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

作　　者：孙程龙[1,2] 杨洋[1,2] 王颖[2] 宫婷[2] 刘祥义 陈奇[2] 王景龙[1,2] 刘晔[2] 张守峰[2] 扈荣良[2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,长春130122 [3]华北制药集团新药研究开发有限公司

出　　处：《中国生物制品学杂志》2012年第5期539-544,共6页Chinese Journal of Biologicals

基　　金：国家863计划项目(2011AA10A212)

摘　　要：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.

关 键 词：狂犬病病毒 SRV9株 糖蛋白 氨基酸 变异 免疫原性 

分 类 号：R373.9[医药卫生—病原生物学] R392.33[医药卫生—基础医学]