FSH-occludin融合蛋白在毕赤酵母中的表达纯化及免疫原性检测

Purification of FSH-occludin protein expressed in Pichia pastoris and analysis of its immunogenicity

目的纯化FSH-occludin融合蛋白,并检测其免疫原性。方法将表达FSH-occludin蛋白的酵母工程菌GS115/pPIC9K接种到YPD培养基中多次传代后,用PCR检测其目的基因。该酵母工程菌在BMMY培养基中经甲醇诱导表达,上清液初步浓缩后上柱进行凝胶过滤层析,并将纯化蛋白免疫Balb/c小鼠,检测其血清抗体水平,以免疫组化的方法分析各组织oc-cludin的表达与定位。结果该酵母工程菌经多次传代后外源基因不丢失,Western blot检测结果表明纯化蛋白具有很好的反应原性,免疫组化证实occludin在实验组高表达。结论表达FSH-occludin蛋白的酵母工程菌具有很好的遗传稳定性,其表达产物可诱导小鼠产生特异性体液免疫应答。

Objective To purify FSH-occludin protein and analyze its immunogenicity. Methods The engineering yeast strain GS115/pPICgK expressing the FSH-occludin protein was inoculated into YPD medium and subcultured continuously, and then the gene-of-interest was detected by PCR. After the engineering yeast strain expression in BMMY medium under the induction of methanol, the supematant of the culture was condensed and loaded into the chromatographic column for gel filtration chromatography. Balb/c mice were immunized with the purified protein and the serum antibody level was measured. Simultaneously, the expression and distribution of occludin were detected by immunohistochemistry. Results The engineering yeast strain did not lose the exogenous gene after the subculture. Western blot analysis showed that the purified protein had good reactogenicity. Occludin was expressed at a very high level in the trial group. The purified protein could induce the specific antibodies in mice. Conclusion The engineering yeast strain expressing the FSH-occludin protein has good genetic stability and its expression products can elicit the specific humoral immune response in mice.