鸡IL-2全基因和去信号肽基因的酵母表达研究

根据已发表的鸡白介素-2（ChIL-2）cDNA编码基因序列设计三条引物,利用RT-PCR技术从经诱导的卢氏鸡胚脾淋巴细胞中扩增出429bp的ChIL-2全长基因cDNA和363bp去除信号肽的ChIL-2基因cDNA。分别克隆到酵母表达载体pPICZαA中,并分别转化巴斯德毕赤酵母X-33中,经甲醇诱导,SDS-PAGE检测,在28kD和24kD处有两条清晰的蛋白带,相同条件下pPICZαA-△SIL-2表达量较高。RNAstructure软件分析表明,pPICZαA-IL-2的序列形成了较为稳定的二级结构,不利于翻译,证明信号肽序列的存在对ChIL-2基因在酵母细胞中的表达有一定的影响。

The P.pastoris Expression of ChIL-2 whole gene and partial gene without singlnal sequence

Three primers were designed according to the ChIL-2 gene sequences in the GenBank and the whole 429bp gene and 363ph gene without signal sequence of ChIL-2 were amplified from the induced spleen lymph ceils of Lushi chicken embryo by using RT-PCR, which were cloned into the P. pastoris express vector pPICZaA respectively to construct the P. pastoris recombinate plasmid pPICZaA-IL-2, pPICZaA-ASIL-2, and then transformed to P. pastofis X-33. IL-2 was expressed under the induction of methanol. There were two clear protein band in the 28 kD and 24 kD, which were identified by SDS-PAGE. Under the same conditions pPICZaA-ASIL-2 expression was higher. RNAstructure software analysis showed that sequences of pPICZaA-IL-2 formed a complex and relatively stable secondary structure. It is not conducive to translation which testifies that the existence the signal peptide sequence has influence to ChIL-2 gene in P. pastoris express.