人白细胞介素10的克隆及分泌表达
人白细胞介素10,,人白细胞介素10；克隆；分泌表达
摘 要 目的：研究人白细胞介素10(hIL-10)的高效分泌表达。方法：根据Genbank报道的hIL-10基因序列，人工合成hIL-10基因，并将某些稀有密码子替换成E.Coli偏爱密码子，克隆至pUC18质粒中，测序结果与预期一致。将hIL-10基因克隆至PET22b，构建分泌表达克隆PET22b-hIL-10，经IPTG诱导在大肠杆菌BL21(DE3)中表达hIL-10天然蛋白。结果：SDS-PGAE电泳及凝胶密度扫描分析显示，重组蛋白分子量约为18 KD和21 KD(含信号肽)，不同温度条件下重组蛋白的表达状态和表达量不同。结论：18 ℃重组蛋白hIL-10的表达量为7.6%。关键词 人白细胞介素10；克隆；分泌表达
The cloning and secreting expression of human interleukin-10 in E.coli
YANG Hong,LUO Shaohong,PENG Lisheng,ZHONG Xiaofen
(Department of Biochemistry,Guangdong College of Pharmacy,Guangzhou，Guangdong 510224;School of Life Science, Zhongshan University,Guangzhou,Guangdong 510275)
Abstract Objective: To study the secreting expression of human interleukin-10(hIL-10). Methods: According to the reported human interleukin-10 sequence,hIL-10 gene was synthesized and replaced some rare codes with hobby codes. The hIL-10 gene was inserted into PUC18 plasmid,identified by DNA sequencing. The expression plasmid referred to as PET22b-hIL-10 was constructed by inserting the cloned hIL-10 genes into secreting expression vector PET22b. The hIL-10 was overexpressed induced by IPTG in E.coli BL21. Results: Detected by densitometry analysis,the MW of recombinant protein was about 18KD and 21KD(with signal peptide),and its expression level varied at different temperatures. Conclusion: The expression level of the recombinant hIL-10 is about 7.6% at 18 ℃. ......
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