人和小鼠白细胞介素-5重组蛋白疫苗的提取

Extraction of Recombination Protein Vaccines of Human and Murine IL-5

利用PCR技术从含有人和小鼠IL-5基因的质粒pORF9-hIL-5和pORF9-m IL-5质粒中扩增出人和小鼠IL-5基因序列,然后利用基因工程技术将人和小鼠IL-5基因序列分别插入原核表达质粒pQE30,获得人和小鼠IL-5重组表达质粒pQE-hIL-5和pQE-m IL-5.用酶切和DNA序列测定方法证实pQE-hIL-5和pQE-m IL-5正确后,转化感受态大肠杆菌JM109.用IPTG诱导表达重组蛋白,表达产物经N i-NTA亲合层析进行分离纯化,最后用SDS-PAGE和免疫印迹法进行鉴定.结果表明:琼脂糖凝胶电泳发现PCR扩增的人和小鼠IL-5基因序列相对分子质量大小和预期值一致,酶切pQE-hIL-5和pQE-m IL-5片段和预期值相符,DNA序列测定插入的人和小鼠IL-5 PCR产物基因序列和阅读框架正确无误.重组蛋白质在大肠杆菌中获得稳定表达,表达的重组蛋白相对分子质量与预期值相一致,纯化的蛋白浓度达93%以上,抗组氨酸抗体可特异性识别hIL-5和m IL-5重组蛋白.

The cDNAs of extracellular domains of human and murine IL-5 were amplified from the genetic particles that contain human and murine genetic particles pORF9-hIL-5 and pORF9-mIL-5 using PCR technology, and then employed genetic engineering technology, human and murine IL-5 genetic sequences were inserted into prokaryotic expression particles respectively, the recombinant expression particles pQE-hIL-5 and pQE-mIL-5 of human and murine IL-5 were obtained. Employed endonuclease and DNA sequence measurement technology, the correctness of pQE-hIL-5 and pQE-mIL-5 were validated, and then the confirmed vectors were transformed into E. coli. JMI09 and recombinant protein expression were induced by Isopropy-B-D-thi nant protein hIL-5 and mIL-5 were obtained and ogalactoside （IPTG）. The corresponding recombipurified by Ni-NTA affinity chromatography under denature conditions. Finally, the purified proteins were further detected by SDS-PAGE and western blot analysis. Results gained from the experience show that two new recombinant expression vectors （pQE-hIL-5 and pQE-mIL-5 ） were constructed successfully. The recombinant hIL-5 and mIL- 5proteins were expressed stably in E. coli. JM109, and these two recombinant proteins were also successfully purified. Further more, hIL-5 and mIL-5 could react with specific antibody against mouse IL-5 as expected. The purity of the final products reached a level higher than 92%.