摘要
为了明确透明质酸酶(Hyl)在鱼源无乳链球菌致病过程中的作用,根据Gen Bank上公布的罗非鱼源无乳链球菌Hyl的全基因序列设计引物,采用PCR方法扩增长度为3 156 bp的hyl B基因片段。经限制性内切酶Eco RⅠ和XhoⅠ消化后克隆至表达载体p ET-32a(+),构建重组表达质粒p ET-32a(+)-hyl B,经双酶切、测序鉴定后转化至大肠杆菌BL21(DE3)中,进行原核表达。结果显示,137.1 ku的目的蛋白获得成功表达,平板法证实该重组蛋白具有良好的降解透明质酸的活性;用纯化的重组蛋白注射ICR小鼠,病理组织学观察显示,肺和脑组织出现明显的出血和炎性细胞浸润等病理变化。结果表明,Hyl在鱼源无乳链球菌引致肺炎和破坏血脑屏障引起脑膜炎过程中发挥重要作用。
To determine the role ofhyaluronidase（Hyl）in the pathogenicity of Streptococcus agalac- tiae, a pair of specific primers was designed according to the hylB gene sequence of piscine S. agalactiae published in GenBank. A 3156bp long whole fragment of hylB was amplified from the genome of S. agalactiae GD201008-001 by PCR. This amplified fragment was inserted into the pET-32a（-F） vector, thus creating a recombinant expression vector pET-32a（-F）-hylRThe recombinant vector was transformed into Escherichia coli BL21 and induced by IPTG to express. SDS-PAGE and Western blotting analyses demonstrated that the target protein of 137.1 ku was obtained. Hyaluronic acid （HA） degradation assays showed that the expressed protein had a good enzymatic activity. Further more, the protein was injected into the ICR mice through the tail vein. Histopathological observation showed that both of the lung and the brain were damaged seriously with manifestations of pulmonary interstitial broadening, the infiltration of inflammatory cells and cerebrovascular hemorrhage. It suggests that Hyl plays an important role in the pathogenicity of pneumonia and meningitis caused by piscine S. agalactiae.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2016年第8期997-1002,共6页
Chinese Veterinary Science
基金
江苏省高校优势学科建设项目(PAPD)
关键词
无乳链球菌
透明质酸酶
原核表达
毒力
Streptococcus aga]actiae
hyaluronidase
prokaryotic expression
virulence
