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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。
Total RNA was extracted from Chinese lymphoid tissue and the mature peptide gene of secondary lymphatic tissuechemokine (SLC) was amplified by RT-PCR. Sequence analysis showed that the cloned SLC gene was only one nucleotide difference reported in the literature, and did not affect the amino acid coding. The recombinant plasmid pET SLC was constructed by inserting the cDNA of SLC into the expression vector pET 2 8a (+) containing the T7 promoter. The recombinant plasmid was transformed into E. coli BL21 (DE3) to screen the expressed strain. The expression strain was induced by 1mmol LIPTG for 3-5h, then it was sonicated and centrifuged. The supernatant was subjected to SDS PAGE, and a clear band of about 18kD was observed. Purification of expression products with Ni2 + affinity chromatography column, the purity of more than 90%.