Establishment of genetic engineering method for Pasteurellaceae bacteria from animal diseases.

通过构建编码高分子量免疫球蛋白结合蛋白的ibpA基因和编码索姆尼主要外膜蛋白(MOMP)基因的等位基因交换突变体，建立了将索姆尼组织杆菌作为巴氏杆菌科模式菌的目的基因序列替换为所需序列的基因工程方法。该方法包括：将突变的DNA序列导入松材线虫，通过利用带有框内缺失的ibpA序列克隆的新的温度敏感质粒，或直接利用通过重叠延伸PCR扩增的DNA片段，即来自供体菌株的MOMP基因加上来自受体菌株的MOMP基因上下游序列侧翼的抗生素抗性标记基因，来诱导后来的同源重组事件。该方法还包括使用与MOMP基因的部分序列及其下游序列克隆的温度敏感质粒来从等位基因交换的MOMP突变体与标记创建无标记突变体。因此，在本研究中，该方法已成功地应用于所有期望的等位基因交换突变体的创建，这表明该工程方法对于创建任何期望的靶序列的未标记突变体是有用的。

Genetic engineering method to replace any target gene sequence on the chromosome with the desired sequence in Histophilus somni as a model bacterium of the family Pasteurellaceae has been established through creating allelic exchanged mutants of ibpA gene encoding high-molecular weight immunoglobulin binding proteins and a gene encoding major outer membrane protein (MOMP) of H. somni. The present method includes introduction of mutated DNA sequence into H. somni in order to induce the later homologous recombination event by use of a new temperature-sensitive plasmid cloned with in-frame deleted ibpA sequences or by direct use of DNA fragment amplified by overlapped extension PCR of the sequence that is MOMP gene from a donor strain plus antibiotic resistance marker gene flanked by upstream and downstream sequences of MOMP gene from a recipient strain. The method also includes use of temperature-sensitive plasmid cloned with partial sequence of MOMP gene plus its downstream sequence to create an unmarked mutant from the allelic exchanged MOMP mutant with a marker. Thus, the method has been successfully applied to create all desired allelic exchanged mutants in this study, indicating the usefulness of this engineering method for creation of unmarked mutants of any desired target sequences.