Cloning and expression of genes encoding Haemophilus somnus antigens
Cloning and expression of genes encoding Haemophilus somnus antigens
复制标题
编码睡眠嗜血杆菌抗原的基因的克隆和表达
DOI：
10.1128/iai.56.10.2736-2742.1988
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发表时间：
1988
影响因子：
3.1
通讯作者：
D. Guiney
中科院分区：
文献类型：
--
作者：
L. Corbeil;G. Chikami;M. Yarnall;Jeffrey W. Smith;D. Guiney
睡眠嗜血杆菌2336的基因组文库，这是一种从患肺炎的小牛中分离的强毒分离株（后来用于繁殖H。睡眠实验性肺炎），构建在粘粒载体pHC 79中。用能与几种H.睡眠对免疫反应菌落进行Western印迹分析，发现5个克隆表达与H.睡眠表面抗原三个克隆（DH 1 pHS 1、pHS 3和pHS 4）表达120千道尔顿（kDa）抗原和76 kDa抗原，一个克隆（DH 1 pHS 2）仅表达76 kDa抗原，第五个克隆（DH 1 pHS 5）表达60 kDa抗原。120-kDa和76-kDa抗原在内部发现，而60-kDa蛋白在DH 1 pHS 5培养物上清液中作为膜泡或不溶性蛋白检测到。H. somnus 120-kDa抗原和重组120-kDa抗原具有免疫球蛋白Fc结合活性。限制性内切酶图谱表明，基因组DNA插入的克隆表达76-kDa的抗原共享一个共同的28.4-内切酶对区域，和三个克隆也表达120-kDa的抗原共享一个额外的7.0-内切酶对区域。表达60-kDa抗原的pHS 5的限制性内切酶图谱与其他四种质粒的图谱不相似。因为这三个H。结果表明，该重组蛋白可与恢复期血清发生免疫反应，为进一步研究牛H.睡眠病
A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease.
DOI：
10.1073/pnas.81.9.2645
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
GRAY, GL;SMITH, DH;HEYNEKER, HL
通讯作者：
HEYNEKER, HL