小反刍兽疫病毒血凝素蛋白与受体蛋白SLAM的相互作用

Interaction between Hemagglutinin Protein of Peste des Petits Ruminants Virus and Signalling Lymphocyte Activation Molecule

旨在利用免疫共沉淀技术验证小反刍兽疫病毒血凝素蛋白和受体蛋白SLAM间的相互作用。鉴于截短的H蛋白仍具有正常的与细胞受体结合的能力,分别构建pcDNA3.1-tH真核表达载体和SLAM及其缺失突变体(m1-胞外区、m2-无信号肽胞外区、m3-胞外区N端29-136位氨基酸和m4-胞外区C端137-240位氨基酸)编码基因的pEGFP-N1系列真核表达载体,将测序正确的重组质粒共转染中国仓鼠卵巢细胞CHO-K1细胞株,利用免疫共沉淀技术验证tH蛋白与SLAM蛋白相互作用的关键氨基酸区段。结果表明,成功构建了预期的重组表达载体,转染CHO细胞后目的蛋白正确表达;免疫共沉淀时,tH能与SLAM、m1、m2和m3蛋白发生反应,但没有与m4蛋白发生反应。由此可知,SLAM N端29-136位氨基酸是决定SLAM与PPRV H蛋白结合的关键氨基酸区段,这与麻疹病毒属其他宿主SLAM受体的研究结果一致。

This experiment was conducted to explore the interaction between PPRV Hemagglutinin(H)protein and signalling lymphocyte activation molecule(SLAM)by co-immunoprecipitation.In view of the truncated H protein still has normal binding capacity to cell receptor,the genes of tH,SLAMand its several deletion mutants which lacked transmembrane and intracellular fragments(m1),a signal peptide fragment(m2)or C-terminal fragment(amino acids 29-136,m3)or N-terminal fragment(amino acids 137-240,m4),were directionally cloned into eukaryotic expression vectors pcDNA3.1and pEGFP-N1,respectively.The recombinant plasmids were co-transfected into CHO-K1cells,and then the key amino acid regions of viral protein and SLAM protein interactions was identified initially by co-immunoprecipitation.The results showed that 1)The recombinant vectors were constructed successfully,and the proteins were expressed correctly in CHO cell;2)tH was identified can interact with SLAM,m1,m2and m3,and can not with m4by co-immunop recipitation.These results indicated that the N-terminal segment(amino acids 29-136)of SLAM was essential to bind to PPRV H protein.This finding is agreed with that of Morbillivirus receptor SLAM.