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基因和编码主要外膜蛋白（MOMP）的基因的等位基因交换突变体，建立了用巴氏杆菌科模式菌睡眠嗜组织菌（Histophilussomni）的目的基因序列替换染色体上任意目的基因序列的基因工程方法。睡觉。本发明的方法包括将突变的DNA序列引入H. somni以诱导随后的同源重组事件，所述同源重组事件是通过使用用读框内缺失的ibpA序列克隆的新的温度敏感性质粒或通过直接使用通过重叠延伸PCR扩增的DNA片段，所述重叠延伸PCR是来自供体菌株的MOMP基因加上侧翼为来自受体菌株的MOMP基因的上游和下游序列的抗生素抗性标记基因的序列。该方法还包括使用克隆有MOMP基因的部分序列及其下游序列的温度敏感性质粒，以从具有标记的等位基因交换的MOMP突变体产生未标记的突变体。因此，在本研究中，该方法已成功地应用于产生所有期望的等位基因交换突变体，表明该工程方法对于产生任何期望的靶序列的未标记突变体的有用性。

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.