Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin.
Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin.
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插入沙门氏菌鞭毛中的链球菌 M 蛋白表位的表达和免疫原性。
DOI：
10.1128/iai.59.6.2158-2165.1991
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发表时间：
1991
影响因子：
3.1
通讯作者：
Beachey,EH
中科院分区：
文献类型：
--
作者：
Newton,SM;Kotb,M;Poirier,TP;Stocker,BA;Beachey,EH
在pLS408(S.M.C.牛顿，C.O.Jacob和B.A.D.Stocker，Science 244：70-72,1989)中沙门氏菌鞭毛蛋白基因48bpEcoRV缺失的位置，通过钝端连接将合成的48bp的寡核苷酸插入化脓性链球菌5型M蛋白的N-端15个氨基酸(加上CTA密码子，以终止基因的反向插入)。通过限制性内切酶阴性的鼠伤寒沙门氏菌从大肠杆菌中转移到依赖芳香族化合物、鞭毛蛋白阴性的都柏林沙门氏菌活疫苗菌株SL7127中，获得了可移动的菌株SL7127。通过免疫印迹和鞭毛菌与相应合成肽S-M5(1-15)产生的抗体的反应(固定化、免疫金标记)，表明插入的表位在鞭毛中的表达和在鞭毛细丝表面的暴露。用由嵌合鞭毛蛋白组成的鞭毛接种活疫苗株或注射这种细菌的浓缩鞭毛免疫的兔，在与S-M5(1-15)和大消化多肽PepM5的酶联免疫吸附试验中产生抗体。这些抗体对5型链球菌有调理作用。注射活体SL7127(6剂，每剂1×10(6)至2×10(6)，持续8周)的小鼠对M5特异性多肽的滴度达到约12,800，对5型链球菌具调理活性，但对24型链球菌不具调理活性。同样用没有插入鞭毛基因的对照活疫苗株免疫的小鼠的血清不与M5特异性抗原发生反应。给予对照菌株的五只小鼠在感染5型链球菌或24型链球菌后全部死亡；相比之下，给予SL7127菌株的五只小鼠中有四只在M5挑战中幸存下来，但接受24型链球菌挑战的五只小鼠中没有一只存活。因此，我们的研究表明，M蛋白表位可以在无关蛋白的背景下表达，并保持其免疫原性。此外，我们还证明，用鞭毛制成的沙门氏菌活疫苗免疫小鼠可以保护小鼠免受5型化脓性链球菌的攻击，鞭毛制成的鞭毛带有携带M5蛋白保护性表位但没有完整蛋白的交叉反应表位的插入物。
A synthetic 48-bp oligonucleotide specifying the N-terminal 15 amino acids of M protein of Streptococcus pyogenes type 5 (plus a CTA codon, to terminate translation of genes with the insert in reverse orientation) was inserted by blunt-end ligation at the site of the 48-bp EcoRV deletion in the Salmonella flagellin gene in plasmid pLS408 (S. M. C. Newton, C. O. Jacob, and B. A. D. Stocker, Science 244: 70-72, 1989). The resulting plasmid was transferred from Escherichia coli via a restriction-negative Salmonella typhimurium strain into an aromatic-compound-dependent, flagellin-negative live-vaccine strain of Salmonella dublin to produce strain SL7127, which was motile. Expression of the inserted epitope in flagellin and its exposure at the flagellar filament surface were shown by immunoblotting and by the reaction of flagellate bacteria (immobilization, immunogold labeling) with antibody raised by injection of the corresponding synthetic peptide, S-M5(1-15). Rabbits immunized by injection of the live-vaccine strain with flagella composed of the chimeric flagellin or by injection of concentrated flagella from such bacteria developed antibodies reactive in an enzyme-linked immunosorbent assay with peptide S-M5(1-15) and with the large peptic-digest peptide pepM5. These antibodies were opsonic for type 5 streptococci. Mice that were given parenteral live SL7127 (six doses, each 1 x 10(6) to 2 x 10(6), over 8 weeks) developed titers of ca. 12,800 for the M5-specific peptides and opsonizing activity for type 5 streptococci but not for type 24 streptococci. Sera from mice similarly immunized with a control live vaccine strain without an insert in the flagellin gene did not react with the M5-specific antigens. All of the five mice given the control strain, without an insert, died after challenge with type 5 streptococci or type 24 streptococci; by contrast, four of the five mice given strain SL7127, with an insert, survived the M5 challenge, but none of the five challenged with the type 24 strain survived. Therefore, our study shows that an M protein epitope can be expressed in the context of an unrelated protein and maintain its immunogenicity. Furthermore, we demonstrate that mice can be protected against a Streptococcus pyogenes type 5 challenge by immunization with a Salmonella live vaccine with flagella made of flagellin with an insert carrying a protective epitope of M5 protein but without the cross-reactive epitopes of the complete protein.