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目的构建携带人白细胞介素12(h IL-12)基因的长双歧杆菌(Bifidobacterium longum,B.longum),观察其表达产物对鸡胚尿囊膜(Chick embryo chorioallantoic membrane,CAM)血管生成的影响。方法以前期构建的表达载体p BBADs为基础,将h IL-12基因克隆入载体中,构建质粒p BBADs-h IL-12,电转化长双歧杆菌NCC2705,L-阿拉伯糖诱导表达后,ELISA法鉴定基因表达。原位摄影后分析照片,田氏法计数血管条数,运用IPP软件计算所选区域血管面积。各组数值经检验服从正态分布,采用单因素方差分析(Bonferroni法)检验。结果成功构建携带h IL-12基因的长双歧杆菌(BL-h IL-12),经过诱导后检测到目的蛋白的表达,诱导24h h IL-12表达水平最高(P<0.05),为最佳诱导时间。BL-h IL-12诱导组与其它组比较,血管数量及面积均减少,差异有统计学意义(P<0.05)。BL-h IL-12未诱导组、普通双歧杆菌组、生理盐水组两两比较,差异均无统计学意义(P>0.05)。结论携带h IL-12的转基因双歧杆菌其诱导表达产物对鸡胚尿囊膜新生血管数目有一定抑制作用。
Objective To construct Bifidobacterium longum (B.longum) harboring human interleukin 12 (hIL-12) gene and investigate its effect on the angiogenesis of chick embryo chorioallantoic membrane (CAM) influences. Methods The recombinant plasmid p BBADs-h IL-12 was constructed by cloning the hIL-12 gene into the vector on the basis of the p BBADs constructed previously. The expression of IL-12 was induced by electroporation of Bifidobacterium longum NCC2705 and L-arabinose, Law identification of gene expression. Photographs were taken after in situ photography and Tia’s method was used to count the number of blood vessels. IPP software was used to calculate the area of blood vessels in the selected area. The values of each group obey the normal distribution, using one-way analysis of variance (Bonferroni method) test. Results BL-h IL-12 carrying h IL-12 gene was successfully constructed and the expression of IL-12 was induced after induction for 24 h, which was the highest (P <0.05) Good induction time. Compared with other groups, the number and area of blood vessels in BL-h IL-12 induced group decreased, with statistical significance (P <0.05). There was no significant difference in any pairwise comparison between BL-h IL-12 non-induced group, Bifidobacterium common group and saline group (P> 0.05). Conclusion The Bifidobacterium that carries hIL-12 can inhibit the number of neovascularization of chick embryo chick embryo.