摘要
为研究鹅β-防御素1(AvBD1)的生物学特性以及初步尝试探寻其抗肠炎沙门菌的作用机理,利用RT-PCR方法从鹅骨髓组织中扩增到鹅AvBD1基因片段,并将该基因亚克隆到原核表达载体pGEX-6p-1的EcoRⅠ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-goose AvBD1。将重组质粒转化大肠杆菌BL21,于37℃用IPTG诱导表达,SDS-PAGE电泳表明,重组鹅AvBD1蛋白在原核高效表达(相对分子质量约31ku)。该重组蛋白经纯化后测定其体外抗菌活性与理化特性。结果显示,鹅AvBD1基因cDNA片段大小为198bp,编码65个氨基酸残基。经相似性分析发现鹅AvBD1氨基酸序列与鸵鸟AvBD1氨基酸序列相似性最高,为77.1%,而且重组鹅AvBD1蛋白具有广谱的抗菌活性,对12种细菌(包括革兰阳性菌和革兰阴性菌)均具有抑菌作用。高盐离子浓度显著降低重组鹅AvBD1蛋白的抗菌活性,且该重组蛋白的溶血活性极低。试验表明,肠炎沙门菌确实会诱导鹅AvBD1基因在鹅骨髓组织中的表达,说明鹅AvBD1起到了抗肠炎沙门菌感染的作用,而这种作用有可能与TLR4介导的信号转导有关。
The objective of this study was to study the biological characteristics of goose AvBD1 and preliminary attempts to explore its resistance to Salmonella enteritidis mechanism. The mR- NA of goose AvBD1 was cloned from bone marrow of the gooses by RT-PCR. The cDNA of goose AvBD1 was sub-cloned into EcoR I and Sal I sites of pGEX-6p-1 vector to construct re- combinant plasmid pGEX-goose AvBD1. The recombinant plasmid was transformed into E. coli BL21 and the bacteria was induced with IPTG. It was demonstrated by SDS-PAGE that a 31 kDa protein which was equal to goose AvBD1 protein in molecular weight was highly expressed. The sequence analysis showed that the gene fragment of mino acids. Homology analysis showed that goose AvBD1 shared the highest percentage of ami- no acid homology （77.1%） with ostrich AvBD1. The purified recombinant goose AvBD1 exhibi- ted extensive antimicrobial activity against twelve investigated bacteria strains, including Gram- positive and Gram-negative. At high salt ions conditions, antimicrobial activity of recombinant goose AvBD1 protein against both Staphylococcus aureus and Proteus mirabilis decreased signifi- cantly and hemolysis activity of the recombinant protein was extremely low. Experimental results show that Salmonella enteritidis did induced goose AvBD1 gene expression in hone marrow, at the same time goose AvBD1 resistance to Salmonella enteritidis infection induces expression of TLR4 may be mediated signal transduction.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2012年第12期1938-1948,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(30972110)
