【摘要】 目的 初步评价基于杆状病毒表达系统制备的诺如病毒（Norovirus,No V） GⅡ.17型病毒样颗粒（virus-like particles,VLPs）的免疫原性。方法 制备并纯化No V GⅡ.17 VLPs，采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecylsulfate-polyacrylamide gel electrophroesis,SDS-PAGE）、免疫印迹法（Western blot）、透射电镜和粒径检测方法对其进行鉴定。GⅡ.17型VLPs采用肌内免疫途径，免疫Balb/C小鼠。研究Al（OH）₃佐剂组4个免疫剂量（0.4、2、10、50μg）及不使用佐剂组3个剂量（0.4、2、10μg）的免疫应答。另外，设PBS、PBS加佐剂2个对照组。将每组的5只SPF级雌性健康Balb/c小鼠（6～8周龄）分别于0、3周进行初次免疫和加强免疫。于免疫前（第0天），初次免疫后3周（第21天），加强免疫后1周（第28天）、3周（第42天）分别眼眶采血。采用ELISA检测VLP-特异性Ig G抗体，组织血型抗原（histo-blood group antigen,HBGA）受体-VLPs结合阻断试验检测50%阻断滴度(50%of block‐ing titer,BT₅₀)。组间特异性Ig G抗体水平和BT₅₀水平进行对数转换后，采用Two-way ANOVA法进行统计学处理。结果 成功制备No V GⅡ.17 VLPs。经SDS-PAGE检测，No V GⅡ.17 VLPs纯度为95%,Western blot分析可见特异性条带，经透射电镜和粒径分析颗粒形态完整，粒径均一。2针肌内免疫后，疫苗不同剂量组（0.4、2、10μg）产生VLP-特异性Ig G抗体水平分别为4 222、67 559、135 118;BT₅₀抗体水平分别为12.5、57.4、174.1,Al（OH）₃作为佐剂的不同剂量组（0.4、2、10μg）产生VLP-特异性Ig G抗体水平分别为12 800、117 627、409 600;BT₅₀抗体水平分别为19.0、100.0、229.7，抗体水平明显高于无佐剂组（P均<0.05）。结论 Al（OH）₃佐剂具有显著的免疫增强作用，肌内注射作为免疫途径，2针免疫，剂量高于2μg可产生较高的抗体Ig G和BT₅₀水平。应用含铝佐剂的GⅡ.17型VLPs免疫小鼠，诱生了较好的HBGA受体结合阻断抗体，可作为诺如病毒疫苗的组分抗原。更多还原

【Abstract】 Objective To evaluate the humoral immunogenicity of virus-like particles （VLPs） of Norovirus（No V） GⅡ.17 genotype assembled based on recombinant baculovirus expression system.Methods The No V GⅡ.17 VLPs in the culture medium were purified.The purity and integrity of GⅡ.17 VLPs were determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis （SDS-PAGE）,Western blot,transmission electron mi‐croscopy and particle size analysis.The No V GⅡ.17 VLPs were administered via intramuscular injection to immu‐nize 6—8 weeks old female Balb/C mice at 4 doses （0.4,2,10,50μg） with adjuvant Al（OH）₃ and 3 doses （0.4,2,10μg） without adjuvant Al（OH）₃.Two control groups were established,one given PBS （p H7.4） and the other given PBS plus Al（OH）₃.The BALB/c mice （5 per/group） were immunized twice,in week 0 and week 3,respec‐tively.Retro-orbital blood was collected before the first immunization （on Day 0）,and on Day 21,Day 28,and Day42 after immunization.VLP-specific Ig G antibody level was assessed using enzyme-linked immunosorbent assay,and50%blocking titer (BT₅₀) using histo-blood group antigen （HBGA）-VLP binding blockade test.The Ig G and BT₅₀levels were log transformed and compared across groups using two-way analysis of variance （ANOVA）.Results No V GⅡ.17 VLPs were successfully prepared.SDS-PAGE analysis indicated that the purity was up to95%.Western blot showed specific doublet band with expected sizes.Transmission electron microscopy and particle size analysis showed that the purified No V GⅡ.17 VLPs were morphologically intact with uniform size.After two in‐tramuscular injections,the levels of VLP-specific Ig G antibody produced by the doses of 0.4,2,and 10μg were4 222,67 559,and 135 118,respectively;the levels of BT₅₀ antibody were 12.5,57.4 and 174.1,respectively.Among the groups immunized with VLPs plus Al（OH）₃ as adjuvant,the levels of VLP-specific Ig G antibody pro‐duced by the doses of 0.4,2 and 10μg were 12 800,117 627 and 409 600,respectively;the antibody levels of BT₅₀were 19.0,100.0 and 229.7,respectively.At the same dose,the levels of VLP-specific Ig G antibody and BT₅₀ in the groups with Al（OH）₃ as adjuvant were significantly higher than those in the groups without adjuvant （P<0.05）.Conclusions The Al（OH）₃ adjuvant has a significant immune-enhancing effect.High titers of VLP-specific Ig G antibody and BT₅₀ may be produced after two intramuscular injections at doses higher than 2μg.The No V GⅡ.17 VLPs in combination with aluminum adjuvant can induce high titers of HBGA receptor blocking anti‐bodies in mice,suggesting that it could be used as a candidate antigen for vaccine against norovirus.更多还原