狂犬病病毒SRV_9疫苗株糖蛋白核酸序列及抗原特性研究

Characteristics of the Glycoprotein cDNA and Antigenicity of Rabies virus Vaccine Strain SRV_9

狂犬病病毒SRV9株是经克隆获得的中等蚀斑口服弱毒疫苗候选株,具有安全和免疫原性较好等优点。本试验根据狂犬病病毒糖蛋白核苷酸5’末端和3’末端序列设计了一对引物,通过反转录-聚合酶链式反应(RT-PCR)分别扩增了 SRV9及其母源株SAD B19糖蛋白的全长cDNA。测序结果表明,SRV9和SAD B19糖蛋白cDNA读框长1575bp,编码524氨基酸残基的多肽。通过和其母源株SAD B19核苷酸序列比较发现,SRV9的158、575、931位碱基分别出现了G→A、A→G和A→G的转换,相应地导致第53位、192位、311位氨基酸出现了Gly→Glu、His→Arg、Thr→Ala突变;和我国现行的犬用疫苗株ERA的核苷酸和氨基酸比较,有10个碱基不同.7个氨基酸发生改变。经Jameson-Wolf抗原表位优势图分析发现,SRV9在192位氨基酸的改变,导致该部位产生了一个新的潜在抗原位点。由于狂犬病病毒糖蛋白是诱导机体产生中和抗体唯一抗原,因此,上述氨基酸的改变可能与其特殊的蚀斑特性和其安全性提高有关。保持对毒株的氨基酸序列分析也是监测其特性的重要手段之一。

Oral vaccine candidate of Rabies virus strain SRV9 is an intermediate plaque subclone of SAD B19 and has been used in field with good safety and immunogenicity. A pair of primers was designed according to the published sequence of rabies virus glycoprotein. Reverse transcription -poly-merase chain reaction (RT-PCR) was emoloyed to amplify the full cDNAs of the SRV9 and its ancestor SAD B19. Sequencing result has shown that the RT-PCR product was 1588bp in length in which a 1575bp open reading frame was included. It encodes a mature polypeptide composing of 505 amino acids. Compared to SAD B19, base transversions occurred at positions 158, 575 and 931, resulting in amino acid mutations at 53, 192 and 311 residues in polypeptide. Compared to the vaccine strain ER-A in ues, ten base differences and 7 amino acid alterations were marked. Antigenic index analyses found that the mutation in 192 amino acids resulted in the formation of a new potential antigenic site. As the glycoprotein of rabies virus is the only antigen inducing neutralizing antibodies, the alteration of His→Arg in 191 position may be the causes of forming a special plaque and further improvement of its safety. Keeping analyses on amino acids of the glycoprotein of rabies virus is also an important approach of surveillance on rabies virus strains.