摘要
利用人工合成的丁型肝炎病毒(HDV)特异引物和PCR法,从本实验室克隆的含我国四川株HDV-cDNA抗原编码区的重组质粒(PGEM/HDC707和PGEM/HDC274)中,获得2个长度分别为653bp(位于1610-957)和220bp(位于1610-1390)的片段,经Klenow DNA聚合酶补平后,平端插入表达载体pBV220的P_RP_L.串连启动子下游的SmaⅠ位点,转化大肠杆菌DH5α。通过原位杂交和快提抗原,分别筛选到表达不同长度抗原的阳性克隆。由核苷酸推导,它们分别由214和74个氨基酸组成。SDS-PAGE和Western blot表明,大抗原的主要分子量为为29kD,另有24kD和17kD的小肽;小抗原是分子量为10kD的单一条带。RNA结合能力试验证明,大抗原具有结合RNA的特性,而小抗原没有这种特性。此外,大抗原还有结合Neo-RNA的活性,其结合机理及其与病毒复制的关系有待进一步研究。本研究证明,保留HDV抗原编码区近N端的57个氨基酸就具有抗原性。这种基因工程表达的丁肝病毒抗原具有很好的免疫原性,用它免疫豚鼠可产生抗HD抗体。中国株丁肝抗原的表达成功和抗体的制备对于发展我国丁肝诊断试剂,研究我国丁肝病毒特征均具有重要意义。
Hepatitis Delta virus is a defective virus which requires hepatitis B virus for its transmission. High prevalence of HBV infection in China is well known, but anti-HDV antibody positive rate in HBV infected po-pulation is very low ( 0-2.7% ) except XinJiang,XiZang ( Tibet) and Nei-MengGu(Inner Mongolia). In order to study if there are variant subtype of HDV in China, two Chinese HDV-cDNA fragments encoding HDAg were isolatd from recombinants by PCR with specific primers. They were inserted into expression plasmid pBV220 and transformed into E .coli DH5 α. The ampicillin-resistant positive clones were selected by in situ hybridi-zetion with specific Digoxigenin labelled HDV-cDNA probe and ELISA. The results show that both fragments encoding HDAg either 214 amino acids or 74 amino acids at the N terminal end of ORF5(according to Wang's report 1986)show the activity of HDAg. The ELISA titer was 1:10000 to l:32000. SDS-PAGE gel electrophoresis and Westem blotting show that the molecular weights were about 29kD and 10kD respectively. The large HDAg has the activity of binding HDV-RNA bit the samll HDAg has lost the site of binding. It is intresting that the large Chinese HDAg ex-pressed ia E. coli also has the ability of binding Neo-RNA. The mecha-nism of this nonspecific reaction and its significience should be studied. These antigens reacted specifically with anti-HD positive serum and anti-HD monoclonal antibodies. These antigens also had high immunogenicity for production of antibody.
出处
《病毒学报》
CAS
CSCD
北大核心
1993年第4期316-324,共9页
Chinese Journal of Virology
基金
国家863高科技生物领域资助
关键词
丁型肝炎病毒
基因表达
抗原
Hepatitis Delta virus, Expression of HDV-RNA, HDAg, E. coli
