CAJ | 학술논문

目的：研究纯化大肠杆菌中 Aβ42蛋白的表达情况，并探索该蛋白接种小鼠后抗体的产生情况。方法构建表达载体pGEX-4T-1-Aβ42，设置不同的诱导温度来进行Aβ42在大肠杆菌中表达。用SDS-PAGE、GST柱及Western blot来鉴定纯化目的蛋白。动物实验中，用福氏佐剂采用背部多点注射的方式免疫BALB/c小鼠3次。间接ELISA法检测免疫小鼠血清和脑组织匀浆上清液中特异性抗体的滴度。结果在25℃下，经1.0 mmol/L的IPTG诱导后该蛋白表达量最多，实现可溶性表达。小鼠实验中，第2次免疫后，Aβ42蛋白组小鼠的免疫血清有抗Aβ42抗体产生，且第三次免疫后，抗体数量增高。同时，在脑组织匀浆上清液中也可检测出低滴度的抗Aβ42抗体。结论在大肠杆菌中可获得了高纯度的可溶性Aβ42蛋白。该蛋白结合福氏佐剂免疫BALB/c小鼠后，可诱导小鼠产生特异性的抗Aβ42抗体。
목적：연구순화대장간균중 Aβ42단백적표체정황，병탐색해단백접충소서후항체적산생정황。방법구건표체재체pGEX-4T-1-Aβ42，설치불동적유도온도래진행Aβ42재대장간균중표체。용SDS-PAGE、GST주급Western blot래감정순화목적단백。동물실험중，용복씨좌제채용배부다점주사적방식면역BALB/c소서3차。간접ELISA법검측면역소서혈청화뇌조직균장상청액중특이성항체적적도。결과재25℃하，경1.0 mmol/L적IPTG유도후해단백표체량최다，실현가용성표체。소서실험중，제2차면역후，Aβ42단백조소서적면역혈청유항Aβ42항체산생，차제삼차면역후，항체수량증고。동시，재뇌조직균장상청액중야가검측출저적도적항Aβ42항체。결론재대장간균중가획득료고순도적가용성Aβ42단백。해단백결합복씨좌제면역BALB/c소서후，가유도소서산생특이성적항Aβ42항체。
Objective In order to purify the Aβ42 protein expressed in E. coli and explore the production of anti-Aβ42 antibody after immunization. Methods Expression vector of pGEX-4T-1-Aβ42 was constructed and different experimental temperature was set to Induce expression of Aβ42 protein in Escherichia coli. The expression product was determined by SDS-PAGE and Western blot analysis. Fusion protein was purified by GST affinity chromatography. BALB/c mice were immunized by subcutaneous injection with the purified fusion protein and the titers of anti-Aβ42 antibodies in sera and supernatants of brain tissue homogenates from the immunized BALB/c mice were detected by indirect ELISA. Results Aβ42 protein reaches the highest level which was induced at 25℃by 1.0 mmol/L IPTG. Western blot analysis indicated that anti-Aβ42 antibody could react specifically against the purified protein. Mice experiment showed that after the third time, the titers of anti-Aβ42 antibodies in the immunized mice by purified protein was significantly higher than the control group. At the same time, in the brain homogenate supernatant can also detect low titers of anti Aβ42 antibody. Conclusion Anti-Aβ42 antibody is induced in BALB/c mice by the Aβ42 protein expressed in E.coli.