带肝细胞受体结合区的HBV表面抗原蛋白性质的研究——preS区有不同缺失的表面抗原蛋白性质的比较

本文用聚合酶链反应(PCR)构建了四对preS区有缺失突变的乙型肝炎病毒表面抗原基因。对这些突变体及我们以前构建的缺失突变体在哺乳动物细胞中的表达和产物性质进行了比较,结果表明,preS区的部分缺失并不影响羧端S区的基本结构和氨端preS区的表面分布性。当缺失preS1区滞留顺序(a.a.2—19)后,preS区对S区带主要抗原决定簇的区域的掩蔽明显减弱。我们发现过长的preS区严重影响表面抗原蛋白从哺乳动物细胞中的分泌,除已知的滞留顺序外,preS1的另一区域(a.a.48—65)可能也有阻滞表面抗原蛋白分泌的作用,而preS2区对LS蛋白分泌的阻滞不起主要作用。preS1的肝细胞受体结合区(a.a.21—47)和S区直接融合所得蛋白性质稳定,分泌良好,有很强的抗preS1抗原性,是值得发展为新型疫苗的带preS1肝细胞受体结合区的表面抗原蛋白。     

带肝细胞受体结合区的乙型肝炎病毒表面抗原蛋白性质的研究——形成表面抗原颗粒和在不同细胞中的分泌

我们用定向缺失突变和体外重组法构建了含有肝细胞受体结合区的乙型肝炎病毒(HBV)表面抗原主蛋白(S蛋白)基因。该基因在猴肾细胞COS-M6中的表达产物(S309蛋白)能形成表面抗原(HBsAg)颗粒并分泌出细胞。它在细胞和培养液中稳定,并能分别被抗HBsAg和抗preSl区的抗血清所沉淀。CsCl密度梯度分析显示S309蛋白形成颗粒的密度比S蛋白略大(1.25g/ml)。S309蛋白在肝癌细胞株中容易分泌,分泌量和S蛋白相近。但它在COS-M6细胞中的分泌和肝细胞株相差悬殊,分泌量只有10％左右,而且在培养液中出现较迟。和S蛋白的同时表达则能明显地提高它的分泌效率。     

一个能分泌出哺乳动物细胞的HBV表面抗原大蛋白

本文用定向突变方法缺失了人乙型肝炎病毒表面抗原氨端位于preS1区的54个碱基对(编码氨基酸2—19).该基因在猴肾细胞株COS-M6中表达的产物(S301蛋白)和野生型表面抗原大蛋白不同.它不但能被分泌出细胞,而且对表面抗原主蛋白分泌的抑制作用大大减弱,提示了缺失区域中含有一个阻止大蛋白分泌的滞留顺序.S301蛋白的分泌依赖于主蛋白的表达,而且它们的分泌有同步性.和野生型表面抗原大蛋白一样,S301蛋白在COS细胞中合成后能转移至内质网膜,并被糖基化.它对热稳定,对蛋白酶不敏感,并保留了大蛋白和主蛋白的抗原性.由于S301蛋白具有易分泌、稳定性好、抗原性和大蛋白相同等特点,它很可能成为新一代的防治肝炎的基因工程疫苗.     

The Properties of an HBV Surface Antigen Protein Carrying the Binding Site for the Receptor of Hepatocytes——Its Formation of Surface Antigen Particles and Secretion From Discrete Cell Lines

A human hepatitis B virus (HBV) gene, which encodes the major surface antigen protein(S protein) carrying the hepatocyte receptor-binding site, was constructed with site-directed mutagenesis and in vitro recombination. When expressed in monkey kidney cell line COS-M6, this gene product (S309 protein) formed surface antigen (HBsAg) particles and secreted from the cells. It was stable within the cells and in the culture medium and could be immunoprecipitated with antisera directed against plasma-derived HBsAg or synthetic preS1 polypeptide. Isopycnic CsCl gradient centrifugation showed that the density of S309 protein particles (1.25 g/ml) was slightly higher than that of S protein particles. The S309 protein was readily secretable from hepatoma cell lines, and the amount secreted was comparable to that of the S protein. By contrast, only about 10% of the S309 protein was secreted from COS-M6 cells, and its appearance in culture medium was delayed. The efficiency of the secretion of the S309 protein can b     

AN HBV LARGE SURFACE ANTIGEN PROTEIN WHICH CAN BE SECRETED FROM MAMMALIAN CELLS

The N-terminal 54 base pairs (encoding amino acid residues 2—19) within the preS1 region of the human hepatitis B virus surface antigen gene were deleted by site-directed mutagenesis. Unlike the wild type large surface antigen protein; when this mutated gene was expressed in monkey kidney cell line COS-M6, the protein product (S301 protein) could be secreted from the cells. Moreover, the inhibition of the secretion of the major surface antigen protein by this altered large surface antigen protein was greatly reduced, suggesting that the deleted region contained a retention sequence which prevented the secretion of the large surface antigen. However, the coexpression of the major S protein was essential for the secretion of the S301 protein. When coexpressed, the secretion of these two proteins was synchronous. Like the wild type large surface antigen protein, the S301 protein could be translocated into ertdoplasmic reticulum and glycosylated after its synthesis in COS cells. The S301 protein was thermo