HGV广东株和香港株NS5区部分基因的克隆及序列分析

Molecular Cloning and Sequencing the NS5 Genomic Region of HGV Guangdong and Hong Kong Strains

采用ＨＧＶＮＳ５特异的２对引物，对两个香港株和一个广东株ＨＧＶＲＮＡ进行逆转录套式ＰＣＲ扩增，ＰＣＲ产物克隆入ｐＵＣ１９，重组质粒转化ＤＨ５α和ＪＭ１０９菌株。ＰＣＲ和酶切法鉴定阳性克隆，双脱氧链末端终止法测定核苷酸序列并进行同源性分析。结果发现核苷酸变异呈散在分布，三株间核苷酸和氨基酸序列同源性分别为９３．３％～９４％及９７％～９９．２％，与已报道的中国株（ＣＮ）相比，则同源性分别为９０％～９１．２％和９４％～９６．３％，与美国株（ＰＮＦ２１６１及Ｒ１０２９１）相比，为８７．１％～８９．５％和９５．２％～９７％，而与西非株（ＧＢＶＣ）相比，则达９１．４％～９３．８％和９７％～９７．９％。提示ＨＧＶＮＳ５区核苷酸和氨基酸序列相对保守，不同ＨＧＶ株存在一定的地区差异。

HGV NS5 cDNA of two Hong Kong strains and one Guangdong strain were amplified by reverse transcription polymerase chain reaction (RT PCR). The products were inserted into pUC19 vectors respectively. After transfecting DH5α and JM109 strains, the recombinants were screened and identified by PCR and digested with restriction endonucleses. The nucleotides were sequenced by the dideoxy chain termination method. The homologies of HGV NS5 region nucleotide and deduced amino acid sequences were 93.3%-94% and 97%-99.2% respectively between each Hong Kong strain and the Guangdong strain, and were 90%-91.2% and 94%-96.3%, 87.1%-89.5% and 95.2%-97%, 91.4%-93.8% and 97%-97.9% respectively compared with the reported China isolate (CN), American isolates (PNF2161 and R10291), and West African isolated (GBV C). The variant sites of the nucleotides appear sporadically. The nucleotide and deduced amino acid sequences of HGV NS5 genomic region are relatively conserved, and it also suggests that the nucleotide variation of HGV may be influenced by geographical factors.