禽流感病毒NP基因的真核表达及抗体检测芯片的建立

摘要

This paper described a protein microarray, which could be used into detect antibody of influenza A virus (AIV)using recombinant NP protein.The NP gene was amplified by RT-PCR from viral RNA and cloned into the eukaryotic expression vector pFastBacHTa to express recombinant protein.The recombinant protein was identified by western blot, purified through Ni+affinity chromatography and spotted on the aldehyde-coated slides to prepare protein microarray.The optimized conditions of the assay were included a spotting NP protein concentration of 2 mg/mL, immobility time of 24 h, 1％ BSA as blocking buffer, 1∶100 serum dilution and 1∶2,000 dilution for second antibody.This assay was used to test on positive sera against AIV subtype 1 to 15,newcastle disease virus, infectious bursal disease virus, and chicken clinic serum samples, compared with HI test.The result showed that this protein microarray was specific and sensitive for AIV antibody detection.This work provided a novel method for AIV antibody detection and laid the foundation for developing protein microarray to detect AIV different subtype and other pathogen antibodies.%为建立一种检测A型禽流感病毒(AIV)抗体的蛋白芯片,本研究以AIV总RNA为模板,RT-PCR方法扩增NP基因,与真核表达载体pFastBacHTa连接,并在昆虫细胞中表达.重组蛋白经纯化及western blot鉴定,点样于醛基包被的载玻片上,制备检测AIV抗体的蛋白芯片.确定芯片反应最佳条件为:点样浓度2 mg/mL,固定24 h,1%BSA进行封闭,一抗稀释度1:100,二抗稀释度1:2000.利用蛋白芯片分别对15个亚型流感病毒免疫血清、SPF鸡血清、抗新城疫病毒血清、抗法氏囊病毒血清和现地血清样品进行检测,通过与血凝抑制试验比较,表明建立的蛋白芯片方法具有良好的灵敏性和特异性,为AIV抗体检测及制备检测AIV不同亚型或多种病原抗体的蛋白芯片提供方法.