Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame
Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame
复制标题
整个pre-s开放阅读框编码的乙型肝炎病毒多肽的鉴定
DOI：
10.1128/jvi.55.1.223-231.1985
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发表时间：
1985
影响因子：
5.4
通讯作者：
John J. SNINSKYl
中科院分区：
文献类型：
--
作者：
T. Wong;Nrapendra;Nath;John J. SNINSKYl
编码乙型肝炎病毒226个氨基酸外壳蛋白（乙型肝炎病毒表面抗原[HBsAg]）的开放阅读框（ORF）有可能编码400个氨基酸的多肽。整个 ORF 将指导多肽的合成，该多肽的 C 端氨基酸代表 HBsAg，N 端有额外的 174 个氨基酸（pre-s）。最近，病毒颗粒已被证明含有与 HBsAg 相对应的多肽，在 N 末端有一个额外的 55 个氨基酸，由紧邻 HBsAg 基因上游的 DNA 序列编码。含有 TAC 启动子、β-半乳糖苷酶基因的前 8 个密码子以及氯霉素乙酰转移酶的整个编码序列的新型 ORF 表达载体在细菌中使用，以表达从 ORF 的 pre-s 部分预测的 174 个氨基酸的决定簇。所得三联蛋白含有由前s编码的108个氨基酸，被表达为携带重组质粒的细菌的主要蛋白之一。通过在氯霉素亲和树脂上分级分离，实现了三杂交体融合蛋白的一步纯化。针对融合蛋白产生的多克隆抗血清能够检测病毒颗粒中的 42 道尔顿和 46 道尔顿多肽；两种多肽也显示含有 HBsAg 决定簇。多克隆抗血清能够从病毒颗粒中识别具有这些特征的多肽，这提供了令人信服的证据，证明整个 ORF 的 DNA 序列表达为包含 HBsAg 的连续多肽。来自这个单一 ORF 的多个启动子和初级翻译产物的存在表明，所编码的多肽的功能和潜在的相互作用在病毒的生命周期中发挥着至关重要的作用。此外，本报告中描述的程序和载体可以应用于其他系统，以促进针对确定的决定簇的抗体的生成，并且应该允许表征现有抗体的表位特异性。
The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.