唾液酸化路易斯-X(sialyl lewis x,Slex)是选择素家族的一个共同糖配体,通过与选择素竞争性地结合炎性细胞,可以抑制炎症反应。克隆表达Slex合成过程中的关键酶,就可以在体外进行Slex的生物合成,从而进行相关生物制剂的开发。α-1,3-岩...唾液酸化路易斯-X(sialyl lewis x,Slex)是选择素家族的一个共同糖配体,通过与选择素竞争性地结合炎性细胞,可以抑制炎症反应。克隆表达Slex合成过程中的关键酶,就可以在体外进行Slex的生物合成,从而进行相关生物制剂的开发。α-1,3-岩藻糖基转移酶(alpha-(1,3)-fucosyltransferase,FT)就是参与Slex生物合成过程的关键酶之一。利用相关软件对牛的FT基因进行了生物信息学的分析,了解了FT的相关理化性质。通过PCR的方法获得了FT基因,构建了重组质粒pMD19-FT,并亚克隆至表达载体pPIC9K。通过电转化将线性化的表达质粒pPIC9K-FT整合到宿主菌Pichia pastoris GS115基因组上,构建了重组酵母GS115-FT。经诱导表达后,SDS-PAGE检测到了目的蛋白质条带,证明了此基因在P.pastoris GS115中能够可溶性表达。展开更多
Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothel...Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothelial cells PEDSV.15 and human α-galactosidase transgenic mice were produced. The Galα1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV.15 cell surface while human α-galactosidase combined with α1,2-fucosyltransferase genes removed Galα1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene. RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galacto-sidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by comp-lement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.展开更多
文摘Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothelial cells PEDSV.15 and human α-galactosidase transgenic mice were produced. The Galα1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV.15 cell surface while human α-galactosidase combined with α1,2-fucosyltransferase genes removed Galα1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene. RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galacto-sidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by comp-lement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.
