鸡TGF-β1对大肠杆菌和鸡白痢沙门菌黏附DF1细胞的影响

Effects of Chicken TGF-β1 on the Adhesion of Escherichia coli and Salmonella pullorum to DF1 Cells

旨在研究鸡转化生长因子-β1(transfer growth factor-β1,TGF-β1)对大肠杆菌和鸡白痢沙门菌黏附DF1细胞的影响。通过ELISA方法检测鸡新城疫病毒(Newcastle disease virus,NDV)感染DF1细胞后鸡TGF-β1表达量的变化,参考GenBank中鸡TGF-β1序列构建鸡TGF-β1的过表达和干扰表达载体,将构建成功的TGF-β1重组表达载体转染DF1细胞后48 h在荧光显微镜下观察其转染效率,荧光定量PCR检测鸡TGF-β1 mRNA水平表达量的变化,ELISA方法检测鸡TGF-β1胞外蛋白表达量的变化,通过黏附试验检测鸡TGF-β1对致病性大肠杆菌和鸡白痢沙门菌黏附DF1细胞的影响。结果显示,NDV感染DF1细胞后,鸡TGF-β1胞外表达量显著高于未感染细胞的表达量(P<0.05),经酶切测序鉴定TGF-β1干扰表达和过表达重组载体构建成功。转染鸡TGF-β1重组过表达载体细胞的TGF-β1 mRNA和胞外蛋白表达量均显著高于未转染细胞(P<0.01),致病性大肠杆菌和鸡白痢沙门菌对细胞的黏附率均显著高于未转染细胞(P<0.01),转染鸡TGF-β1重组干扰表达载体细胞的mRNA和胞外蛋白表达量均显著低于未转染细胞(P<0.01),致病性大肠杆菌和鸡白痢沙门菌对细胞的黏附率均显著低于未转染细胞(P<0.01)。综上,NDV感染DF1细胞后,鸡TGF-β1的表达量增加,鸡TGF-β1可促进致病性大肠杆菌和鸡白痢沙门菌黏附DF1细胞,这为进一步研究鸡TGF-β1在家禽病毒感染继发细菌性疾病中的作用提供试验基础。

The aim of this experiment was to study the effects of chicken transfer growth factor-β1(TGF-β1) on the adhesion of Escherichia coli and Salmonella pullorum to DF1 cells.The expression of chicken TGF-β1 was detected by ELISA after DF1 cells were infected with Newcastle disease virus(NDV),then the overexpression and inhibitory expression vectors of chicken TGF-β1 were constructed by referring to the chicken TGF-β1 sequence in GenBank.The transfection efficiency of the recombinant TGF-β1 expression vector was observed under fluorescence microscope 48 h after transfection of DF1 cells.The mRNA level changes of chicken TGF-β1 in DF1 cells were detected by fluorescence quantitative PCR,and the changes of chicken TGF-β1 extracellar protein expression level were detected by ELISA.The effects of chicken TGF-β1 on the adhesion of pathogenic Escherichia coli and Salmonella pullorum to DF1 cells were detected by adhesion assay.The results showed that the extracellular expression level of TGF-β1 in DF1 cells infected with NDV was significantly higher than that in uninfected cells(P<0.05).Enzyme digestion and sequencing confirmed that the recombinant vectors with TGF-β1 inhibitory expression and overexpression were successfully constructed.The expression levels of TGF-β1 mRNA and extracellular protein in transfected chicken TGF-β1 recombinant overexpressed vector cells were significantly higher than those of untransfected cells(P<0.01),and the adhesion rates of pathogenic Escherichia coli and Salmonella pullorum to cells were significantly higher than those of untransfected cells(P<0.01).The expression levels of TGF-β1 mRNA and extracellular protein in transfected chicken TGF-β1 recombinant inhibitory vector cells were significantly lower than those of untransfected cells(P<0.01),and the adhesion rates of pathogenic Escherichia coli and Salmonella pullorum to cells were significantly lower than those of untransfected cells(P<0.01).In conclusion,the expression level of chicken TGF-β1 increased after NDV infection of DF1 cells,and chicken TGF-β1 can promote the adhesion of pathogenic Escherichia coli and Salmonella pullorum to DF1 cells,which provides experimental basis for further study on the role of chicken TGF-β1 in the bacterial disease secondary to poultry virus infection.