中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2013年
4期
270-275
,共6页
诺如病毒%原核表达系统%P颗粒%P二聚体%HBGAs受体
諾如病毒%原覈錶達繫統%P顆粒%P二聚體%HBGAs受體
낙여병독%원핵표체계통%P과립%P이취체%HBGAs수체
Norovirus%Prokaryotic expression system%P particle%P dimer%HBGAs receptor
目的 建立我国流行优势株GⅡ.4型诺如病毒(GZ121)P蛋白(包括P颗粒和P二聚体)的原核表达系统,并明确其结合HBGAs受体的能力和方式.方法 从GⅡ.4型诺如病毒GZ121株基因组中克隆P区域基因片段,通过构建P区域氨基酸序列进化树,确定其基因簇.构建含铰链Hinge-P和不含铰链P-CDCRGDCFC的pGEX4T-1原核表达质粒,将构建的原核表达载体导入大肠杆菌(E coliBL21,用0.6 mmol/L IPTG(isoProPylthio-β-D-galaetoside)过夜诱导P颗粒(不含铰链)和P二聚体(含铰链)在BL21中表达.目的重组蛋白经溶血酶酶切和FPLC鉴定分析,用EIA法检测P颗粒和P二聚体与HBGAs的结合特征.结果 GZ121株诺如病毒为GⅡ.4基因型2004簇(GⅡ.4/2004 cluster).SDS-PAGE分析确定P颗粒和P二聚体相对分子质量(Mr)约为36×103,大小与报道相符.重组P颗粒经FPLC证实,其在Mr约为830×103处形成特异的P颗粒波峰,Western blot技术证实了重组P蛋白的特异性.EIA分析结果表明,GZ121株P蛋白与A、B、O型唾液均能结合,与非分泌型唾液不结合,而P颗粒结合HBGAs受体能力是P二聚体的80～ 100倍,与96簇VA387 P颗粒相比,其结合O型能力增强,而结合A型能力稍弱.结论 我国优势流行株GⅡ.4型诺如病毒P蛋白的表达成功及其与HBGAs受体结合模型的建立,为今后研究我国诺如病毒的流行进化规律及疫苗的开发研究奠定了实验基础.
目的 建立我國流行優勢株GⅡ.4型諾如病毒(GZ121)P蛋白(包括P顆粒和P二聚體)的原覈錶達繫統,併明確其結閤HBGAs受體的能力和方式.方法 從GⅡ.4型諾如病毒GZ121株基因組中剋隆P區域基因片段,通過構建P區域氨基痠序列進化樹,確定其基因簇.構建含鉸鏈Hinge-P和不含鉸鏈P-CDCRGDCFC的pGEX4T-1原覈錶達質粒,將構建的原覈錶達載體導入大腸桿菌(E coliBL21,用0.6 mmol/L IPTG(isoProPylthio-β-D-galaetoside)過夜誘導P顆粒(不含鉸鏈)和P二聚體(含鉸鏈)在BL21中錶達.目的重組蛋白經溶血酶酶切和FPLC鑒定分析,用EIA法檢測P顆粒和P二聚體與HBGAs的結閤特徵.結果 GZ121株諾如病毒為GⅡ.4基因型2004簇(GⅡ.4/2004 cluster).SDS-PAGE分析確定P顆粒和P二聚體相對分子質量(Mr)約為36×103,大小與報道相符.重組P顆粒經FPLC證實,其在Mr約為830×103處形成特異的P顆粒波峰,Western blot技術證實瞭重組P蛋白的特異性.EIA分析結果錶明,GZ121株P蛋白與A、B、O型唾液均能結閤,與非分泌型唾液不結閤,而P顆粒結閤HBGAs受體能力是P二聚體的80～ 100倍,與96簇VA387 P顆粒相比,其結閤O型能力增彊,而結閤A型能力稍弱.結論 我國優勢流行株GⅡ.4型諾如病毒P蛋白的錶達成功及其與HBGAs受體結閤模型的建立,為今後研究我國諾如病毒的流行進化規律及疫苗的開髮研究奠定瞭實驗基礎.
목적 건립아국류행우세주GⅡ.4형낙여병독(GZ121)P단백(포괄P과립화P이취체)적원핵표체계통,병명학기결합HBGAs수체적능력화방식.방법 종GⅡ.4형낙여병독GZ121주기인조중극륭P구역기인편단,통과구건P구역안기산서렬진화수,학정기기인족.구건함교련Hinge-P화불함교련P-CDCRGDCFC적pGEX4T-1원핵표체질립,장구건적원핵표체재체도입대장간균(E coliBL21,용0.6 mmol/L IPTG(isoProPylthio-β-D-galaetoside)과야유도P과립(불함교련)화P이취체(함교련)재BL21중표체.목적중조단백경용혈매매절화FPLC감정분석,용EIA법검측P과립화P이취체여HBGAs적결합특정.결과 GZ121주낙여병독위GⅡ.4기인형2004족(GⅡ.4/2004 cluster).SDS-PAGE분석학정P과립화P이취체상대분자질량(Mr)약위36×103,대소여보도상부.중조P과립경FPLC증실,기재Mr약위830×103처형성특이적P과립파봉,Western blot기술증실료중조P단백적특이성.EIA분석결과표명,GZ121주P단백여A、B、O형타액균능결합,여비분비형타액불결합,이P과립결합HBGAs수체능력시P이취체적80～ 100배,여96족VA387 P과립상비,기결합O형능력증강,이결합A형능력초약.결론 아국우세류행주GⅡ.4형낙여병독P단백적표체성공급기여HBGAs수체결합모형적건립,위금후연구아국낙여병독적류행진화규률급역묘적개발연구전정료실험기출.
Objective To construct a prokaryotic expression system of norovirus (NoⅤ) G Ⅱ-4 strain P protein (P particle and P dimer) and to explore its binding activity and patterns with HBGAs receptor.Methods P domain sequence of GZ121 NoⅤ ORF2 gene was cloned and its phylogenic tree was constructed to identify the gene cluster.The pGEX-4T-1-based expression plasmids were constructed respectively by inserting P domain gene fragments with hinge and P-CDCRGDCFC without hinge,and then transformed into BL21 to express fusion proteins,which was induced with 0.6 mmol/L IPTG at 22℃ overnight.P proteins were purified by thrombin cutting and characterized by FPLC.The binding patterns of NoⅤ P protein to HBGAs antigens were analyzed by EIA.Results P region gene of GZ121 belonged to genotype G Ⅱ.4/2004 cluster.SDS-PAGE analysis showed the relative molecular weight of P particle and P dimer was about 36×103,which was consistent with other reports.The peak appeared at 830×103 confirmed the formation of P particle by FPLC.The expression of P protein was further confirmed by Western blot.The EIA results showed that GZ121 P protein could bind to saliva of A-group,B-group and O-group secretors,but not to nonsecretor.The binding affinity of P particle was 80-100 times higher than that of P dimer.Compared with VA387 P particle,it showed stronger binding affinity to O-group,but weaker to A-group.Conclusion The NoⅤ GⅡ-4 GZ121 P proteins including P particle and P dimer were successfully expressed and HBGAs receptor binding assays were established.This pave the way for further studies on the evolution dynamics of NoⅤ G Ⅱ.4 strains and the development of NoⅤ vaccines.
