猪白细胞介素4/6与牛融合抗菌肽共表达重组毕赤酵母的构建及其对小鼠的免疫协同效应

Construction of recombinant Pichia pastoris co-expressing pig il-4/6 and fusion bovine cathelicidins and its synergistic effect on the immunity of mice

以2A自剪接技术构建猪白细胞介素4／6与牛融合抗菌肽（FBC）共表达重组毕赤酵母，为研究重组酵母对小鼠体内外的免疫协同效应，首先采用猪淋巴细胞增殖试验和抑菌试验研究重组毕赤酵母的体外生物学活性；再以发酵的重组酵母菌液进行小鼠灌胃试验，在试验的28d用致病性大肠杆菌和金黄色葡萄球菌进行腹腔攻毒，每周采血检测小鼠的生长和免疫功能变化。体外试验结果显示，试验组比对照组显著刺激猪淋巴细胞增殖；明显抑制大肠杆菌标准菌、大肠杆菌耐药菌、金黄色葡萄球菌标准菌和金黄色葡萄球菌耐药菌的生长（P〈0．05）。小鼠灌胃试验结果表明，试验组小鼠外周血的白细胞、Th和Tc细胞含量显著多于对照组；试验组tlrs（tlr-1，4，6，9），il-1，il2，il-4，il-6，il-7，il-15，il-23和cd62l基因的表达明显高于对照组；试验组血清中的IgG、IgGl和IgG2a的含量比对照组显著增加。此外，试验组小鼠的体质量比对照组明显增加。攻毒后试验组80％的小鼠存活，而对照组小鼠则有明显的炎症及临床症状，最后全部死于感染，这表明试验组的存活率显著高于对照组（P〈0．05）。白细胞介素4／6与牛融合抗菌肽共表达重组毕赤酵母能够有效提高小鼠免疫功能，为探索研制增强动物免疫机能的安全高效的免疫调节剂提供新途径。

To develop a effective and safe immunomodulator to enhance immunity,the recombinant Pichia pastoris co-expressing pig fused interleukin-4/6 （il-4/6） and bovine cathelicidins （FBC） genes was firstly constructed by the 2A self cleavage technique, and then evaluate synergistic effecton immunity of mice in vivo. The in vitro bioactivity of recombinant Pichia pastoris was detected by the proliferation of lymphocytes cells of pig and directly antimicrobial activity. Then 21-day-old female ICR mice were orally inoculated with recombinant yeast and challenged intraperitoneally with virulent E. coli and S. aureus at 28 day post-inoculation. The blood was weekly collected to e-valuate the immune and growth of mice in vivo. The results showed that proliferation of pig lymphocytes were raised significantly,and the viability of gram positive and negative bacteria Was in-hibited dramatically（P〈0. 05）. Compared with the control mice, the leukocytes, Th and Tccells obviously increased in the blood of mice treated with the recombinant yeast co-expressing fused il-4/6 and FBC,and the levels of IgG,IgG1 and IgG2a also dramatically elevated in the seraof treated mice （P〈0. 05） ,so did the expression of tlrs （tit-l,4,6,9） ,il-1,il-2,il-4,il-6,il-7,il- 15,il-23 and cd62l genes of the treated mice （P〈0.05）. Meanwhile,80% of challenged mice sur-vived,whereas the control mice manifested evident clinical symptoms and lesions and all died of in- fection. These results indicate that in addition to antimicrobial activity, recombinant yeast co-ex-pressing fused il4/6 and FBC genes effectively improved immunity of mice via oral inoculation, which can be served as a novel valid immunomodulator to enhance immunity of animals against infectious disease.