Effect of amino acid variation at site 333 on immunogenicity of glycoprotein of rabies virus SRV9 strain

Objective: To investigate the effect of mutation of amino acid at site 333 from Ser (S) to Arg (R) or Glu (E) on immunogenicity of glycoprotein (G) of rabies virus (RV) SRV9 strain. Methods: G gene of SRV9 strain was amplified by RT-PCR and identified by sequencing, of which the nucleotide encoding S at site 333 was mutated to those encoding R and E respectively. The constructed replication-defective recombinant adenoviruses type 5, i. e. rAd5-SRV9-G333E, rAd5-SRV9-G333S and rAd5-SRV9-G333R were observed for morphology, identified by RT-PCR and direct IFA, and evaluated for immunogenicity in mice by determination of level and positive rate of neutralizing antibody. Results: Correct sequences of the G gene and its mutant of SRV9 strain were proved by sequencing. The constructed recombinant adenoviruses showed typical appearance of adenovirus under electron microscope, which showed obvious CPE in infected HEK293AD cells. RT-PCR and direct IFA showed that all the three recombinant adenoviruses were successfully expressed in HEK293AD cells. Both the titer and positive rate of neutralizing antibody induced by rAd5-SRV9-G333E were significantly higher than those by rAd5-SRV9-G333S (P < 0. 05). Conclusion: The mutation of S at site 333 to E enhanced the immunogenicity of glycoprotein of RV SRV9 strain.