摘要
HBeAg和抗-HBe是乙型肝炎的经典标志物。HBeAg基因与HBcAg基因在DNA C区上相重叠。为要获得重组DNA衍生的HBeAg,本文从adw_2亚型HBV 3.2 kb全长DNA上切取带有HBc基因的BamHI-EcoRI片段,用Bal31和Hpa Ⅱ酶处理产生一系列长度不同的片段,与载体pUR222和pUC 18构成不同的重组质粒,转化到大肠杆菌。通过平板筛选、质粒酶切图谱和抗原抗体反应等筛到高效表达HBeAg的转化株YG301和YG302,中和试验证明了重组HBeAg的血清学特异性。用凝胶过滤、离子交换层析和亲和层析对其进行提纯并用聚丙烯酰胺凝胶电泳结合Western印迹法进行纯度鉴定、含量分析和分子量测定。重组HBeAg制品具有高活性、稳定和安全等特点,实验证明它可以代替血源HBeAg制成酶联药盒用于乙肝诊断检测。
The HBeAg/anti-HBe system are now widely used in clinical and epidemiological study of hepatitis B. HBeAg is now considered to be coded by HBc gene. In this paper, fragment carrying HBc gene from adw_2 HBV DNA was digested with Bal31 and Hpa Ⅱ, a series of fragments was produced and cloned to pUR222 and pUC18 vectors to construct various recombinant plasmids. After transferming to E. coli e selected the recombinants which direct the synthesis of HBcAg at high level in E. coli. The derived HBeAg was purified by chromatographying the crude extract through DEAE column and affinity chromatography column, the preparation obtained have been identified by electrophoresis, Western blot and immunochemical analysis. The rccombinant HBeAg is uscful in the development of the diagnostic agent for detecting anti-HBe and HBeAg.
出处
《生物工程学报》
CAS
CSCD
北大核心
1990年第3期199-204,共6页
Chinese Journal of Biotechnology
