狂犬病病毒SRV9株糖蛋白333位氨基酸变异对其免疫原性的影响

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

目的探讨狂犬病病毒(Rabies virus,RV)SRV9株糖蛋白(G)333位丝氨酸(S)分别突变为精氨酸(R)和谷氨酸(E)后,对其免疫原性的影响。方法采用RT-PCR法扩增RV SRV9株G蛋白基因,经测序鉴定正确后,通过PCR将编码糖蛋白333位丝氨酸的核苷酸分别突变为编码精氨酸和谷氨酸的核苷酸,并分别构建复制缺陷型重组人5型腺病毒rAd5-SRV9-G333E、rAd5-SRV9-G333S和rAd5-SRV9-G333R,经形态学观察、RT-PCR检测及直接免疫荧光试验鉴定重组腺病毒,并免疫小鼠,检测小鼠中和抗体水平及阳转率,评价3种重组腺病毒免疫原性的差异。结果 SRV9株G蛋白基因及其突变体经测序鉴定正确;制备的重组腺病毒镜下可见典型的腺病毒外形特征,感染HEK293AD细胞后出现明显的细胞病变;RT-PCR及直接免疫荧光试验结果显示,3种重组腺病毒在HEK293AD细胞中均已成功表达;rAd5-SRV9-G333E诱导小鼠产生的中和抗体效价和阳转率均明显高于rAd5-SRV9-G333S(P<0.05)。结论 RV SRV9株糖蛋白333位丝氨酸突变为谷氨酸后,可显著增强其免疫原性。

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.