伪狂犬病病毒gH糖蛋白在哺乳动物细胞中的表达及其免疫原性

Expression and immunogenicity of glycoprotein H of pseudorabies virus in mammalian cells

目的 在哺乳动物细胞中表达伪狂犬病病毒(pseudorabies virus,PRV)gH糖蛋白,并检测其免疫原性。方法PCR扩增PRV-XiangA株gH基因片段,插入哺乳动物细胞表达载体pIRES-neo3中,构建重组表达质粒pIRES-gH,将其转染至HEK-293F细胞中,悬浮培养5 d。取细胞培养上清,进行Western blot鉴定后,经镍离子层析柱纯化。将纯化的gH糖蛋白作为免疫抗原与ISA 201 VG佐剂乳化后免疫8只雌性ICR小鼠,对照组8只小鼠免疫等量佐剂。初次免疫后5和8周各加强免疫1次,并分别于初次免疫后4、7、10周采血,检测小鼠血清特异性抗体及中和抗体效价;第3次采血2 d后,使用PRV-XiangA毒株(1.5×10^(4)TCID_(50))对小鼠进行滴鼻攻毒,每日观察小鼠发病及死亡情况。结果 重组表达质粒pIRES-gH经测序鉴定构建正确。gH糖蛋白成功在哺乳动物细胞中表达并进行了糖基化修饰,且反应原性良好,在100 mL培养体积下可纯化到约625μg蛋白。免疫3次后,小鼠能产生高水平的特异性抗体,且具有中和PRV的作用,其中和抗体效价最高可达1∶256。攻毒试验中对照组小鼠全部发病并死亡;而gH免疫组有一半小鼠未发病,存活率为50%。结论 在哺乳动物细胞中成功表达了PRV gH糖蛋白,并首次证实其具有免疫保护作用,为gH糖蛋白的进一步研究及应用提供了实验依据,也为PRV亚单位疫苗的研发提供了新的思路。

Objective To express glycoprotein H(gH)of pseudorabies virus(PRV)in mammalian cells and detect its immunogenicity.Methods The gH gene fragment of PRV-XiangA strain was amplified by PCR and inserted into mammalian cell expression vector pIRES-neo3 to construct recombinant expression plasmid pIRES-gH,which was transfected to HEK-293F cells and cultured in suspension for 5 d.The cell culture supernatant was identified by Western blot and purified by nickel ion chromatography column.The purified gH was emulsified with ISA 201 VG adjuvant to immunize 8 female ICR mice,and 8 mice in control group were immunized with the same amount of adjuvant,which was strengthened at 5 and 8 weeks after the first dose respectively.The blood samples were collected at 4,7 and 10 weeks after the first dose and detected for the titer of specific antibody and neutralizing antibody in serum of mice;The mice were challenged with PRVXiangA strain(1.5×10^(4) TCID_(50))by nasal drops 2 d after the third blood collection,and observed for the morbidity and mortality daily.Results The recombinant expression plasmid pIRES-gH was constructed correctly as identified by sequencing.The gH protein was successfully expressed and modified by glycosylation in mammalian cells with good reactivity,and about 625μg purified protein was obtained under 100 mL culture volume.After three times of immunization,mice produced high level of specific antibody and showed the effect of neutralizing PRV,and the titer of neutralizing antibody reached 1∶256.In the challenge test,all the mice in control group became ill and died,while half of the mice in gH immunized group did not get sick with a survival rate of 50%.Conclusion PRV gH was successfully expressed in mammalian cells,and its immune protection was confirmed for the first time,which provided experimental basis for the further research and application of gH,and also provided a new idea for the development of PRV subunit vaccine.