopsis cumingii

作　　者：李雪男 冯上乐 王贺 申晓雅 陈一格 白志毅[1,2] 李文娟[1,2] LI Xuenan;FENG Shangle;WANG He;SHEN Xiaoya;CHEN Yige;BAI Zhiyi;LI Wenjuan(National Experimental Teaching Demonstration Center of Aquatic Science,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Freshwater Aquatic Germplasm Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai201306,China)

机构地区：[1]上海海洋大学水产科学国家级实验教学示范中心,上海201306 [2]上海海洋大学农业农村部淡水水产种质资源重点实验室,上海201306

出　　处：《上海海洋大学学报》2022年第6期1335-1341,共7页Journal of Shanghai Ocean University

基　　金：国家重点研发计划(2018YFD0901406);国家自然科学基金(31872565);上海市学术研究带头人计划(19XD1421500)。

摘　　要：胰岛素样生长因子2(insulin-like growth factors 2, IGF2)在多种细胞生长、分化和合成代谢的调节中发挥重要的调控作用。本研究成功构建了淡水珍珠贝三角帆蚌IGF2的重组质粒pEGFP-IGF2和pET32a-IGF2,通过pEGFP-IGF2-细胞转染,表明该蛋白定位在细胞质与细胞核中。将pET32a-IGF2转化到BL21 (DE3)中进行蛋白诱导表达,成功得到其原核表达载体,SDS-PAGE检测显示,在约25ku处有一条清晰的条带,符合目的条带大小(26.27 ku),且主要表达于上清液中。为研究重组蛋白IGF2对三角帆蚌细胞活性的作用,通过CCK8细胞活性测定,进一步探讨了添加不同质量浓度重组目的蛋白(0、2.5、5.0、7.5、 12.5、17.5、 22.5μg/mL)培养的三角帆蚌外套膜细胞增殖活性。结果显示,三角帆蚌IGF2重组蛋白能显著促进三角帆蚌外套膜细胞的增殖活性(P<0.05),且在其质量浓度为12.5μg/mL时对三角帆蚌外套膜细胞的促生长效果最佳,这为促进三角帆蚌外套膜细胞传代、细胞系建立奠定了基础,也为下一步探究IGF2在贝类中的功能提供依据。Insulin-like growth factors 2(IGF2) plays an important regulatory role in the regulation of growth, differentiation and anabolism in a variety of cells. In this study, the recombinant plasmids pEGFP-IGF2 and PET32 a-IGF2 of freshwater pearl mussel Hyriopsis cumingii were successfully constructed, and showed that the protein was localized in the cytoplasm and nucleus by pEGFP-IGF2-cell transfection. The pET32 a-IGF2 was transformed into BL21(DE3) for protein induction expression, and its prokaryotic expression vector was successfully obtained. SDS-PAGE showed a clear band at about 25 ku, which matched the size of the target band(26.27 ku), and was mainly expressed in the supernatant. In order to investigate the effect of recombinant protein IGF2 on the cellular activity of Hyriopsis cumingii, the proliferation activity of Hyriopsis cumingii mantle cells cultured with different mass concentrations of recombinant target protein(0, 2.5, 5.0, 7.5, 12.5, 17.5, 22.5 μg/mL) was further investigated by CCK8 cell activity assay. The results showed that IGF2 significantly promoted the proliferation activity of Hyriopsis cumingii mantle cells(P<0.05), and the best pro-growth effect was achieved at the mass concentration of 12.5 μg/mL, which laid the foundation for the promotion of Hyriopsis cumingii mantle cells and cell line establishment, and also provided the basis for the next investigation of the function of IGF2 in shellfish.

关 键 词：三角帆蚌 IGF2 亚细胞定位 重组蛋白 细胞培养 生物活性 

分 类 号：S917[农业科学—水产科学]