中文摘要：

目的预测人偏肺病毒（hMPV）融合蛋白N一糖基化位点，构建N-糖基化修饰突变体。方法NetNGlycl．0Server神经网络软件预测hMPV4个亚型代表株F蛋白氨基酸序列的N-糖基化位点；根据预测结果针对性地设计突变引物，应用基因工程技术进行定点突变，从而获得特定位点去糖基化的F基因，包括单位点突变体、双位点突变体及三位点突变体；双酶切鉴定、基因序列分析重组质粒，明确N-糖基化修饰突变体构建是否成功。结果hMPV4个亚型代表株F蛋白氨基酸序列从N端起第57、172、353位均存在可能性大于50％的N-糖基化位点。构建hMPVF蛋白7个不同的N-糖基化修饰突变体，并通过序列分析测定证实重组突变质粒构建成功。结论hMPVF蛋白的N-基化位点保守，不同亚型的代表株有相同的糖基化位点，利用定点突变技术成功构建了糖基化突变体重组质粒。

英文摘要：

Abstract： Objective To predict the N - glycosylation sites of human metapneumovirus （hMPV） fusion protein, and construct various mutants with modified N - glycosylation. Methods Position and score of N - glyeosylation sites in hMPV fusion protein of 4 subtype repre- sentative strains were predicted with NetNGlyel. 0 Server neural network software. Mutagenic primers were designed according to the possible sites,and with site- directed mutation technique,F gene with specific sites deglyeosylation was obtained,including mono -glycosylation sites mu- tants,dipl -glyeosylation sites mutants, and tri - glycosylation sites mutated. Recombinant plasmids were verified by digestion of double re- striction enzyme and analyzed by gene sequencing, to identify whether the mutants with modified N - glycosylation were successfully construe- ted. Results Fusion protein of hMPV 4 subtype representative strains showed conservative N - glycosylation sites with possibility exceeding 50% at the 57th,172nd ,353st from N- terminal. Seven various mutants with modified N -glycosylation were successfully constructed,which were verified by gene sequencing. Conclusions The N - glycosylation sites locating at 4 subtypes of hMPV fusion protein were conservative. The mutants have modified glyeosylation of hMPV fusion protein,which are successfully constructed.