Grundy F J, Plaut A, Wright A
Department of Molecular Biology and Microbiology, Tufts University Health Sciences Campus, Boston, Massachusetts.
J Bacteriol. 1987 Oct;169(10):4442-50. doi: 10.1128/jb.169.10.4442-4450.1987.
Many bacteria which establish infections after invasion at human mucosal surfaces produce enzymes which cleave immunoglobulin A (IgA), the primary immunoglobulin involved with protection at these sites. Bacterial species such as Haemophilus influenzae which produce IgA1 proteases secrete this enzyme into their environment. However, when the gene encoding this protein was isolated from H. influenzae serotype d and introduced into Escherichia coli, the activity was not secreted into the medium but was localized in the periplasmic space. In this study, the IgA1 protease gene (iga) from an H. influenzae serotype c strain was isolated and the gene from the serotype d strain was reisolated. The IgA1 proteases produced in E. coli from these genes were secreted into the growth medium. A sequence linked to the carboxyl terminus of the iga gene but not present in the original clone was shown to be necessary to achieve normal secretion. Tn5 mutagenesis of the additional carboxyl-terminal region was used to define a 75- to 100-kilodalton coding region required for complete secretion of IgA1 protease but nonessential for protease activity. The iga genes were isolated by a plasmid integration-excision procedure. In this method a derivative of plasmid pBR322 containing a portion of the protease gene and the kanamycin resistance determinant of Tn5 was introduced into H. influenzae by transformation. The kanamycin resistance gene was expressed in H. influenzae, but since pBR322 derivatives are unable to replicate in this organism, kanamycin-resistant transformants arose by integration of the plasmid into the Haemophilus chromosome by homologous recombination. The plasmid, together with the adjoining DNA encoding IgA1 protease, was then excised from the chromosome with DNA restriction enzymes, religated, and reintroduced into E. coli. Comparisons between the H. influenzae protease genes were initiated which are useful in locating functional domains of these enzymes.
许多在侵入人体黏膜表面后引发感染的细菌会产生裂解免疫球蛋白A（IgA）的酶，IgA是参与这些部位保护作用的主要免疫球蛋白。诸如流感嗜血杆菌等产生IgA1蛋白酶的细菌会将这种酶分泌到周围环境中。然而，当从d型流感嗜血杆菌中分离出编码该蛋白的基因并导入大肠杆菌时，该活性并未分泌到培养基中，而是定位于周质空间。在本研究中，从c型流感嗜血杆菌菌株中分离出IgA1蛋白酶基因（iga），并重新分离出d型菌株的基因。从这些基因在大肠杆菌中产生的IgA1蛋白酶被分泌到生长培养基中。结果表明，与iga基因羧基末端相连但在原始克隆中不存在的一个序列对于实现正常分泌是必需的。利用Tn5对额外的羧基末端区域进行诱变，以确定一个75至100千道尔顿的编码区域，该区域是IgA1蛋白酶完全分泌所必需的，但对蛋白酶活性并非必不可少。iga基因通过质粒整合 - 切除程序分离得到。在该方法中，通过转化将含有部分蛋白酶基因和Tn5卡那霉素抗性决定簇的质粒pBR322衍生物导入流感嗜血杆菌。卡那霉素抗性基因在流感嗜血杆菌中表达，但由于pBR322衍生物无法在该生物体中复制，卡那霉素抗性转化体是通过质粒通过同源重组整合到嗜血杆菌染色体中产生的。然后用DNA限制性内切酶从染色体上切下质粒以及相邻的编码IgA1蛋白酶的DNA，重新连接，并重新导入大肠杆菌。开始了对流感嗜血杆菌蛋白酶基因之间的比较，这对于定位这些酶的功能结构域很有用。
