EXPRESSION AND CHARACTERIZATION OF WILD TYPE AND MUTANT PRES1 PEPTIDES AND HBSAG

如上一份年度报告所述，最近的筹备工作 我们的(野生型)重组preS1肽(RpreS1)含有两个肽 只有通过SDS-PAGE才能将其分离。当这种rpreS1制剂是 通过与125I-Bolton-Hunter试剂偶联用作标记配体，NO 与人肝细胞制备的任一种质膜的特异性结合 或观察到Hep G2细胞。已寄出此rpreS1制剂的样本 对Neurath医生来说，但在他的体内检测出竞争能力为阴性 乙型肝炎表面抗原与HepG2细胞的独特结合系统。因此，点突变是 进行并生产了一种突变型重组质粒，可表达一种 含有90个氨基酸突变的前S1肽的融合蛋白在大肠杆菌中。 在Xa因子消化后，这个突变的rpreS1肽，它包含Tyr12 Tyr13，而不是野生型phe12phe13和C-末端Gly90 释放了。突变株rpreS1经Mono-Q柱层析纯化 柱层析，确认了18个N-端氨基酸残基 通过氨基酸序列分析。这个突变体rpreS1可以标记为 无载体的Na~(125)I通过碘素法，因为 酪氨酸残留量的可用性。约束性研究正在进行中。

As described in the previous annual report, the more recent preparations of our (wild type) recombinant preS1 peptide (rpreS1) contained two peptides which are only separable by SDS-PAGE. When this rpreS1 preparation was used as a labeled ligand by conjugating with 125 I-Bolton-Hunter reagent,no specific binding to either plasma membranes prepared from human hepatocytes or Hep G2 cells was observed. A sample of this rpreS1 preparation was sent to Dr. Neurath but tested negative in its competition capability in his unique binding system of HBsAg to HepG2 cells. Hence, point mutations were performed and produced a mutant recombinant plasmid which can express a fusion protein containing a 90 amino acid mutant preS1 peptide in E. coli. Upon Factor Xa digestion, this mutant rpreS1 peptide, which contains tyr12 tyr13 rather than a wild type phe12 phe13 and a C-terminal gly90, was released. The mutant rpreS1 has been purified by mono-Q column chromatography, and 18 N-terminal amino acid residues have been confirmed by amino acid sequence analysis. This mutant rpreS1 can be labeled with carrier-free Na 125I by means of the Iodogen method because of the availability of tyrosine residues. Binding studies are in progress.