为建立可同时检测新疆出血热病毒(CCHFV)、裂谷热病毒(RVFV)、拉沙病毒(LSV)的荧光基因芯片,分剐针对CCHFV和RVFV的S基因、LSV的NP基因,应用生物学软件设计了3对引物与6条探针.运用多重PCR扩增和标记目的片段,将其与基因芯片杂交,最后扫描分析结果.分别对杂交温度与杂交时间进行条件优化,最后对基因芯片的特异性、敏感性和重复性进行评估.结果显示,设计的所有探针都可以与目的片段特异性杂交,探针1a与1b可检测到CCHFV,探针2a与2b可检测到RVFV,探针3a与3b可检测到LSV.相比多重PCR,荧光基因芯片检测CCHFV的灵敏度提高了10倍,检测RVFV的灵敏度提高了100倍,检测LSV的灵敏度提高了100倍.结果表明,本试验建立的同时检测3种出血热病毒的荧光基因芯片检测方法特异性高、敏感性强,可用于这3种病毒的快速检测与甄别.

To construct American serotype porcine reproductive and respiratory syndrome virus(PRRSV)vaccine containing glycosylated protein GP5 antigenic epitope,hepatitis B virus core protein(HBc)particle was used as a vector.The codons of HBc sequence were optimized and cloned into the pET-28a(+)to gain plasmid pEO-NEW.The GP5 gene was inserted into the major immunodominant region of HBc to gain recombinant expression plasmid pEO-LEORF5.It was transformed into Escherichia coli BL21(DE3)for expression under induction of IPTG at 22 ℃.SDS-PAGE,Western-blot and the transmission electron microscope were utilized to analyze the character and structure of the recombinant protein.After ultrasonic broken,fusion protein was purified by sucrose gradient centrifugation.The results showed that the recombinant expression plasmid pEO-LEORF5 was constructed correctly.The expressed fusion protein,with a molecular mass of about 32 ku,existed in both soluble and inclusion body forms.After purification,the fusion protein reached a purity of 67% and could be assembled automatically into virus-like particles(VLPs).The constructed HBc-GP5 VLPs candidate vaccine provided a new way of developing the PRRS vaccine.