中国医药生物技术
中國醫藥生物技術
중국의약생물기술
Chinese Medicinal Biotechnology
2015年
5期
398-404
,共7页
吴守振%李海龙%段明玥%王宁
吳守振%李海龍%段明玥%王寧
오수진%리해룡%단명모%왕저
哮喘%受体,白细胞介素 4%T细胞表位%自身菌苗
哮喘%受體,白細胞介素 4%T細胞錶位%自身菌苗
효천%수체,백세포개소 4%T세포표위%자신균묘
Asthma%Receptors,interleukin-4%Epitopes,T-lymphocyte%Autovaccines
目的针对鼠 IL-4Rα胞外段制备含 TT830-844表位的 mIL-4Rα-EX-TT自体疫苗，观察其对小鼠哮喘的治疗。<br> 方法全基因合成 mIL-4Rα-EX-TT，并插入 pET-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 mIL-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，mIL-4Rα-EX-TT自体疫苗治疗， PA S 染色检测气道杯状细胞数量及黏液分泌。<br> 结果制备 mIL-4Rα-EX-TT蛋白并纯化，其纯度为96%， Western blot表明纯化的 mIL-4Rα-EX-TT蛋白能与其抗体特异性结合。mIL-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，mIL-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。<br> 结论制备的 mIL-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。
目的針對鼠 IL-4Rα胞外段製備含 TT830-844錶位的 mIL-4Rα-EX-TT自體疫苗，觀察其對小鼠哮喘的治療。<br> 方法全基因閤成 mIL-4Rα-EX-TT，併插入 pET-28a(+)原覈錶達載體，轉化 BL21，異丙基硫代半乳糖苷（IPTG）誘導錶達，利用陰離子和分子篩兩步層析分離。SDS-PAGE、Western blot鑒定重組蛋白，HPLC分析蛋白純度。以 mIL-4Rα-EX-TT蛋白免疫 BALB/c小鼠，ELISA測定其效價。以鷄卵白蛋白構建 BALB/c哮喘模型，mIL-4Rα-EX-TT自體疫苗治療， PA S 染色檢測氣道杯狀細胞數量及黏液分泌。<br> 結果製備 mIL-4Rα-EX-TT蛋白併純化，其純度為96%， Western blot錶明純化的 mIL-4Rα-EX-TT蛋白能與其抗體特異性結閤。mIL-4Rα-EX-TT蛋白免疫 BALB/c小鼠能夠產生抗體，其效價為1：10000。與對照組比較，mIL-4Rα-EX-TT蛋白榦預組明顯減少瞭哮喘小鼠氣道上皮的數量和黏液分泌。<br> 結論製備的 mIL-4Rα-EX-TT自體疫苗減少瞭哮喘小鼠氣道上皮數量和黏液分泌。
목적침대서 IL-4Rα포외단제비함 TT830-844표위적 mIL-4Rα-EX-TT자체역묘，관찰기대소서효천적치료。<br> 방법전기인합성 mIL-4Rα-EX-TT，병삽입 pET-28a(+)원핵표체재체，전화 BL21，이병기류대반유당감（IPTG）유도표체，이용음리자화분자사량보층석분리。SDS-PAGE、Western blot감정중조단백，HPLC분석단백순도。이 mIL-4Rα-EX-TT단백면역 BALB/c소서，ELISA측정기효개。이계란백단백구건 BALB/c효천모형，mIL-4Rα-EX-TT자체역묘치료， PA S 염색검측기도배상세포수량급점액분비。<br> 결과제비 mIL-4Rα-EX-TT단백병순화，기순도위96%， Western blot표명순화적 mIL-4Rα-EX-TT단백능여기항체특이성결합。mIL-4Rα-EX-TT단백면역 BALB/c소서능구산생항체，기효개위1：10000。여대조조비교，mIL-4Rα-EX-TT단백간예조명현감소료효천소서기도상피적수량화점액분비。<br> 결론제비적 mIL-4Rα-EX-TT자체역묘감소료효천소서기도상피수량화점액분비。
Objective To prepare an autovaccine of extracellular fraction IL-4Rα recombined with T cell helper epitope (TT830-844) mIL-4Rα-EX-TT and observe the therapeutic effect of the autovaccine in mice asthma model. <br> 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 Western blot. The purification of mIL-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. <br> 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-4Rα antibody using Western blot. The titer of mIL-4Rα-EX-TT autovaccine 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 obviously decreased the numbers of goblet cells in airway and mucin secretion in mice asthma model. <br> 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.
