鸭疫里默氏杆菌RIA0940基因缺失株的构建及主要生物学特性测定  

Construction and Main Biological Characteristics Determination of RIA0940 Gene Deleted Strain of Riemerella anatipestifer

作　　者：李林林 董嘉文 张俊勤 黄允真 孙敏华 LI Linlin;DONG Jiawen;ZHANG Junqin;HUANG Yunzhen;SUN Minhua(Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases,Ministry of Agriculture and Rural Affairs,Key Laboratory of Livestock Disease Prevention and Treatment of Guangdong Province,Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China)

机构地区：[1]广东省农业科学院动物卫生研究所,农业农村部禽流感等家禽重大疾病防控重点实验室,广东省畜禽疫病防治研究重点实验室,广州510640

出　　处：《中国畜牧兽医》2022年第12期1464-1472,共9页China Animal Husbandry & Veterinary Medicine

基　　金：科技创新战略专项资金(202110TD、R2020PY-JC001、R2020PY-JX014、R2020QD-049);广东省农业厅基础性长期性监测项目(2021KJ156);广东省动物疫病野外科学观测研究站项目(2021B1212050021);江门市科技计划项目(2021030102450004518)。

摘　　要：【目的】通过构建鸭疫里默氏杆菌RIA_0940基因缺失株并测定其生物学特性,探讨RIA_0940基因的潜在功能。【方法】以鸭疫里默氏杆菌RA-GD株为亲本株,扩增其左右同源臂及红霉素抗性基因(ermR)盒片段,构建左同源臂-ermR抗性基因盒-右同源臂融合片段,通过自然转化的方法缺失RA-GD株RIA_0940基因,并利用PCR筛选、鉴定RA-GDΔRIA_0940基因缺失株。分别对亲本株RA-GD和缺失株RA-GDΔRIA_0940的生长特性、对雏鸭的半数致死量、感染鸭血液和组织载菌量及对Vero细胞的黏附和入侵性能进行比较分析。【结果】试验成功构建了RA-GD株的RIA_0940基因缺失株(RA-GDΔRIA_0940);生物学特性检测结果显示,RA-GDΔRIA_0940株体外生长能力较亲本株低,基因缺失株在生长后期生长受到显著或极显著抑制(P<0.05;P<0.01);RA-GDΔRIA_0940株对雏鸭的半数致死量是亲本株的114倍;与亲本株相比,缺失株感染鸭血液和组织的载菌量显著或极显著下降(P<0.05;P<0.01);缺失株对Vero细胞的黏附和入侵性能均极显著低于亲本株(P<0.01)。【结论】本试验成功构建RA-GD株RIA_0940基因缺失株,该缺失株在体外培养条件下生长能力较亲本株低,对雏鸭的致病力、感染鸭血液和组织载菌量及对Vero细胞的黏附和入侵性能均显著或极显著低于亲本株。本试验结果为深入研究鸭疫里默氏杆菌的分子致病机理和研制基因工程疫苗奠定了基础。【Objective】 In this study, Riemerella anatipestifer RIA_0940 gene deletion strain was constructed, and its main biological characteristics were studied to explore potential functions of RIA_0940 gene.【Method】 Taking the isolated Riemerella anatipestifer RA-GD strain as the parent strain, the left and right homologous sequences and erythromycin resistance gene fragments were amplified, and the left homologous arm-ermR resistance gene box--right homologous arm fusion fragment was constructed.The RIA_0940 gene of RA-GD was deleted by natural transformation, and the gene deleted strains RA-GDΔRIA_ 0940 were screened and identified by PCR.Growth curves, LD_(50) to ducklings, blood and tissues bacterial load and the ability of cell adhesion and invasion were tested and analyzed of wild-type strain RA-GD and deletion mutant RA-GDΔRIA_0940,respectively.【Result】 The results showed that the RA-GD RIA_0940 gene deletion strain was successfully constructed.The results of biological characteristics showed that the growth ability of strain RA-GDΔRIA_0940 in vitro was significantly or extremely significantly inhibited compared with its wild-type(P<0.05;P<0.01),the LD_(50) of RA-GDΔRIA_0940 was 114 times that of wild-type strain.Compared with wild-type strain, the blood and tissues bacterial load of RA-GDΔRIA_0940 infected ducks were significantly or extremely significantly decreased(P<0.05;P<0.01);The adherence and invasion capacities of RA-GDΔRIA_0940 to Vero cells were extremely significantly decreased(P<0.01).【Conclusion】 In summary, RA-GDΔRIA_0940 deletion mutant was constructed successfully.The growth trend of the deletion strain was slow under the condition of in vitro culture.The pathogenicity to ducklings, the amount of bacteria in infected duck blood fluid and tissues, and the ability of adhesion and invasion to Vero cells were significantly or extremely significantly lower than those of the wild-type strain.The results laid a foundation for the further study of the molecular pathogenesis of Riem

关 键 词：鸭疫里默氏杆菌 RIA_0940基因 基因缺失 生物学特性 毒力因子 

分 类 号：S852.61[农业科学—基础兽医学]