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为建立1株猪源2009/H1N1流感病毒A/swine/Heilongjiang/44/2009(HLJ44)的反向遗传系统,利用双向表达质粒p HW2000,分别构建了该病毒株8个基因节段的重组质粒,将其共转染于293T和MDCK混合培养的细胞,拯救出重组病毒R-HLJ44。测序结果表明,R-HLJ44与亲本病毒HLJ44的核苷酸序列完全一致;二者在细胞上具有相似的增殖特性;抗原性未发生变化;分别以106TCID50的剂量鼻腔感染BALB/c小鼠,结果显示R-HLJ44与亲本HLJ44在小鼠脏器中的复制滴度也基本一致。以上结果表明拯救的重组病毒保持了与亲本病毒一致的生物学特性。该病毒反向遗传系统的建立,为进一步研究H1N1亚型流感病毒的致病分子机制及新型疫苗研制等奠定了基础。
In order to establish a reverse genetic system of a swine 2009 / H1N1 influenza virus A / swine / Heilongjiang / 44/2009 (HLJ44), two recombinant plasmids were constructed by using the bidirectional expression plasmid p HW2000. , And co-transfected them into 293T and MDCK mixed culture cells to rescue the recombinant virus R-HLJ44. The sequencing results showed that the nucleotide sequence of R-HLJ44 was identical with that of the parental strain HLJ44; both of them had similar proliferation characteristics on the cells; the antigenicity did not change; BALB / c mice were respectively infected with 106TCID50 nasal cavity. Results The results showed that the replication titer of R-HLJ44 and its parental HLJ44 in mouse organs were also basically the same. The above results indicate that the rescued recombinant virus retains the biological characteristics consistent with the parental virus. The reverse genetics system of the virus established for further study of H1N1 influenza virus pathogenic molecular mechanism and the development of a new vaccine laid the foundation.