抗HBcAg人源性单链抗体细胞内表达及其抗病毒复制的实验研究

应用人源性抗HBcAg单链抗体细胞内表达技术,探讨抗HBV复制基因治疗的应用价值.应用噬菌体展示和基因重组技术,从HBV感染的外周血淋巴细胞克隆了人源性抗HBcAg单链抗体,并重组至逆转录病毒载体.以人肝癌细胞smmc-7721和PLC/PRF/5为靶细胞进行基因共转染,分别测定实验组细胞上清中的HBsAg和HBeAg,与对照组做比较,观察抗HBcAg单链抗体细胞内表达的抗病毒治疗作用.结果显示,在急性HBV感染的细胞株中,抑制病毒复制效率为49%～61%,在慢性病毒感染细胞,抑制率为41%～54%.实验结果表明,应用单链抗体细胞内表达技术,在抗病毒治疗研究中具有潜在的应用价值.应对HBV的4个开放阅读框架编码产物进行全面的对比研究,以发现抑制效率高、实用价值大的靶基因.

Intracellular Expression of Human Single Chain Anti-HBcAg Antibody and Its Inhibitory Effect on Hepatitis B Virus

This study was to explore the applicable value of intracellular expressed human single chain anti-HBcAg antibody in inhibiting the hepatitis B virus replication.The cDNA of human single chain antibody(sFv),anti-HBcAg,was cloned from the peripheral blood mononuclear cells of a hepatitis B virus infected individual using techniques of phage display and molecular cloning.The pLNSX and pLNCX were used as shuttle vectors.The retroviral vectors which expressed the anti-HBcAg sFv were transfected to human hepatocellular carcinoma cell lines smmc-7721 and PLC/PRF/5 respectively.The culture supernatants were collected after transfection and the concentrations of HBsAg and HBeAg were determined by an HBV antigen captured enzyme-linked immunosorbent assay.The results showed that the full length fragment of HBcAg sFv consisted of 788 bp and expressed effectively in target cells after transfection.The inhibited ratio of HBV replication was approximately 49% to 61% in the acute infectious cells smmc-7721 and 41% to 54% in the latent infectious cells PLC/PRF/5.These data suggest that the intracellular expressed single chain anti-HBcAg antibody has a potential therapeutic value in inhibiting hepatitis B virus replication.But it is important to search a more effective target gene among the four open reading frames of hepatitis B virus to promote the inhibitory effect of virus replication.