人胰岛素基因在乳酸菌中的表达及其对非肥胖糖尿病(NOD)小鼠的作用

Expression of human insulin in lactic acid bacteria and its oral administration in non-obese diabetic mice

为实现人胰岛素基因在乳酸菌中的表达及探索其用作口服疫苗治疗Ⅰ型糖尿病(T1DM)的可行性,首先将人胰岛素基因密码子替换为乳酸菌偏爱密码子,同时在A,B链序列间加入连接短肽序列,经引物退火拼接合成人胰岛素基因。克隆至乳酸菌表达载体中后,利用电击转化法实现了带信号肽SPUsp45的人胰岛素基因在乳酸乳球菌(Lactococcus lactis)MG1363和干酪乳杆菌(Lactobacillus casei)ATCC27092中的表达。Western blot检测显示重组胰岛素位于细胞壁上,当菌体生长到OD600为0.4时达到最大表达量。用含有表达重组人胰岛素的Lactobacillus caseiATCC27092/pSW501菌体饲喂非肥胖糖尿病(NOD)小鼠,发现可刺激小鼠产生特异性抗体,同时使与免疫耐受相关的细胞因子IL-4水平明显升高(38.583±2.083pg/mL,P<0.05),提示其对NOD小鼠产生免疫耐受有一定的作用,为研制乳酸菌口服疫苗防治T1DM的可行性进行了有益的探索。

Type 1 diabetes mellitus（T1DM） is an auto-immune disease while oral administrating its autoantigens could be a treatment of T1DM. To express human insulin gene （ins） in lactic acid bacteria（LAB） for oral vaccine, ins gene was replaced by LAB bias codon and an 8-amino-acid-residue linker peptide forming a β-turn was designed to link insulin chain A and B. After synthesized by primer annealing method, the whole ins gene was fused with signal peptide sequence SPUsp45 ,subcloned into a LAB secretory expressive vector pSW501 and then introduced to Lactococcus lactis （L. lactis ） MG1363 and Lactobacillus casei（ Lb. casei） ATCC27092 respectively. Western blot showed that the expression product （SPUsp45-INS protein） targeted mainly at the cell wall while little was found in cytoplasm or supernatant. The highest expression level emerged in exponential phase when the optical density at 600nm of the culture was 0.4. The culture of the recombinant strain Lb. casei/pSW501 was administered to non-obese diabetic （NOD） mice orally. ELISA and Western blot results showed that the recombinant strain could induce SPUsp45-INS -specific antibodies and raise IL-4 level （38.583 ±2.083pg/mL, P 〈 0.05） in the mice＇ s sera. Expression of insulin in the food-grade vehicle LAB could induce oral immune tolerance in NOD mice and protect it from pancreas injury, suggesting it might be a new way to the treatment of T1DM.