摘要
为构建副猪嗜血杆菌Hfq基因缺失株,以副猪嗜血杆菌S2261基因组为模板扩增Hfq基因的上、下游同源臂,用融合PCR的方法融合拼接两条同源臂。以Eco RⅠ+HindⅢ分别对融合片段与p K18mobSac B质粒进行双酶切并用T4 DNA连接酶连接以构建同源重组质粒p K18mob Sac B-Hfq-ud。以大肠杆菌S17-1-pir作为供体菌,副猪嗜血杆菌S2261作为受体菌,通过接合转移法成功构建了副猪嗜血杆菌Hfq基因缺失株。绘制了S2261野生株与Hfq缺失株的生长曲线以及测定了LD50,结果显示,相比于S2261野生株,Hfq缺失株在对数期的生长速度下降;S2261野生株与Hfq缺失株的LD50分别为2.9×108与3.16×1010CFU,这说明相比野生株,Hfq缺失株导致小鼠半数死亡所需的剂量显著升高,相对的其毒力则明显下降。药敏试验结果显示,Hfq缺失株对氧氟沙星和诺氟沙星的敏感性明显增加。以上结果说明在副猪嗜血杆菌中,Hfq基因对该菌的生长速度、毒力以及药物敏感性具有调控作用。这为副猪嗜血杆菌Hfq基因功能以及该菌相关的疫苗研究奠定了基础。
To construct the Hfq deletion strain of Haemophilus parasuis,upstream and downstream homology arms were amplified from H.parasuis strain S2261.Two of these flanking sequences were connected with overlapping PCR. Both of the assembled fragments and p K18 mob Sac B plasmid were.digested with Eco RⅠand HindⅢ, purified and ligated with T4 DNA ligase to construct the homologous recombinant plasmid p K18 mob Sac B-Hfq-ud.The method of conjugative transfer was applied to construct the Hfq deletion strain of H.parasuis with a donor strain S17-1-pir and a recipient strain S2261. The growth curve and LD50 of wild-type strain S2261 and Hfq deletion strain were measured. And the growth rate of Hfq deletion strain was decreased, compared to that of wild-type strain S2261. The LD50 of wild-type strain S2261 and Hfq deletion strain were 2.9×10^8and 3.16×10^10,respectively,which demonstrated the LD50 of Hfq deletion strain was increased while its virulence to mice was sharply reduced. The data of antibiotics sensitivity test showed an increase in ofloxacin and norfloxacin of Hfq deletion strain. All of these results indicated that Hfq plays a key role in regulation of growth,virulence and antibiotics sensitivity in H.parasuis,which laid a foundation for further study on Hfq gene function and vaccine development of H.parasuis.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2016年第2期203-209,共7页
Chinese Veterinary Science
基金
"十二五"国家科技支撑计划项目(2013BAD12B04)
