中国白兔IL-6基因的分子克隆及其表达研究

运用RT-PCR技术对用ConA刺激的中国白兔外周血淋巴细胞(PMBCr)进行了扩增,将纯化后的PCR产物克隆入pMD18-T中进行核苷酸序列测定.结果中国白兔IL-6基因全长726 bp,编码242个氨基酸,其中前26个氨基酸残基构成信号肽序列.与不同物种IL-6基因相比,核苷酸和推导的氨基酸序列有一定的差异.在推导的中国白兔IL-6氨基酸序列中,在108-110位存在1个潜在的N-联糖基化位点,同时存在9个Cys残基.将pTIL-6双酶切,回收目的基因片段克隆到大肠杆菌表达载体pET28a中构建了重组质粒pETIL-6,转化大肠杆菌BL21(DE3),并用IPTG进行了诱导.结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子量为29.5kDa的重组目的蛋白.经凝胶薄层扫描,目的蛋白表达量可占菌体蛋白的16.2%.

Molecular Cloning and Expression of Chinese White Rabbit Interleukin-6 Gene

Chinese white rabbit IL-6 gene was amplified by RTPCR method from the PMBCr stimulated by ConA.Then the PCR product was purified and ligatured with pMD18-T vector.The positive recombinant was used for sequencing.The complete length of Chinese white rabbit IL-6 gene was 726 bp,which encoded 242 amino acids.The front 26 amino acids consist of signal peptide.Compared the IL-6 genes with other species,there were some differences in the nucleotide sequence and deduced amino acid sequence.There were one N-glycosylation sites and 9 Cys in the deduced amino acid sequence.Meanwhile,the IL-6 gene was subcloned into the prokaryotic expressing vector pET28a and transformed into host E.coli strain BL21(DE3) for expression under the induction of IPTG..The expression of IL-6 protein was detected by SDS-PAGE.The results revealed that it had a molecular weight of 29.5 kDa,which amounts to 16.2 % in the total protein of the induced bacteria by the assaying of gel scanning.