小鼠釉丛蛋白在大肠杆菌中的表达,纯化及抗血清的制备

Expression of mouse tuftelin in E. coli and preparation of antiserum against purified tuftelin

目的 :以大肠杆菌为工程菌制备釉丛蛋白 ,纯化并免疫制备抗血清。方法 :用酶切、连接的方法将已克隆至 pBluescriptKS +载体中的TuftelincDNA片段 (编码 36 5个氨基酸 )用T4DNA连接酶连入载体pPRoEXTMHTc中 ,转化大肠杆菌 ,IPTG诱导。收集菌体 ,裂解后SDS -PAGE电泳 ,表达出分子量Mr 4 2×10 3 的融合蛋白。割胶后免疫新西兰大白兔 ,制备抗体。结果 :表达出分子量Mr 4 2× 10 3 的融合蛋白与预期一致。 8周以后 ,Western检测抗原抗体特异性及抗体效价达 1:10 0 0 0 0。结论 :认为成功的表达出釉丛蛋白并制备出了高效价多克隆抗体。

AIM:To express tuftelin and purify it from E.coli and prepare its antiserum. METHODS :The plasmid pPRoEX TM HTc and the sequence of Tuftelin cDNA(365 amino acid)cloning in the plasmid pBluescript KS+ were cut by the restricted endonucleas.Then the two fragment were ligased by T4 DNA ligasing and the expression plasmid of fusion protein was constructed.The product was transformed into E.coli . After the induce of IPTG, the E.coli was centrifuged and breakaged by SDS loading buffer. The purified tuftelin was mixed with incomplete adjuvant, and then used to immunize rabbits. RESULTS:The product of M r 42×10 3 fusion protein was in accordance with the anticipation. 8 weeks later. the antiserum titer of 1:100 000 could be showed by the test of Western blot. CONCLUSION :The tuftelin is expressed successfully and the antiserum is prepared.