【摘要】 用DNA重组技术 ,将已克隆的猪IL - 6cDNA片段插入pGEX 1λT质粒 ,转化大肠杆菌 ,以BamHI和PstI酶切鉴定重组子 ,以IPTG诱导融合蛋白表达 ,并作RT PCR及SDS PAGE分析表达情况 ,从聚丙烯酰胺制备凝胶中回收融合蛋白 ,用MTT法测定重组蛋白刺激小鼠脾淋巴母细胞增殖反应的活性 ,并免疫家兔制备高免血清 ,得到高效表达猪IL 6的质粒pGPIL 6 ,RT PCR确证 pGPIL 6在大肠杆菌中能正确转录 ,经SDS PAGE分析表达蛋白分子量为 4 9kD ,融合蛋白具有较高的促小鼠淋巴细胞增殖反应活性 ,以此免疫家兔 ,可制备滴度为 1∶12 80 0 0的兔高免血清更多还原

【Abstract】 The experiment was conducted to achieve expression of PIL 6 gene in E.coli ,to separate fusion protein which contains GST and PIL 6,to test it’s bioactivity and prepare rabbit anti PIL6 serum.Methods:The PIL 6 cDNA fragment was inserted into pGEX 1λT and the recombinant plasmid was identified by BamH I and Pst I digest.The expression product was assayed by RT PCR and SDS PAGE.The fusion protein was recovered from polyacrylamide gel and it’s bioactivity was tested by stimulating the proliferation of lymphoblast cells from the spleen of mice.Rabbit was immunized with the fusion protein.Result:The recombinant plasmid expressing PIL 6 in a high level was obtained,a special DNA band of 740bp was observed in the electophoresis result of RT PCR product,a new protein band was found with molecular weight of about 49kD in SDS PAGE.The PIL 6 fusion protein significantly promoted the proliferation of lymphoblast cells from mice,in comparison with the control,and the titer of rabbit anti PIL 6 serum was 1∶128 000.更多还原