THE MOLECULAR BIOLOGICAL STUDY OF THE RELATIONSHIP BETWEEN THE ACTIVE STAGE OF CHRONIC HEPATITIS B AND THE MUTATION OF THE HEPATITIS B VIRUS

慢性乙型肝炎活动期与乙型肝炎病毒突变关系的分子生物学研究

• 批准号: 63480203
• 负责人: OHTA Yasuyuki
• 金额: $ 3.84万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480203/
• 关键词: HBVDNA; PGR(Polymerase chain reaction); Mutation; PCR; mutation; B型慢性肝炎; PCR法

We have reported that the presence of sublobular necrosis of the hepatocytes is important in the progress of chronic hepatitis to liver cirrhosis. It has been evident that the necrosis is caused by the immunological response of the host to the hapatocytes infected by hepatitis B virus (HBV). But it has not been clear why the necrosis of hepatocytes are repeated even if the HBV-infected hepatocytes are cleared once.We report here, the result of the study of the presence of mutation at the C region of a HBV clone in cases of chronic active hepatitis B, and the HBV clone with the stop codon at amino acid position 28 in the precore region of HBVDNA is prominently present in the serum of some of HBeAg-positive patients. Four serum samples from two patients with chronic hepatitis B were examined for the cloning or HBV. Serum samples were collected before and after seroconversion. from HBeAg to anti-HBe in one patient, and at two different Points in the HBeAg-positive stage of another patient. DNA was extracted from 370ul of serum by the alkaline lysis method, and HBVDNA fragment was amplified by PCR (Polymerase Chain reaction). Amplified fragments were inserted into a pUC vector, and 4 independent clones possessing HBVDNA were isolated, the sequences of which were determined by the dideoxy chain termination method. In the two patients there are no mutations before and after the elevation or the transaminase, but the stop codon at the precore region was present in all 12 clones derived from the HBeAg- Positive stages of both patients, and also in all 4 clones derived from the anti-HBe-positive stage of the first patient.These results suggest that mutant HBV with the stop codon, and Wild-type HBV coexist and that a small population of wild-type HBV among a large population of mutant HBV was producing in our cases.

我们曾报道肝细胞小叶下坏死的存在在慢性肝炎向肝硬化的进展中是重要的。研究表明，肝细胞坏死是由宿主对B型肝炎病毒（HBV）感染的肝细胞产生的免疫反应引起的。但是，为什么即使HBV感染的肝细胞被清除一次，肝细胞的坏死也会重复，这一点还不清楚。我们在这里报告，在慢性活动性肝炎B病例中，HBV克隆的C区存在突变的研究结果，在HBVDNA前C区28位氨基酸终止密码子的HBV克隆主要存在于一些HBeAg阳性的人血清中，阳性患者对2例慢性B型肝炎患者的4份血清标本进行了HBV克隆性检测。在血清转换前后采集血清样品。1例患者HBeAg转为抗-HBe，另1例患者HBeAg阳性时在两个不同的时间点。用碱裂解法从370 μ l血清中提取DNA，用聚合酶链反应（PCR）扩增HBVDNA。将扩增的片段插入pUC载体，分离出4个含有HBVDNA的独立克隆，用双脱氧链终止法测定其序列。在这两名患者中，转氨酶升高前后均无突变，但在两名患者HBeAg阳性阶段的所有12个克隆中以及在第一名患者抗-HBe阳性阶段的所有4个克隆中均存在前C区的终止密码子。和野生型HBV共存，并且在我们的病例中，在大量突变型HBV中产生了少量野生型HBV。

项目成果

Takashi NONAKA et al.: "Presence of a translational stop codon in the precore region of HBVDNA in the HBeAg positive stage of CHB" Gostroenterologia Japonica. 25. (1990)

Takashi NONAKA 等人：“CHB HBeAg 阳性阶段 HBVDNA 前核心区域存在翻译终止密码子”Gostroenterologia Japonica。

Takahi NONAKA, et al: "Presence of a translational stop codon in precore region of HBV in the HBeAg positive stages of chronic hepatitis B." Gastroenterologia Japonica.25, 1990.

Takahi NONAKA 等人：“慢性乙型肝炎 HBeAg 阳性阶段的 HBV 前核心区存在翻译终止密码子。”

Takashi NONAKA,et al: "Presence of a translational stop codon in precore region of HBV in the HBeAg positive stages of chronic hepatitis B." Gastroenterologia Japonica.(印刷中). 25. (1990)

Takashi NONAKA 等人：“慢性乙型肝炎 HBeAg 阳性阶段中存在翻译终止密码子。”（1990 年出版）。