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目的分离人甲型H1N1流感病毒,探讨血凝素基因的生物学特征。方法采集流感患者鼻咽分泌物,MDCK细胞进行病毒分离,血凝抑制实验测定病毒滴度,提取病毒总RNA,设计特异性引物,RT-PCR扩增HA基因ORF开放阅读框cDNA,克隆到pUCm-T载体,转化大肠杆菌XL1-Blue,经蓝白斑筛选阳性克隆,PCR鉴定后进行基因序列测定,通过生物信息学软件对HA的基因组结构和生物学特征进行分析。结果通过MDCK细胞培养成功分离出人甲型H1N1流感病毒,并于接种后24 h出现CPE,第二代病毒培养物滴度为256;成功扩增出产物大小为1 701 bp的H1N1流感病毒HA基因;构建了pUCm-T/HA载体并测序成功;BLAST结果显示与A/California/04/2009(H1N1)病毒株核苷酸与氨基酸同源性均为99%,所分离的毒株包含20种566个氨基酸。结论甲型H1N1流感病毒的分离和HA基因的测序,为后续HA蛋白的功能学研究奠定了坚实基础。
Objective To isolate human influenza A (H1N1) virus and explore the biological characteristics of hemagglutinin gene. Methods Nasopharyngeal secretions and MDCK cells from patients with influenza were collected for viral isolation. The titer of virus was determined by hemagglutination inhibition assay. The total RNA was extracted and specific primers were designed. The ORF open reading frame of HA gene was amplified by RT-PCR and cloned into pUCm -T vector and transformed into E.coli XL1-Blue. The positive clones were screened by blue and white stains. After identification by PCR, the gene sequences were determined. The genome structure and biological characteristics of HA were analyzed by bioinformatics software. Results The human influenza A (H1N1) virus was successfully isolated from MDCK cells and CPE appeared 24 h after inoculation. The titer of the second generation virus was 256, and H1N1 influenza virus HA with a product size of 1 701 bp The pUCm-T / HA vector was constructed and sequenced successfully. The BLAST results showed that the nucleotide and amino acid homology of the strain was 99% with the A / California / 04/2009 (H1N1) 566 amino acids. Conclusion The isolation of influenza A (H1N1) virus and the sequencing of HA gene lay a solid foundation for the functional study of HA protein.