CAJ | 학술논문

目的建立H5N1禽流感假病毒的体外中和试验技术平台,并系统评价2份H5N1人禽流感患者恢复期血清的中和效价.方法通过磷酸钙沉淀法将转移载体pHR-Luc、包装载体pCMV△8.2和包膜载体CMV/R-SZ(或CMV/R-TH)共转染人胚肾细胞(293T),72 h后收集假病毒上清液,免疫印迹(Western blot)鉴定假病毒颗粒HA、P24蛋白的表达,并取200μL假病毒上清液测定感染活性.收集制备假病毒过程中整合HA蛋白的293T细胞,通过流式细胞术测定2份恢复期血清中的HA抗体.利用2份恢复期血清分别与深圳株和泰国株假病毒进行中和试验,通过计算IC5o判断不同血清对2株假病毒中和效价的差异.结果构建的2株假病毒颗粒不仅含有HIV基质蛋白P24,还可以整合HA蛋白,并且能够由前体蛋白HA0裂解为具有生物活性的HA1和HA2.200μL假病毒上清液感染靶细胞后RLA值约为2×104,具有较高的感染效价.FACS检测结果显示2份血清中均含有HA抗体,而且对不同分枝的假病毒具有交叉反应性.中和试验表明2份血清对深圳株和泰国株假病毒均具有中和能力,而且对不同分枝的假病毒具有交叉中和活性,但对深圳株的中和效价均高于泰国株.结论本研究成功构建具有感染活性的深圳株和泰国株H5N1禽流感假病毒,并可以快速、安全、高效的评价血清的中和效价,具有广阔的应用前景.
목적 건립H5N1금류감가병독적체외중화시험기술평태,병계통평개2빈H5N1인금류감환자회복기혈청적중화효개.방법 통과린산개침정법장전이재체pHR-Luc、포장재체pCMV△8.2화포막재체CMV/R-SZ(혹CMV/R-TH)공전염인배신세포(293T),72 h후수집가병독상청액,면역인적(Western blot)감정가병독과립HA、P24단백적표체,병취200μL가병독상청액측정감염활성.수집제비가병독과정중정합HA단백적293T세포,통과류식세포술측정2빈회복기혈청중적HA항체.이용2빈회복기혈청분별여심수주화태국주가병독진행중화시험,통과계산IC5o판단불동혈청대2주가병독중화효개적차이.결과 구건적2주가병독과립불부함유HIV기질단백P24,환가이정합HA단백,병차능구유전체단백HA0렬해위구유생물활성적HA1화HA2.200μL가병독상청액감염파세포후RLA치약위2×104,구유교고적감염효개.FACS검측결과현시2빈혈청중균함유HA항체,이차대불동분지적가병독구유교차반응성.중화시험표명2빈혈청대심수주화태국주가병독균구유중화능력,이차대불동분지적가병독구유교차중화활성,단대심수주적중화효개균고우태국주.결론 본연구성공구건구유감염활성적심수주화태국주H5N1금류감가병독,병가이쾌속、안전、고효적평개혈청적중화효개,구유엄활적응용전경.
Objective To establish neutralization assay based on H5N1 avian influenza pseudotyped virus in vitro and to evaluate neutralizing titer of convalescent serum from 2 patients with H5N1 avian influenza.Methods pHR-Luc,pCMV△8.2 and CMV/R-SH or CMV/R-TH were cotransfected into 293T cell by co-precipitation with calcium phosphate.Pseudotyped virus supernatant was harvested 72 h posttranofection and identified the expression of HA and P24 by Western blot,and then we analyzed infective activity of 200 μL supernatant of pseudotyped virus.293T cell integrated HA was prepared and anti-HA antibodies in convalescent serum were measured with FACS assay.Neutralizing titers of convalescent serums against Shenzhen and Thailand pseudotyped virus were determined based on calculating IC50 with neutralizing assay.Results Pseudotyped virus involved P24 and HA,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Pseudotyped virus possessed better infective activity,and RLA value was about 2 × 104 with 200 μL supernatant.Both convalescent serums contained anti-HA antibodies and had cross-reactivity against different virus clades with FACS assay.Both convalescent serums had neutralizingactivity and could cross-neutralize different virus clades.However,both serums'neutralizing titers against Shenzhen virus were higher than Thailand.Conclusion We successfully constructed infectious pseudotyped virus which integrated HA of Shenzhen or Thailand virus,and it could be used for evaluation of serum neutralizing activity fast,efficiently and safely with broadly application prospect.