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为了研究人副流感病毒3型(hPIV3)HN糖蛋白N-糖链的功能,采用基因定点突变技术构建糖基化位点突变体,然后检测各突变株的蛋白电泳速率、细胞表面表达量、受体结合活性、神经氨酸酶活性和促细胞融合活性。HN分子的G1、G2、G3和G4 4个糖基化位点分别和联合突变后发现G1、G2和G4及其联合突变株(G12、G14、G24和G124)电泳速率加快,而G3突变株电泳速率没有变化。各突变株的表达效率,神经氨酸酶活性与野毒株相比差别无统计学意义(P>0.05),但受体结合活性和促细胞融合活性均有不同程度的降低(P<0.05)。G1、G2和G4位点突变后受体结合活性分别为突变前的83.94%、76.45%和55.32%,而促细胞融合活性降为突变前的80.84%、77.83%和64.16%。联合突变株G12、G14、G24和G124血吸附活性进一步降低,为突变前的33.07%、20.67%、19.96%和15.11%,促细胞融合活性进一步降低为突变前的46.36%、12.04%、13.43%和4.05%。结果表明:hPIV3HN糖蛋白的糖链对HN糖蛋白的受体结合活性和促细胞融合活性有重要影响,推断糖链的丢失可能会引起HN糖蛋白头部结构(受体结合活性位点所在区域)或者方向的改变或者无法与宿主细胞膜表面的凝集素受体(一种与N-糖链结合的受体)结合,进而导致受体结合活性和促细胞融合活性的降低。
In order to study the function of N-glycans of HN glycoprotein of human parainfluenza virus type 3 (hPIV3), site-directed mutagenesis was used to construct glycosylation site mutants. Then the protein electrophoresis rates, cell surface expression, Receptor binding activity, neuraminidase activity and cell fusion activity. The G1, G2, G4 and G144 mutants (G12, G14, G24 and G124) were found to be faster and faster at the four glycosylation sites G1, G2, G3 and G4 of the HN molecule, respectively. The electrophoresis rate has not changed. The expression efficiency and neuraminidase activity of the mutants were not significantly different from those of the wild type (P> 0.05), but the receptor binding activity and the cell fusion activity were decreased to some extent (P <0.05). The receptor binding activities of G1, G2 and G4 mutants were 83.94%, 76.45% and 55.32% of those before mutation, respectively, while the cell fusion activity was reduced to 80.84%, 77.83% and 64.16% of that before mutation. The blood adsorption activity of the combined mutant G12, G14, G24 and G124 was further reduced to 33.07%, 20.67%, 19.96% and 15.11% before mutation, and the cell fusion activity was further reduced to 46.36%, 12.04% and 13.43% And 4.05%. The results showed that the sugar chain of hPIV3HN glycoprotein has an important influence on the receptor binding activity and cell fusion activity of HN glycoprotein. It is concluded that the loss of sugar chain may cause the head structure of HN glycoprotein (the region where the receptor binds to the active site ) Or change in direction or fail to bind to the lectin receptor (a receptor that binds to N-glycan) on the surface of the host cell membrane, leading to a decrease in receptor-binding activity and cell-fusion activity.