牛乳铁蛋白肽(LfcinB)在苏云金芽胞杆菌中的融合表达

牛乳铁蛋白肽（bovine lactoferrincin，LfcinB）来源于牛乳铁蛋白（bovine lactoferrin），是目前已知所有乳铁蛋白肽中活性最高的。前期研究表明，可以通过大肠杆菌表达体系和毕赤酵母表达体系表达有活性的LfcinB，但得到的产物难以纯化，产量也不理想，所以研究构建新型的LfcinB表达体系有很重要的意义。苏云金芽胞杆菌（Bacillus thuringiensis, Bt）能在芽胞形成的同时产生一种δ-内毒素组装成的杀虫晶体，在发酵完成后细胞裂解，将芽胞和晶体释放到培养基中。基于Bt的这种优势，将LfcinB与分子量为35kDa的Cry60Ba晶体蛋白作融合表达。通过PCR扩增和酶切连接，将LfcinB基因连接到 cry60Ba 的下游，构建融合基因并转入无晶体Bt菌株中表达，得到了工程菌4Q7/pPFT60Ba-LfcinB。利用GYS培养发酵48h，再经过超声破碎、包涵体纯化，SDS-PAGE分析表明，工程菌表达了一条38kDa左右的蛋白质条带。将纯化的融合蛋白盐酸水解后进行SDS-PAGE分析，得到近似于3kDa的蛋白质条带，挖取目的条带进行胶内酶解和质谱分析，质谱结果显示，挖取的目的条带样品为LfcinB蛋白的特异性肽段，说明在酸水解过后，融合蛋白Cry60Ba-LfcinB中的Cry60Ba和LfcinB分离开，得到单独的LfcinB蛋白。由此可见，利用晶体蛋白在苏云金芽胞杆菌中进行融合表达可能作为一种新型有效的LfcinB的高效表达体系，为采用基因工程的方法大量生产具有活性的牛乳铁蛋白肽LfcinB蛋白奠定基础。

The Fusion Expression of Bovine Lactoferricin (LfcinB) in Bacillus thuringiensis

LfcinB is a small piece of peptide derived from bovine lactoferrin. It was reported that antibacterial activity of LfcinB stands its leading place among all kinds of lactoferricin. Preliminary research showed that, activated LfcinB can be expressed through E.coli expression system and Pichia pastoris expression system, but with limited yield and difficulty in purification. So building a new LfcinB expression system is significative. Baciullus thuringiensis can form ICPs consisting of plenty of δ-endotoxins during spore formation. It was subjected to lysis after fermentation,releasing spore and crystal into culture medium. Based on the above advantages of Bt, a fusion protein of LfcinB and Cry60Ba about 35kDa was constructed. The fusion gene was constructed by fusing the gene encoding LfcinB to the 3' terminus of the intact cry60Ba gene. The recombinant vector containing the fusion gene was transformed into acrystalliferous Baciullus thuringiensis strain 4Q7 recombinant strain 4Q7/pPFT60Ba-LfcinB was obtained. After cultivated in GYS medium for 48 h, the recombinant strain produced parasporal crystal, which was collected and subjected to SDS-PAGE analysis. The result showed that the expected protein band about 38 kDa was obtained. The purified fusion protein was hydrolyzed with HCl and subjected to SDS-PAGE analysis. The result showed that 3kDa target protein was obtained, which was then excised and was subjected to LC-MS/MS analysis. The result showeds that Cry60Ba and LfcinB from fusion protein was separated after hydrolysis and single LfcinB was obtained.This indicated that expressing fusion protein of crystal protein and LfcinB in Baciullus thuringiensis may be a efficient expression system of LfcinB, which laid the foundation for massively producting active LfcinB through genetic engineering methods.