Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain.
Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain.
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与白喉毒素羧基末端片段融合的破伤风毒素片段 C 在伤寒沙门氏菌 CVD 908 疫苗株中的表达。
DOI：
10.1016/0264-410x(95)00094-h
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发表时间：
1995
期刊：
影响因子：
5.5
通讯作者：
Levine,MM
中科院分区：
文献类型：
--
作者：
Gómez-Duarte,OG;Galen,J;Chatfield,SN;Rappuoli,R;Eidels,L;Levine,MM
我们报告了破伤风毒素C片段与白喉毒素真核细胞结合域（羧基端）的融合蛋白（FC-bDt融合蛋白）在减毒伤寒沙门氏菌活载体疫苗株CVD 908中的表达。使用质粒pTETnir 15构建FC-bDt蛋白融合体，该质粒携带在nirB启动子（nirBP）控制下的编码FC的基因。对FC的开放阅读框进行了修饰，在FC基因的3′端引入了一个符合读框的甘氨酸-脯氨酸铰链区和一组四个限制性位点。将编码白喉毒素真核细胞结合域的482 bp DNA片段插入到修饰后的FC基因的3′端，构建了符合读框的FC-bDt融合基因。所得到的质粒pOG 215能够在大肠杆菌DH 5a和S.伤寒CVD 908，如通过使用抗FC和抗C-末端白喉毒素单克隆抗体的Western免疫印迹所证实的。通过在巯基乙酸盐肉汤的低氧化还原电位下，即在激活irBP并驱动FC-bDt融合基因转录的条件下，使CVD 908（pOG 215）生长，实现FC-bDt融合蛋白的最大表达。而最大表达的FC单独使用巯基乙酸盐肉汤也观察到，表达的bDt单独是不成功的使用各种生长条件。FC融合物构成了一种策略，以“拯救”否则难以表达的蛋白质的表达。
We report the expression of fragment C of tetanus toxin (FC) fused to the eukaryotic cell binding domain (the carboxyl-terminus) of diphtheria toxin (FC-bDt fusion) in attenuated Salmonella typhi live vector vaccine strain CVD 908. The FC-bDt protein fusion was constructed using plasmid p TETnir15 which carries the gene encoding FC under control of the nirB promoter (nirBP). The open reading frame for FC was modified to incorporate an in-frame glycine-proline hinge region and a set of four restriction sites at the 3′ end of the FC gene. A 482 bp DNA fragment encoding the eukaryotic cell binding domain of diphtheria toxin was then inserted at the 3′ end of the modified FC gene to create an in-frame FC-bDt fusion gene. The resulting plasmid, pOG215, was able to express the FC-bDt fusion protein in both Escherichia coli DH5a and S. typhi CVD 908, as evidenced by Western immunoblots using anti-FC and anti-C-terminal diphtheria toxin monoclonal antibodies. Maximum expression of the FC-bDt fusion protein was achieved by growing CVD 908 (pOG215) at the low oxidation-reduction potential of thioglycollate broth, i.e. in conditions that activatenirBP and drive transcription of the FC-bDt fusion gene. Whereas maximum expression of FC alone was also observed using thioglycollate broth, expression of bDt alone was unsuccessful using a variety of growth conditions. FC fusions constitute one strategy to “rescue” expression of proteins which are otherwise difficult to express.