牛IFN-α基因在食品级乳酸乳球菌中的表达及鉴定

Expression and identification of bovine interferon-α gene in Lactococcus lactis

本研究旨在构建能够表达牛α干扰素(IFN-α)基因的重组乳酸乳球菌。根据乳酸乳球菌密码子的偏好性对牛IFN-α的成熟肽编码基因序列进行了优化合成,得到IFN-α基因3′端添加6个组氨酸密码子的重组质粒pUC57-IFN-α,以此重组质粒为模板,通过PCR扩增得到上下游分别带有NcoⅠ和SpeⅠ酶切位点的目的片段,双酶切后与表达载体pNZ8149连接,连接产物转化乳酸乳球菌NZ3900感受态细胞,得到的重组质粒通过PCR扩增、酶切鉴定分析以及测序分析表明,重组表达载体构建成功,将重组菌命名为NZ3900/pNZ8149-IFN-α。对重组菌使用nisin诱导表达,表达产物经SDS-PAGE分析,可见在约20 ku处有明显差异性条带,Western-blotting检测其为特异性条带,间接免疫荧光检测重组菌有特异性荧光。本试验成功构建了重组表达载体pNZ8149-IFN-α,并使其在乳酸乳球菌中获得表达,这为牛IFN-α作为口服抗病毒药物和免疫增强剂的开发以及应用奠定了基础。

In order to express the bovine interferon-α（IFN-α） gene in Lactococcus lactis, the se- quence of mature polypeptide of bovine IFN-α was optimized according to the codon bias of Lactococcus Lactis, with six codons of histidine added to the 3＇- end, and synthesized before being cloned into pUC57 vector. Special primers were designed for PCR amplification and enzymatically cloned into pNZSI49 vec- tor. The recombinant plasmids were transformed into Lactococcus lactis strain NZ3900 and identified by PCR amplification, double enzyme digestion and nucleotide sequencing. SDS-PAGE and Western blotting showed that the fusion protein was about 20 ku, and green fluorescence was observed in the indirect im- munofluorescence assay（IFA）, indicating that the Lactococcus lactis expression vector was success- fully constructed and induced to express the purpose protein. This study provides the foundation for the development of bovine IFN-α in prevention and treatment of the mueosal-related viral diseases as oral preparation.