牦牛B细胞淋巴瘤2相关蛋白A1的原核表达及体外活性  

The prokaryotic expression and vitro activity of yak B cell lymphoma 2 related protein A1

作　　者：梅群弟 王海鹏 王利[1] 傅芳 李娟[1] 郑姚 张玲[1] MEI Qundi;WANG Haipeng;WANG Li;FU Fang;LI Juan;ZHENG Yao;ZHANG Ling(Key Laboratory of Animal Science of State Ethnic Affairs Commission,Southwest Minzu University,Chengdu 610041,China)

机构地区：[1]西南民族大学动物科学国家民委重点实验室,成都610041

出　　处：《兽类学报》2022年第2期196-203,共8页Acta Theriologica Sinica

基　　金：西南民族大学中央高校基本科研业务费专项资金项目(2021NYYXS90);四川省留学人员科技活动择优资助项目(2019)。

摘　　要：为研究牦牛(Bos grunniens) B细胞淋巴瘤2相关蛋白A1 (B cell lymphoma 2 related protein A1, BCL2A1)的原核表达及体外活性。采用原核表达载体构建、细胞划痕实验、CCK-8法、透射电镜和实时荧光定量PCR等方法。结果显示,成功构建pET-32a-BCL2A1重组质粒,表达获得约33 kDa的BCL2A1。终浓度为0.02μg/mL、0.2μg/mL、2.0μg/mL的BCL2A1均能使HepG2细胞活性显著降低(P <0.05),并在一定程度上抑制细胞的迁移。2.0μg/mL BCL2A1导致HepG2细胞核固缩,胞质中高密度物质聚集,溶酶体吞噬形成致密的凋亡小体等。此外,细胞凋亡相关基因CASP9的mRNA水平在2.0μg/mL组中显著上调(P <0.05),CASP8的mRNA水平在0.2μg/mL和2.0μg/mL组中显著上调(P <0.05),而CASP3和Cyt c的mRNA水平在3个浓度处理组均显著上调(P <0.05)。这提示BCL2A1可能通过凋亡途径影响HepG2细胞活性,为进一步探索牦牛BCL2A1基因功能积累资料。The aim of this study was to explore the expression and vitro viability of Bos grunniens B cell lymphoma 2 related protein A1(BCL2A1). To this end, prokaryotic expression vector construction, cell scratch test, CCK-8 kit,transmission electron microscope(TEM) and RT-qPCR were used in this experiment. The results showed that recombinant plasmid pET-32 a-BCL2A1 was successfully constructed and a 33 kDa protein was expressed. The viability of HepG2 cells was significantly decreased after treating with BCL2A1(0. 02 μg/mL, 0. 2 μg/mL, and 2. 0 μg/mL)(P <0. 05), and cell migration was inhibited to some extent. HepG2 cells that were treated with BCL2 A1(2. 0 μg/mL)showed karyopyknosis, the electron-dense nuclear material characteristically aggregated in cytoplasm, and lysosome phagocytic organelles formed apoptotic bodies. In addition, the mRNA level of apoptosis-related gene CASP9 was significantly upregulated by 2. 0 μg/mL BCL2A1(P < 0. 05), mRNA level of CASP8 was significantly upregulated by 0. 2 μg/mL and 2. 0 μg/mL BCL2 A1(P < 0. 05), mRNA level of CASP3 and Cyt c were significantly upregulated at different concentration treatment groups(P < 0. 05). It is indicated that BCL2A1 may affect the viability of HepG2 cells through the apoptosis pathway. These results benefit for further study of the function of yak’s BCL2A1.

关 键 词：B细胞淋巴瘤2相关蛋白A1(BCL2A1) HEPG2细胞 细胞凋亡 

分 类 号：Q753[生物学—分子生物学]