ZHU Fu-xiang, LIU Ze-long, MIAO Jing, QU Hui-ge, CHI Xiao-yan
Biomedical Institute, Life Science College of Ludong University, Yantai 264025, China.
Zhonghua Yi Xue Za Zhi. 2010 Sep 28;90(36):2570-4.
to investigate the effect of glycosylation modification on secretion of intein spliced B-domain-deleted FVIII (BDD-FVIII).
a total of 226 amino acid residues of FVIII B domain with six potential asparagines-linked glycosylation sites (N6) were incorporated into heavy chain of BDD-FVIII. By dual-vector co-transfer of heavy and light chain genes with fused intein (N6HCIntN and IntCLC) into cultured 293 cells, the amounts of spliced BDD-FVIII protein and coagulation activity in culture supernatant were observed by ELISA and Coatest method respectively.
the amounts of spliced BDD-FVIII protein and activity were up to (123 ± 18) ng/ml and (0.94 ± 0.11) U/ml in supernatant from cells co-transfected with N6HCIntN and IntCLC genes. And they were higher than those of cells co-transfected with intein-fused heavy (HCIntN) and light chain (IntCLC) genes [(86 ± 12) ng/ml and (0.65 ± 0.07) U/ml, both P < 0.05]. Spliced BDD-FVIII protein and activity could also be detected in the supernatant from mixed cells individually transfected with N6HCIntN and IntCLC genes [(18 ± 6) ng/ml and (0.15 ± 0.05) U/ml].
it demonstrated that the glycosylation modified heavy chain can improve the secretion of intein spliced BDD-FVIII and the protein splicing can occur independent of cellular mechanism.
研究糖基化修饰对内含肽剪接的B结构域缺失的FVIII（BDD-FVIII）分泌的影响。
将含有六个潜在天冬酰胺连接糖基化位点（N6）的FVIII B结构域的226个氨基酸残基掺入BDD-FVIII的重链中。通过将重链和轻链基因与融合内含肽（N6HCIntN和IntCLC）双载体共转染到培养的293细胞中，分别采用ELISA和Coatest方法观察培养上清液中剪接的BDD-FVIII蛋白量和凝血活性。
用N6HCIntN和IntCLC基因共转染的细胞上清液中，剪接的BDD-FVIII蛋白量和活性分别高达（123±18）ng/ml和（0.94±0.11）U/ml。它们高于用内含肽融合的重链（HCIntN）和轻链（IntCLC）基因共转染的细胞[（86±12）ng/ml和（0.65±0.07）U/ml，均P<0.05]。在分别用N6HCIntN和IntCLC基因转染的混合细胞的上清液中也可检测到剪接的BDD-FVIII蛋白和活性[（18±6）ng/ml和（0.15±0.05）U/ml]。
表明糖基化修饰的重链可改善内含肽剪接的BDD-FVIII的分泌，且蛋白剪接可独立于细胞机制发生。
