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

如前一份年度报告所述， 我们的（野生型）重组preS 1肽（rpreS 1）含有两个肽 它们只能通过SDS-PAGE分离。 当这种rpreS 1制备被 通过与125 I-Bolton-Hunter试剂缀合用作标记配体， 与人肝细胞制备的质膜特异性结合 或Hep G2细胞。 将该rpreS 1制剂的样品 但在其竞争能力测试中呈阴性， HBsAg与HepG 2细胞的独特结合系统。 因此，点突变是 进行并产生了突变重组质粒，其可以表达 在大肠杆菌中表达含有90个氨基酸的突变前S1肽的融合蛋白。杆菌 在Xa因子消化后，这种含有tyr 12的突变体rpreS 1肽 tyr 13而不是野生型phe 12 phe 13和C-末端gly 90， 发布 用mono-Q柱纯化了突变体rpreS 1 经高效液相色谱法纯化，得到18个N-末端氨基酸残基 通过氨基酸序列分析。 该突变体rpreS 1可以用 无载体Na 125 I通过Iodogen方法，因为 酪氨酸残基的可用性。 约束力研究正在进行中。

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.