摘要
目的针对鼠IL-4Rα胞外段制备含TT830-844表位的mI L-4Rα-EX-TT自体疫苗,观察其对小鼠哮喘的治疗。方法全基因合成m IL-4Rα-EX-TT,并插入p ET-28a(+)原核表达载体,转化BL21,异丙基硫代半乳糖苷(IPTG)诱导表达,利用阴离子和分子筛两步层析分离。SDS-PAGE、Western blot鉴定重组蛋白,HPLC分析蛋白纯度。以mI L-4Rα-EX-TT蛋白免疫BALB/c小鼠,ELISA测定其效价。以鸡卵白蛋白构建BALB/c哮喘模型,mI L-4Rα-EX-TT自体疫苗治疗,PAS染色检测气道杯状细胞数量及黏液分泌。结果制备mI L-4Rα-EX-TT蛋白并纯化,其纯度为96%,Western blot表明纯化的mI L-4Rα-EX-TT蛋白能与其抗体特异性结合。mI L-4Rα-EX-TT蛋白免疫BALB/c小鼠能够产生抗体,其效价为1:10000。与对照组比较,mI L-4Rα-EX-TT蛋白干预组明显减少了哮喘小鼠气道上皮的数量和黏液分泌。结论制备的mI L-4Rα-EX-TT自体疫苗减少了哮喘小鼠气道上皮数量和黏液分泌。
Objective To prepare an autovaccine of extracellular fraction IL-4R α recombined with T cell helper epitope （TT830-844） mlL-4Rα-EX-TT and observe the therapeutic effect of the autovaccine in mice asthma model. Methods The mIL-4Rα-EX-TT gene was synthesized and inserted into pET-28a（＋） plasmid. Expression of recombinant mIL-4Rα-EX-TT was induced by IPTG when pET-28a-mIL-4Rα-EX-TT vector was transformed into BL21. Then, mIL-4Rα-EX-TT protein was separated by anion exchange chromatography and gel filtration chromatography, respectively. The recombinant mIL-4Rα-EX-TT was identified by SDS-PAGE and Westem blot. The purification ofmIL-4Rα-EX-TT was analyzed by HPLC. BALB/c mice were immunized by the recombinant mIL-4Rα-EX-TT. Then, the titer of antiserum was measured by ELISA. The mice asthma model was constructed by OVA and treated with mIL-4Rα-EX-TT autovaccine. The numbers of goblet cell in airway and mucin secretion were analyzed by PAS staining. Results Purification of recombinant mIL-4Rα-EX-TT was more than 96% by HPLC. The purified mIL-4Rα-EX-TT could bind with anti mIL-4Ra antibody using Western blot. The titer of mIL-4Rα-EX-TTautovaccine was about 1：10000 in the anti-serum from the mice immunized by recombinant mIL-4Rα-EX-TT. Compared with control group,mIL-4Rα-EX-TT autovaccine obviouslydecreased the numbers of goblet cells in airway and mucin secretion in mice asthma model Conclusion mIL-4Rα-EX-TT autovaccine prepared by bio-engineering decreased the numbers of goblet cells in airway and mucin secretion in mice asthma model
出处
《中国医药生物技术》
2015年第5期398-404,共7页
Chinese Medicinal Biotechnology
关键词
哮喘
受体
白细胞介素4
T细胞表位
自身菌苗
Asthma
Receptors, interleukin-4
Epitopes, T-lymphocyte
Autovaccines
