狂犬病毒5aG株核蛋白基因的克隆及序列分析

Cloning and sequencing of nucleoprotein gene of Chinese rabies virus vaccine strain (5aG)

用ＲＴ、ＲＡＣＥ、ＰＣＲ等方法，从中国狂犬疫苗株（５ａＧ）病毒感染的鼠脑组织中扩增病毒核蛋白基因，用双链测序法进行了全基因核酸序列分析。结果表明：该基因开放阅读框架全长１３５３ｂｐ，编码４５０个氨基酸。与其它３株狂犬病毒核蛋白相比，核酸序列同源性为９０％～９６％，氨基酸序列同源性为９５％～９６％。表明核蛋白保守性较好。

Rabies virus 5aG strain (5aGRV) is used to produce primary hamster kidney tissue culture vaccine against Rabies in China. Nucleoprotein (NP), an important constituent of rabies virus, can intrigue Th cell and induce B cell to produce antibody. In order to study the molecular and biological characteristics of 5aG NP, we have cloned and sequenced the nucleoprotein gene. First, total RNA was extracted from infected mouse brain by Guanidine Isothiocyanate one-step method, then the first strand cDNA was synthesized by reverse transcription with oligo (dT) 18 as primer. After RACE (Rapid Amplification of cDNA Ends), one pair of oligonucleotide primers designed referring to the nucleoprotein gene of other strains, were used in PCR to amplify the fragments of nucleoprotein gene (1.4kb). When these fragments were inserted into vector pUC18, sequences of the nucleoprotein gene were obtained by double-stranded sequencing. Comparing the nucleic acid and deduced amino acid sequence of 5aGRV nucleoprotein gene with available NP sequences of other RV strains, we found that the nucleoprotein has less variation than glycoprotein, so nucleoprotein is more suitable to be used as a diagnostic reagent and a subunit vaccine.