重组鸡β-防御素12蛋白的原核表达及其生物学特性分析

摘要

The objective of the study was to investigate bioactivity of chicken avian β-defensin (AvBD) 12 protein. AvBD12 gene was amplified from spleen tissue of chicken by RT-PCR. The cDNA of the gene consisted of 198 bp nucleotide, encoding a polypeptide of 65 amino acids. The cDNA was sub-cloned into pProex HTa vector and induced to express fusion protein. It was demonstrated by Tricine-SDS-PAGE that 14 ku protein was highly expressed. Antimicrobial activity of the recombinant fusion protein was measured in vitro. The recombinant protein exhibited high antimicrobial activity against Staphylococcus aureus and Bordetella bronchiseptica, low antimicrobial activity against Salmonell choleraesuis and Lactobacillus. The antibacterial activity of AvBD12 significantly decreased in high salt concentration. In addition, recombinant chicken AvBD12 showed little hemolytic activity against chicken erythrocytes. Results of real-time PCR showed that AvBD12 was widely expressed in all of the 15 tissues investigated.%为探讨鸡β-防御素12（AvBD12）的生物学特性，试验采用RT-PCR方法从鸡脾脏组织中扩增到鸡AvBD12基因，其cDNA大小为198 bp，编码65个氨基酸。将该基因亚克隆到大肠杆菌原核表达载体pProex HTa上，进行原核表达。Tricine-SDS-PAGE电泳表明，重组鸡AvBD12融合蛋白分子质量约为14 ku。对该重组蛋白进行纯化与体外抗菌活性的测定，结果显示，纯化后的重组鸡AvBD12融合蛋白对金黄色葡萄球菌和兔波氏杆菌有较高抗菌活性，对猪霍乱沙门氏菌和乳酸菌的抗菌活性较弱。高盐浓度对其抗菌活性有显著影响。该重组蛋白对鸡红细胞无显著溶血活性。荧光定量PCR结果表明，AvBD12基因在所测定的15个鸡组织器官中均有表达。