鸭β-防御素16的分离、鉴定及其抗病毒机制

Isolation,Characterization and Mechanism of Anti-viral Activity of Duck Avian Beta-defensin 16

【研究目的】旨在克隆与表达鸭β-防御素16(AvBD16)基因及测定其生物学特性,监测了鸭肝炎病毒感染后麻鸭不同组织AvBD16与TLR-7的动态变化。【方法】采用RT-PCR方法,从鸭骨髓组织中扩增到鸭AvBD16,并将该基因克隆到原核表达载体pGEX-6p-1上进行原核表达,对其重组和合成蛋白进行生物学活性测定。采用荧光定量PCR方法,分别检测了鸭肝炎病毒对鸭AvBD16和TLR-7在不同组织中表达量的影响。【结果】鸭AvBD16 cDNA大小为155bp,编码50个氨基酸残基,与鸡AvBD3氨基酸同源性最高,为62%。重组和合成鸭AvBD16蛋白对12种细菌均有不同程度的抑制作用,高盐浓度对其抗菌活性有一定影响,且溶血活性极低。重组AvBD16蛋白具有体外抗病毒活性。经鸭肝炎病毒诱导后,鸭AvBD16在肝脏及其他组织中被诱导表达或表达量显著上调,且与TLR7的表达量呈正相关。【结论】成功克隆并表达了鸭AvBD16基因,重组和合成AvBD16蛋白具有广谱的抗菌活性且不具有溶血特性。重组AvBD16蛋白具有抗鸭肝炎病毒的活性,体内抗病毒作用可能受TLR-7通路调节。

[Objective] The objective of the present study was to clone, express, and characterize the antimicrobial activity of duck avian beta-defensin （AvBD16）. AvBD16 and TLR-7 dynamic changes were evaluated in different duck organizations after duck hepatitis virus infection. [Method] The specific eDNA was obtained by PCR fxom bone marrow of ducks. The eDNA of duck AvBD16 was cloned into pGEX-6p-1 vector to construct recombinant plasmid, which were transformed into E. coli BL21 and the bacteria were induced with IPTG. Furthermore, the recombinant protein was purified. Peptides were synthesized according to the gene sequence. The antimicrobial activity of both recombinant and synthetic AvBDs was investigated in vitro. In addition, the mRNA expressions of AvBD16 and TLR-7 in tissues after duck hepatitis virus （DI-IV） infection were examined. [Result] Duck AvBD16 consisted of 155 bp encoding 50 amino acids and shared an amino acid homology （62%） with chicken AvBD3. Both recombinant and synthetic duck AvBD 16 showed similar antibacterial activities. In high salt ions conditions, antibacterial activity of duck AvBD16 protein was decreased. In addition, the hemolysis activity of the peptide was extremely low. The duck AvBD16 exhibited significant antiviral activity against DHV in vitro. The mRNA expression of AvBD16 and TLR-7 in most tissues, including immune organs and liver, was upregulated or induced in response to DHV infection at different time points. [Conclusion] The duck AvBD16 gene from duck was successfully cloned, expressed in E. coil and recombinant and synthetic AvBD16 protein showed antimicrobial activity and has no hemolytic properties. Duck AvBD 16 exhibits significant antiviral activity against DHV in vitro. This result suggests that the antiviral activity against DHV of the AvBD 16 is partially via a TLR7-dependent mechanism.