Galán J E, Timoney J F
Department of Veterinary Microbiology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853.
Infect Immun. 1987 Dec;55(12):3181-7. doi: 10.1128/iai.55.12.3181-3187.1987.
A Streptococcus equi gene bank was constructed in the bacteriophage lambda gt11 cloning vector, and hybrid phage plaques were screened with S. equi M protein antiserum. A hybrid phage expressing the S. equi M protein (lambda gt11/SEM7) was identified and lysogenized into Escherichia coli Y1089. The cloned M protein appeared in immunoblots as three polypeptides with relative molecular weights of 58,000, 53,000, and 50,000. When reacted with S. equi M protein antiserum in an agar double-diffusion assay, the cloned M protein formed a line of identity with a protein in an acid extract of S. equi. Furthermore, lambda gt11/SEM7 protein inhibited opsonization of S. equi by antiserum to S. equi M protein. In addition, the recombinant protein expressed determinants of the antigen in the immune complexes of purpura hemorrhagica. Native M protein obtained from S. equi and recombinant M protein showed very similar molecular weight distributions on immunoblots, appearing as multiple closely spaced bands with molecular weights ranging from 52,000 to 60,000. Antisera prepared separately against each of the acid-extracted polypeptides shown to be important in serum bactericidal responses (molecular weight, 29,000) and nasopharyngeal local antibody responses (molecular weights, 41,000 and 46,000) of the horse each reacted with all three polypeptides in an acid extract. Moreover, antisera against protoplasts and against recombinant M protein of S. equi also reacted with these polypeptides. These results suggest that the entire M protein molecule of S. equi is present in these preparations and that the fragments in acid extracts carry overlapping segments.
用噬菌体λgt11克隆载体构建了马链球菌基因文库，并用马链球菌M蛋白抗血清筛选杂交噬菌体噬菌斑。鉴定出一个表达马链球菌M蛋白的杂交噬菌体（λgt11/SEM7），并使其溶源化到大肠杆菌Y1089中。克隆的M蛋白在免疫印迹中表现为三种相对分子质量分别为58,000、53,000和50,000的多肽。在琼脂双向扩散试验中，当与马链球菌M蛋白抗血清反应时，克隆的M蛋白与马链球菌酸提取物中的一种蛋白形成一条同一线。此外，λgt11/SEM7蛋白抑制了马链球菌抗血清对马链球菌M蛋白的调理作用。另外，重组蛋白在过敏性紫癜免疫复合物中表达了抗原决定簇。从马链球菌获得的天然M蛋白和重组M蛋白在免疫印迹上显示出非常相似的分子量分布，表现为多条紧密间隔的条带，分子量范围为52,00至60,000。分别针对在马的血清杀菌反应（分子量29,000）和鼻咽局部抗体反应（分子量41,000和46,000）中显示重要的每种酸提取多肽制备的抗血清，均与酸提取物中的所有三种多肽发生反应。此外，针对原生质体和马链球菌重组M蛋白的抗血清也与这些多肽发生反应。这些结果表明，这些制剂中存在马链球菌的整个M蛋白分子，并且酸提取物中的片段携带重叠片段。
