摘要
目的:探索在E.coli BL21中表达hTPO抗原决定簇片段(AA568~AA874)。方法:将hTPO抗原决定簇基因(1702~2622,编号相对于起始密码子)连接到表达型载体pGEX-4T-3,构建重组表达质粒。然后转化E.coli BL21,IPTG诱导表达。表达产物采用亲和层析方法纯化,SDS-PAGE法测定其分子质量,ELISA法鉴定免疫学活性,考马斯亮蓝染色法定量。结果:成功地表达了hTPO抗原决定簇基因。纯化的GST-hTPO融合蛋白分子质量为61.4 ku,hTPO片段分子质量为37.6 ku。以此作为抗原按ELISA法分别与TPOAb阳性血清或阴性血清反应,结果表明GST-hTPO及hTPO片段均与TPOAb特异性结合。纯化的GST-hTPO产量为10 mg/L培养基;hTPO片段产量为1.2 mg/L培养基。结论:利用基因工程技术可获得高纯度的GST-hTPO融合蛋白及hTPO片段,产物具有良好的免疫学活性。
Objective: To express hTPO epitopes gene efficiently in E. coli BL21. Methods: hTPO epitopes gene (1 702-2 622) was inserted into expression vector pGEX-4T-3. Expression of foreign protein was induced by IPTG after the recombined plasmid was transformed into E. coli BL21. The products were purified with Glutatione Sapharose 4B and analyzed with SDS-PAGE and ELISA. The yields were estimated with CBG method. Results: Molecular weight of GST-hTPO fusion protein and hTPO fragment was 61.4 ku and 37.6 ku respectively. Datum of ELISA demonstrated the capacity of GST-hTPO fragment binding with TPOAb. 10 mg GST-hTPO or 1.2 mg hTPO could be got from 1 L culture medium. Conclusion: Pure GST-hTPO and hTPO fragment with immunoactivity can be obtained through gene engineering technic at high level.
出处
《天津医药》
CAS
北大核心
2001年第12期732-735,共4页
Tianjin Medical Journal
基金
天津市科委科技攻关资助项目 项目编号:963108411