摘要
将 Eco R1和 Sal I酶切的人脑髓鞘碱性蛋白 ( MBP)基因 c DNA克隆片段与表达载体 p GEX- 5 T重组后转化大肠杆菌 ,经筛选 ,增殖和 IPTG诱导 ,阳性克隆 SDS- PAGE结果证实表达一条特异 42 KDa区带 ,Western印记杂交证实该区带具 MBP抗原特异性 ,免疫斑点杂交和 EL ISA检测表达产量占菌体可溶性蛋白含量 6% ,达414.6m g/ L 菌液。将含可溶性 MBP蛋白的菌液行 SDS- PAGE分离纯化得重组 MBP抗原 ,对新西兰兔进行背部皮下多点注射 ,5次免疫后以琼脂板免疫双扩法检测抗体效价达 1∶ 16,并通过免疫斑点杂交和 Western印迹杂交证实该抗体具抗
We constructed the expression vector by inserting 21.5 KDa MBP human brain full-length cDNA coding sequence digested with restriction enzyme EcoR I and Sal I into downstream of pGEX-5T expression vector.The recombinant vector p5TMP was transformed into E.coli and the positive clonies were selected and incubated in LB medium induced by IPTG(isopropyl- -D-thiogalactoside). A new polypeptide band with apparent molecular weight 42KDa was detected in transformed cell lysates by SDS-PAGE.Western blotting analysis confirmed that this fusion protein reacted specifically with antibodies to MBP,the expression level of MBP was about 414.6 mg/L medium estimated by immuno-dot blot,ELISA and absorbance scanning.Newzealand rabbits were immunized by subcutaneous injection of the purified recombinant MBP. The titer was obtained at 1∶16 after 5 injections. The specificity of the antibody to MBP was confirmed by immuno-blot and Western blotting.
出处
《生物医学工程学杂志》
EI
CAS
CSCD
2003年第1期64-67,共4页
Journal of Biomedical Engineering
