Gentry-Weeks C R, Hultsch A L, Kelly S M, Keith J M, Curtiss R
Laboratory of Microbial Ecology, National Institute of Dental Research, Bethesda, Maryland 20892.
J Bacteriol. 1992 Dec;174(23):7729-42. doi: 10.1128/jb.174.23.7729-7742.1992.
Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.
利用黏粒载体pCP13和pCP13的衍生物、具有壮观霉素抗性的pYA2329，在大肠杆菌LE392中构建了禽博德特氏菌197株DNA的三个基因文库。用恢复期抗禽博德特氏菌火鸡血清和抗禽博德特氏菌197株外膜蛋白的兔多克隆抗血清对黏粒文库进行筛选。一个大肠杆菌重组克隆产生了一种56 kDa的蛋白，它能与感染了禽博德特氏菌197株的火鸡的恢复期血清发生反应。此外，还鉴定出五个大肠杆菌重组克隆，它们产生了分子量分别为21、38、40、43和48 kDa的禽博德特氏菌外膜蛋白。这些大肠杆菌克隆中至少有一个编码21 kDa蛋白的克隆，能与火鸡恢复期血清以及抗禽博德特氏菌197株外膜蛋白的抗体发生反应。通过Tn5seq1诱变对21 kDa外膜蛋白的基因进行定位，并通过双脱氧测序确定其核苷酸序列。对21 kDa蛋白的DNA序列分析显示有一个582个碱基的开放阅读框，可产生一个由194个氨基酸组成的预测蛋白。将编码21 kDa外膜蛋白的基因的预测氨基酸序列与国家生物医学研究基金会蛋白质序列数据库中的蛋白质序列进行比较，发现它与痢疾志贺氏菌、产气肠杆菌、大肠杆菌和鼠伤寒沙门氏菌的OmpA蛋白以及淋病奈瑟氏菌外膜蛋白III、流感嗜血杆菌蛋白P6和铜绿假单胞菌孔蛋白F有显著同源性。编码禽博德特氏菌21 kDa蛋白的基因（ompA）与来自百日咳博德特氏菌和支气管败血博德特氏菌经EcoRI消化的染色体DNA的4.1 kb DNA片段杂交，分别与来自禽博德特氏菌和类禽博德特氏菌DNA经EcoRI消化的染色体DNA的6.0 kb和3.2 kb DNA片段杂交。将一个编码禽博德特氏菌21 kDa蛋白的6.75 kb DNA片段亚克隆到Asd+载体pYA292中，并将构建体导入无毒的ΔcyaΔcrpΔasd鼠伤寒沙门氏菌chi 3987中，用于禽类的口服免疫。编码21 kDa蛋白的基因在禽博德特氏菌197株、Δasd大肠杆菌chi 6097和鼠伤寒沙门氏菌chi 3987中表达水平相当，且主要定位于细胞质膜和外膜。在对用表达编码禽博德特氏菌21 kDa蛋白基因的鼠伤寒沙门氏菌chi 3987口服接种小火鸡的初步研究中，发现单剂量的重组沙门氏菌疫苗未能引发针对21 kDa蛋白的血清抗体，用野生型禽博德特氏菌197株攻击后，气管和胸腺被禽博德特氏菌197株定植。
