人源性甲型流感病毒(H1N1)分离株血凝素生物学特征分析
摘要
Objective To isolate the influenza A (H1N1) virus in vitro, and to explore the biological characteristics of the hemagglutinin gene. Methods Nasopharyngeal secretion was collected from the influenza virus sufferers and viral isolation was carried out by MDCK cells. The total RNA was extracted. One step RT-PCR was utilized to amplify the ORF fragment of HA gene with specific primers, and then the product was cloned to pUCm-T vector and pUCm-T/HA was transferred to Escherichia coli XL1-Blue. After the positive clone was identified by blue-white selection assay and PCR, the sequencing was analyzed by ABI sequencer 3730 and the structure characteristics were analyzed by Blast, DNAStar or BioEdit software. Results The influenza A virus was successfully isolated through MDCK cells culture and the cells appeared to CPE after 24 hours, the second generation of the virus' titer was 256 by hemagglutination inhibition assay (HAI). The PCR product was about 1, 701 bp and the pUCm-T/HA vector was successfully constructed. The homology of nucleotide and amino acid was 99% similar to the strain of A/California/04/2009 (H1N1), and the isolated strain contained 566 amino acid which composed by 20 types of amino acid. Conclusions The successful isolation and gene sequence analysis will be of great benefit to the later research of HA protein.