犬瘟热病毒野毒株H蛋白基因的序列分析

Nucleotide Sequence Analysis of the Haemagglutinin Protein Gene of LIU Strain of Canine Distemper Virus

对引起长春某犬场犬死亡的犬瘟热病毒（ Ｃ Ｄ Ｖ）野毒株（ Ｌ Ｉ Ｕ）附着或血凝蛋白（ Ｈ）基因进行了全基因序列测定，并与疫苗弱毒 Ｏｎｄｅｒｓｔｅｐｏｏｒｔ 株作了比较。用 ４ 对引物对 Ｌ Ｉ Ｕ 株进行了 Ｒ Ｔ Ｐ Ｃ Ｒ 扩增，将扩增得到的 ４ 个阳性 Ｐ Ｃ Ｒ 片段纯化后直接测序。将所测序列拼接后，去掉侧翼的 Ｆ 基因和 Ｌ 基因序列，得到了 Ｈ蛋白的全基因序列。该基因全长为 １ ９４６ ｂｐ，开放阅读框架（ Ｏ Ｒ Ｆ）始于 ２１～２３ 位的 Ａ Ｔ Ｇ，终止于 １ ８４２～１ ８４４位的 Ｔ Ｇ Ａ，编码 ６０７ 个氨基酸。将 Ｌ Ｉ Ｕ 毒株与疫苗弱毒 Ｏｎｄｅｒｓｔｅｐｏｏｒｔ 株进行比较，二者 Ｈ 基因核苷酸序列的同源性为 ９１．７％ ，推导的 Ｈ 蛋白氨基酸序列的同源性为 ９０．２％ 。 Ｏｎｄｅｒｓｔｅｐｏｏｒｔ 株的 Ｈ 蛋白潜在的 Ｎ 联糖基化位点为 ４ 个，而 Ｌ Ｉ Ｕ 株 Ｈ 蛋白为 ８ 个。糖基化位点的不同可能对 Ｌ Ｉ Ｕ 株 Ｈ 蛋白的抗原性产生影响。２ 个毒株 Ｈ 蛋白的半胱氨酸残基数目均为 １２ 个，且位置是保守的；疏水性有一定的不同，但推测的穿膜区位置是相同的，位于 ３５～５５ 位氨基酸之间。

Complete sequence analysis of the haemagglutinin (H) protein gene of LIU strain was conducted from Changchun dogs.Four pairs of primers were designed and synthesized. The PCR products obtained using P1 and P2, P3 and P4, P5 and P6 or P7 and P8 from the LIU strain were directly sequenced after they were purified. The total sequence determined consists of 154 nt at the 3′ end of the F gene, 1 946 nt of the complete H gene and 74 nt at 5′ end of the L gene. The complete H gene sequences of the LIU strain showed that the largest open reading frame (ORF) encoding a protein of 607 amino acid residues started at the ATG at position 21 23 and extended to a termination codon (TGA) at position 1 842 1 844. The deduced amino acid sequence of the H protein of the LIU strain was compared with the CDV vaccine Onderstepoort strain extracted from the GenBank database. The sequence of the H gene and dedeuced H protein of the LIU strain showed an autoploidy of 91.7% and 90.2% with Onderstepoort strain, respectively. All of the 12 cysteine residues found in the LIU strain H protein were conserved in Onderstepoort strain. Although predicted hydrophobicity of the LIU strain differed from that of the Onderstepoort strain, the predicted transmembrane position at 35 55 amino acid residues was conserved in the two strains. Out of the 4 potential N linked glycosylation sites of Onderstepoort H protein, two located in the N terminal 2/3 of the protein (one located in the putative cytoplasmic part and is probably not used), while the remaining 2 sites located in the C terminal 1/3 of the protein. Four of the 8 potential N linked glycosylation sites of LIU H protein were conserved in the Onderstepoort, but the other four sites were unique to LIU, the extra glycosylation sites could influence antigenicity of the LIU.