牦牛B细胞淋巴瘤2相关蛋白A1的原核表达及体外活性

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33kDa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2.0 μg/mL组中显著上调(P < 0.05),CASP8的mRNA水平在0.2 μg/mL和2.0 μg/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 prokaryotic expression and vitro activity of yak B cell lymphoma 2 related protein A1

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-32a-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 BCL2A1(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 BCL2A1(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.