CONSTRUCTION OF A NONTOXIC FUSION PEPTIDE FOR IMMUNIZATION AGAINST ESCHERICHIA-COLI STRAINS THAT PRODUCE HEAT-LABILE AND HEAT-STABLE ENTEROTOXINS
CONSTRUCTION OF A NONTOXIC FUSION PEPTIDE FOR IMMUNIZATION AGAINST ESCHERICHIA-COLI STRAINS THAT PRODUCE HEAT-LABILE AND HEAT-STABLE ENTEROTOXINS
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DOI：
10.1128/iai.58.5.1159-1166.1990
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发表时间：
1990-05-01
影响因子：
3.1
通讯作者：
CLEMENTS, JD
中科院分区：
文献类型：
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作者：
CLEMENTS, JD
将编码耐热性肠毒素基因(ST)的5‘端与编码不耐热性肠毒素结合亚单位(LT-B)的基因3’端进行了基因融合。用于这些研究的ST编码基因是人工构建的，带有适当的限制位点，以允许寡聚体在框内、下游插入。对于这种构建，当LT-B和ST部分之间包含七个氨基酸、含Pro的连接体时，ST抗原性的表达达到最大。用亲和层析法纯化LT-B-ST融合多肽，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法鉴定为一条表观分子量为18000的多肽链。当在十二烷基硫酸钠或十二硫苏糖醇的十二烷基硫酸钠中煮沸时，没有多聚体形成的证据，也没有改变融合肽的迁移率。LT-B-ST融合肽是无毒的，LT和ST的免疫决定簇都能被天然毒素的抗体识别。更重要的是，LT-B-ST融合多肽具有免疫原性。用含有杂化分子的粗制剂或纯化制剂免疫的动物在体外产生能够识别天然毒素的抗体。值得注意的是，这些抗体能够中和天然ST的生物活性。
The 5'' terminus of the gene that codes for the heat-stable enterotoxin of Escherichia coli (ST) was genetically fused to the 3'' terminus of the gene that codes for the binding subunit of the heat-labile enterotoxin of E. coli (LT-B). The ST-encoding gene used for these studies was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion of the oligomer. For this construction, maximum expression of ST antigenicity was obtained when a seven-amino-acid, proline-containing linker was included between the LT-B and ST moieties. The LT-B-ST fusion peptides was purified by affinity chromatography and consisted of a single polypeptide chain with an apparent molecular weight of 18,000 when examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. There was no evidence of multimer formation and no change in the mobility of the fusion peptide when it was boiled in SDS or in SDS with dithiothreitol. The LT-B-ST fusion peptide was nontoxic, and immunologic determinants of both LT and ST were recognized by antibodies to the native toxins. More importantly, the LT-B-ST fusion peptide was immunogenic. Animals immunized with crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro. Significantly, these antibodies were able to neutralize the biological activity of native ST.