丙型肝炎病毒准种在NS2区的变异状况初探

Preliminary Study of Hepatitis C Viral Quasispecies in NS2 Region in Patients with Hepatitis C Virus Infection

探讨HCV准种在NS2区的基因结构特征及变异状况。利用逆转录 巢式PCR从 1份HCV慢性携带者的阳性血清及 1份丙肝患者的血清中获得HCVNS2全长cDNA ,将其克隆于T载体 ,各随机挑取 5个阳性克隆进行序列测定。结果显示克隆到HCVNS2全长基因 ,所测克隆在核苷酸水平和氨基酸水平互不相同。该慢性携带者HCVNS2区序列以完整读码框架 (ORF)为主 ,一个于HCV多聚蛋白第 835位氨基酸的位置出现终止信号 ,而该丙型肝炎患者以NS2N端发现终止信号的序列为主 ,其中三个于第 835位氨基酸的位置出现终止信号 ,一个于第 887位氨基酸的位置出现终止信号 ,仅一个克隆的序列为完整ORF。对ORF完整的序列进行比较 ,发现丙型肝炎患者氨基酸变异主要集中于N端 ,蛋白二级结构模拟显示丙肝患者NS2与慢性携带者的优势二级结构类似。研究表明从我们选择的两例感染者的HCVNS2序列看 ,不同临床类型的HCV病人体内的HCV准种在NS2区存在差异 ,这种差异可能与病毒存在于机体的状态有一定的一致性。

To explore the genetic variation of nonstructural(NS)2 region and its gene structural characteristics in hepatitis C virus quasispecies,NS2 genes of HCV from a HCV asymptomatic carrier and a patient with abnormal ALT level were amplified by RT-nested-PCR respectively and cloned into T vector.5 positive clones form each of them were selected randomly for sequencing.Every clone was unique in nucleotide sequence and amino acid sequence.Almost all the NS2 gene clones from the asymptomatic carrier had complete ORF except one clone had a stop codon at 835aa in HCV polyprotein.But when it came to the patient with abnormal ALT level,3 clones of them had stop codons at 835aa and 1 clone had a stop codon at 887aa in polyprotein,only 1 clone had complete ORF.Comparing this ORF with the sequence of the asymptomatic carrier,most of the amino acid changes were confined to the N-terminal.Simulating the sceondary structures of NS2 proteins with intact open reading frame demonstrated that the structure of the HCV patient with abnormal ALT level was similar to the dominant structure of the asymptomatic carrier.It could be seen from the two patients with HCV infection that Hepatitis C viral quasispecies in NS2 region had an obvious diversity between HCV asymptomatic carrier and HCV patient with abnormal ALT level.The diversity suggested that the state of HCV quasispecies in NS2 region was likely to correlate with the progression of liver disease.