h5n1禽流感假病毒体外中和试验技术平台的建立与应用

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. abstract： 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.     

整合深圳人源H5N1禽流感病毒株血凝素蛋白的假型逆转录病毒的构建

目的 构建整合深圳人源H5N1禽流感病毒(avian influenza virus,AIV)株血凝素(HA)的假型逆转录病毒.方法 利用逆转录聚合酶链反应(RT-PCR)技术扩增深圳人源H5N1禽流感病毒株HA基因,将扩增产物与pGEM-T载体连接,在经过酶切及测序鉴定后,通过Sal Ⅰ与BamHⅠ酶切位点将HA基因克隆至真核表达载体CMV/R,然后协同转移载体pHR-Luc、包装载体pCMV&8.2通过磷酸钙共沉淀法转染人胚肾细胞(293T),72 h后收集假病毒上清,超速离心后通过Western blot鉴定假病毒颗粒HA、P24蛋白的表达,同时测定HIV-HA假病毒对犬肾传代细胞(MDCK)、宫颈癌上皮细胞(HeLa)、中国仓鼠卵母细胞(CHO)和293T等4种不同细胞株的感染活性.结果 深圳人源HSN1禽流感病毒株A/Shenzhen/406H/06属于subelade2.3,其HA基因开放读码框编码567个氨基酸,在GenBank登录号为EF137706.经Sal Ⅰ与BamH Ⅰ双酶切鉴定HA基因成功克隆至真核表达载体CMV/R.将pHR-Luc、pCMV&8.2、CMV-HA共转染293T细胞后,所收集的假病毒经Western blot证实假病毒颗粒不仅含有HIV基质蛋白P24,还可以整合AIV 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蛋白.HIV-HA假病毒可以感染MDCK、HeLa、CHO、293T等4种靶细胞,表明所构建的假病毒具有感染活力,且具有广泛的宿主范围.结论 本研究成功构建整合A/Shenzhert/406H/06 HA蛋白的假型逆转录病毒,并且HIV-HA假病毒具有良好的感染活性,从而为下一步筛选中和抗体及抗原表位分析奠定基础.     

CD19和CD22双靶点CAR-T细胞对肿瘤细胞的杀伤作用

目的：设计并制备一种分别靶向B细胞表面抗原CD19和CD22的CAR-T细胞,检测其对肿瘤细胞的体内外杀伤效果。方法：将含有人源化CD19 Sc Fv的二代CAR分子和带有CD3ε链作为共刺激结构域的CD22 Sc Fv CAR分子以P2A自剪切肽连接,序列连接于慢病毒载体p LTR-CMV-MCS中,以HEK-293T细胞包装相应的慢病毒载体,感染健康志愿者提供的T细胞制备CAR-19-22-T细胞,同时以相同二代结构分别构建单靶向CAR-T细胞作为参照。构建表达荧光素酶、CD19和/或CD22的前列腺癌3M细胞（靶细胞）。将各种CAR-T细胞与靶细胞共同培养,采用荧光素酶化学发光法和ELISA法检测其对靶细胞的杀伤能力和细胞因子的分泌水平。通过尾静脉注射Raji-Luc细胞构建NOD-SCID免疫缺陷小鼠白血病模型,分别注射各组CAR-T细胞进行治疗并评估其疗效。结果：培养7 d的CAR-19-22-T细胞的CAR-19表达率为13.7%,CAR-22表达率为14.3%。CAR-19-22-T细胞在10∶1效靶比时,对3M-CD19-Luc、3M-CD22-Luc和3M-CD19-...