Aβ42基因在大肠杆菌中的表达及诱导BALB/c小鼠的抗体产生  

The expression of Aβ42 gene in Escherichia coli and antibody production in BALB/c mice induced by the protein

作　　者：牟玲燕 沈蒋君 孙爱群[1] 舒建洪[1] 李司[1] 

机构地区：[1]浙江理工大学生命科学学院浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《中华临床医师杂志（电子版）》2014年第10期74-77,共4页Chinese Journal of Clinicians(Electronic Edition)

基　　金：大学生科技创新项目(2013R406018);浙江省自然科学基金面上项目(Y3110354)

摘　　要：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。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.

关 键 词：大肠杆菌 AΒ42 可溶性蛋白 

分 类 号：R378[医药卫生—病原生物学]