中国狂犬病毒疫苗株（5aG株）糖蛋白基因的克隆与序列分析

Cloning and Sequencing of Glycoprotein Gene of Chinese Vaccine Strain(5aG Strain) of Rabies Virus

用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法从中国狂犬疫苗株（５ａＧ株）病毒感染细胞中扩增得到该株病毒糖蛋白基因，并进行序列测定。结果表明该基因开放阅读框架全长１５７５ｂｐ，编码５０５个氨基酸的成熟糖蛋白及Ｎ－端１９个氨基酸的信号肽，该基因与其他株系相应基因比较，核酸序列同源性为８８％～９１％，氨基酸序列同源性为８７％～９０％，其中膜外区同源性高于膜内区及跨膜区同源性。糖蛋白中重要功能区段嗜乙酰胆碱受体区段、抗原位点３等在个株系间高度保守。

Rabies virus 5aG strain(5aGRV) is used for production of primary hamster kindey tissue culture vaccine against Rabies in china. Glycoprotein, one kind of transmembrane protein on the surface of rabies virus particle, is the sole antigen which can induce the neutralizing antibody against rabies. We have cloned and sequenced this glycoprotein gene of 5aGRV using reverse transcription-polymerase chain reaction (RT-PCR) method. Firstly, total RNA was extracted from primary hamster kindey cell infected with 5aGRV by guanidine isothiocyanate one-step methods, then the first-strand cDNA was systhesised by reverse transcription using oligo(dT) 18 as primer. Two pairs of primers were used in PCR to amplify two overlapping fragments of glycoprotein gene. These two fragments were inserted into vectors respectively and sequenced. The results show that the length of 5aGRV glycoprotein gene ORF is 1, 575bp, which encoded 524 aa including 505 aa of mature glycoprotein and 19aa of signal peptide.Comparing the nucleic acid and deduced amino acid sequences of 5aGRV glycoprotein gene with those of other strains,we found that the sequence of signal peptide region and ectodomain were more conserved than that of transmembrane domain and cytoplasmic domain (homology of nucleotide: 87-93% 80-85%,respectively; homology of amino acid: 84-92%,63-74%, respectively). There are three regions in ectodomain which are more divergent than adjacent regions. The homology of the antigen 3 domain (349aa-357aa), and the AChR binding domain (208aa-229aa) are highly implying that these two domains are related to the basic function of glycoprotein.