目的:筛选一套灵敏度高、特异性强的引物探针,用于建立检测HBV病毒载量的荧光定量PCR方法。方法:通过PCR方法扩增HBV基因组片段,与T载体连接,制备工作标准品,并运用设计的荧光定量PCR对HBV DNA国家标准品进行检测。结果:以pMD18-T-HBc作为标准品,HBcF和HBcR为上下游引物,HBcP为探针所建立的荧光定量PCR检测HBV病毒载量方法的线性范围为109copies/mL到103copies/mL,批内重复性变异系数为0.20%～1.44%,批间重复性变异系数为3.08%～5.63%,检测国家阴阳性标准品的特异性灵敏度为100%,国家线性标准品的检测值与理论值的线性相关系数R为0.998。结论:成功筛选出一套荧光定量PCR检测HBV DNA的引物探针,并已建立了一种特异性灵敏性均非常高的荧光定量PCR检测HBV DNA的方法。
本试验旨在建立一种快速准确、灵敏特异的HBV-DNA定量检测方法,并对该方法进行评价.利用基凶重组技术制备外标准品,采用Taqman探针检测HBV-DNA国家参考品和HBsAg(+)血浆,并与商品化试剂盒检测结果进行相关性分析,成功构建了外标准品.且标准曲线相关系数R2为0.9987.最低检测限为100拷贝/mL,批内变异系数(CV)为1.55%～2.88%,批间CV为2.32%～3.78%.与HBV-DNA国家参考品阳件符合率、阴性符合率均为100%,线性参考品检测结果的线性相关系数R2为0.996.与HBV-DNA商品化试剂盒相比,灵敏度为100%,特异性为85.4%,准确度为94.1%,检测结果呈显著的等级相关性.本研究成功建立了一种比商品化试剂盒检测灵敏度更高且更加快速准确的HBV-DNA检测方法.
乙肝血清标志物(HBV-M)对于乙肝筛查、诊断、分期、疗效监测及预后判断等具有苇要价值.目前,检测乙肝血清标志物主要是采用酶联免疫吸附试验(ELISA),该方法仪能单独检测,不能满足临床中多指标检测分析的要求.液相芯片法是一种新型生物芯片技术平台,其最大优势在于可同时对同一样本中多达100种不同目的分子进行分析.
To subclone and express the gene encoding the immunoglobulin binding protein(IGBP) of Rhipicephalus haemaphysaloides tick and analyze its immune protective effect,the IGBP gene was cloned into vector pGEX-4T-1,then transformed to Escherichia coli BL21 cells and expressed by the induction with IPTG.The expressed product was purified and used as antigen to immunize rabbits in order to evaluate its immune protective effect.The rabbits in the vaccinated group were immunized with 500 μg of the recombinant protein emulsified with 206-adjuvant,while those in the control group injected with PBS emulsified with 206 adjuvant.The immunization consisted of the triple injections at weeks 0,2 and 4,respectively.All the rabbits were challenged with R.haenaphysaioides ticks 2 weeks after the final injection in order to evaluate the immune effect.It was found that the fusion protein GST-IGBP was highly expressed in E.coli in the form of the inclusion bodies.There were significant differences in tick weights and mortalities between the vaccinated and control groups.It is evident that the IGBP gene can be highly expressed in E.coli and the fusion proteins GST-IGBP can produce the anti-tick protective immunity to a certain degree.
