次级淋巴趋化因子SLC的克隆及其在原核系统中的表达  

Cloning of Secondary Lymphoid-tissue Chemokine (SLC) and Its Expression in Prokaryotic System

作　　者：王东宁[1] 李明峰[2] 曾而良 张惟杰[1] 吴祥甫[2] 

机构地区：[1]上海交通大学生命科学与技术学院,上海200030 [2]中国科学院上海生命科学研究院生物化学与细胞生物学研究所

出　　处：《生物工程学报》2001年第4期392-395,共4页Chinese Journal of Biotechnology

摘　　要：以中国人的淋巴组织为材料抽提总RNA ,用RT PCR的方法扩增了次级淋巴组织趋化因子 (Secondarylym phoid tissuechemokine,SLC)的成熟肽基因。序列分析表明 ,我们克隆的SLC基因与文献报道的仅有一个核苷酸的差异 ,且并不影响氨基酸的编码。将SLC的cDNA插入含T7启动子的表达载体pET 2 8a(+)中构建重组质粒pET SLC ,转化大肠杆菌BL2 1(DE3)筛选表达菌株。表达菌株经 1mmol LIPTG诱导表达 3～ 5h后 ,超声破菌 ,离心后将上清进行SDS PAGE ,可以看到在 18kD左右处有明显的表达条带。用Ni2 + 亲和层析柱纯化表达产物 ,纯度达到 90 %以上。The total RNA from lymphoid tissue in Chinese was extracted,and the gene encoding the mature peptide of secondary lymphoid-tissue chemokine (SLC) was cloned by RT-PCR.Nucleotide sequence analysis showed that there is only one nucleotide different from that reported,but it doesn′t alter the amino acid encoded.The SLC cDNA was inserted into an expression vector pET-28a(+) under T7 promoter and constructed recombinant plasmid pET28a-SLC.pET28a-SLC was transformed to \%E.coli\% BL21(DE3) and the expression strain was gotten.After inducing with IPTG for 3～5 hours the bacterium were sonicated.After centrifuging the supernatant was analysed by SDS-PAGE.An obvious expression band about 18kD can be seen.The expressed product was purified by Ni 2+ affinity chromatography column,and the purity is up to 90 percent.

关 键 词：次级淋巴组织 趋化因子 克隆 基因表达 趋化作用 

分 类 号：Q789[生物学—分子生物学] R392.11[医药卫生—免疫学]