Mine O M, Carnegie P R
State Agricultural Biotechnology Centre, Murdoch University, Perth, Australia.
Immunol Cell Biol. 1997 Oct;75(5):484-91. doi: 10.1038/icb.1997.75.
Serine proteases are thought to be involved in the initial attack on sheep skin by Dermatophilus congolensis and are obvious antigens for inclusion in a vaccine to prevent lumpy wool disease (dermatophilosis). Degenerate primers were designed after alignment of seven bacterial serine proteases. Inosine was incorporated into the primers at positions of three- and four-base redundancy, and this reduced the complexity of the primer mixtures from several thousand to sixteen different sequences for each primer. The primers were validated by production and sequencing of amplicons from serine protease genes in Bacillus subtilis and Serratia marcescens. The primers were used with heat-soaked polymerase chain reaction (PCR) to produce amplicons from two D. congolensis strains, AG and MB. In the amplicon codons for arginine, rather than the expected serine, were found where inosine was used for both the first and third positions for a codon in the primer. A search with the deduced amino acid sequences of the amplicons showed significant similarity to a keratinase and other serine proteases from various organisms. Similarity was most apparent around the active site residues and other essential secondary structural elements.
丝氨酸蛋白酶被认为参与刚果嗜皮菌对绵羊皮肤的初始攻击，并且是预防羊毛结块病（嗜皮菌病）疫苗中明显的抗原。在对七种细菌丝氨酸蛋白酶进行比对后设计了简并引物。肌苷被掺入到引物中三个和四个碱基冗余的位置，这将引物混合物的复杂性从数千种降低到每个引物有十六种不同序列。通过从枯草芽孢杆菌和粘质沙雷氏菌的丝氨酸蛋白酶基因产生扩增子并进行测序来验证引物。这些引物与热启动聚合酶链反应（PCR）一起使用，从两种刚果嗜皮菌菌株AG和MB中产生扩增子。在扩增子中，发现精氨酸的密码子而非预期的丝氨酸密码子，其中肌苷用于引物中密码子的第一位和第三位。用扩增子推导的氨基酸序列进行搜索，结果显示与来自各种生物体的角蛋白酶和其他丝氨酸蛋白酶具有显著相似性。在活性位点残基和其他必需的二级结构元件周围，相似性最为明显。
