鸡Tgfβ1干扰表达载体的构建及在MDCC-MSB1细胞中的干扰表达

Construction of Chicken Tgfβ1 Interfering Expression Vector and Interfering Expression in MDCC-MSB1 Cells

构建鸡转化生长因子β1(Tgfβ1)干扰表达载体,在马立克病毒转化的肿瘤细胞系(MDCC-MSB1)中鉴定其抑制表达效果。根据GenBank中鸡Tgfβ1序列以及pG1.2载体序列,设计合成3条干扰鸡Tgfβ1表达的短发夹核糖核酸(shRNA)干扰序列,分别将其插入表达载体pG1.2,构建pG1.2-Tgfβ1干扰表达载体。将构建的质粒转染MDCC-MSB1细胞后,用qRT-PCR和Western blot检测MDCC-MSB1细胞鸡Tgfβ1表达量。研究结果表明:pG1.2载体中被插入了设计合成的鸡Tgfβ1 shRNA干扰序列,构建了pG1.2-Tgfβ1干扰表达载体。pG1.2-Tgfβ1干扰表达载体转染细胞后,鸡Tgfβ1的表达量显著降低。本文构建的鸡Tgfβ1的干扰表达载体,可有效干扰MDCC-MSB1细胞中Tgfβ1的表达。

The transfer growth factor β1(Tgfβ1)interfering expression vector was constructed,and its inhibitory effect in marek's virus transformed tumor cell line(MDCC-MSB1)was identified.According to the sequence of chicken Tgfβ1 in Gen Bank and pG1.2 vector sequence,three short hairpin RNA(shRNA)interference sequences were designed and synthesized to interfere with chicken Tgfβ1 expression,and were respectively inserted into pG1.2 expression vector to construct pG1.2-Tgfβ1 interference expression vector.After the constructed plasmid was transfected into MDCC-MSB1 cells,the expression of Tgfβ1 in chicken MDCC-MSB1 cells was detected by qRT-PCR and Western blot.The results show that the designed and synthesized chicken Tgfβ1 short hairpin RNA interference sequence is inserted into pG1.2 vector,and the pG1.2-Tgfβ1 interference expression vector is constructed.After transfection with pG1.2-Tgfβ1 interference expression vector,the expression level of chicken Tgfβ1 is significantly reduced.The constructed interfering expression vector of chicken Tgfβ1 can effectively interfere the expression of Tgfβ1 in MDCC-MSB1 cells.