高迁移率族蛋白B1cDNA的克隆与表达

目的克隆人高迁移率族蛋白B1（HMGB1）cDNA，构建重组原核表达载体，对其诱导表达并鉴定，为设计HMGB1 cDNA突变体及诱导表达可竞争性抑制HMGB1炎症效应的突变体蛋白奠定基础。方法提取人新鲜扁桃体组织总RNA，经RT-PCR扩增出人HMGB1 cDNA序列，并克隆至载体pUC18进行序列测定，随后构建于原核表达载体pQE-80L中，经IPTG诱导4小时后，可表达Mr约30 000的蛋白。Western Blotting鉴定所表达的目的蛋白。结果经RT—PCR扩增得到了648bp的cDNA，经序列分析与GenBank中报道的已知序列完全一致，构建了HMGB1蛋白的重组表达质粒，诱导表达了目的蛋白，经Western Blotting证实，在Mr约为30000处有一条清楚的蛋白带。结论获得了HMGB1 cDNA的克隆与原核表达载体。

Cloning and expression of high mobility group box1 protein(HMGB1) cDNA

Objective The study aims to clone human HMGB1 cDNA, reconstruct recombinant prokaryotic expression vector and induce its expression. This study is helpful for the design of mutant HMGB1 cDNAs and the expression of corresponding mutant proteins which may competitively inhibit the inflammatory effect of HMGB1. Methods Cell RNA was extracted from fresh human tonsil tissue. HMGB1 cDNA was amplified by RT-PCR and was cloned into vector pUC18 for sequencing. HMGB1 cDNA then was reconstructed into prokaryotic expression vector pQE-80L. After 4 hours of induction by IPTG,a protein of which Mr was about 30 000 was expressed. The protein of interest was identified by western blotting. Results cDNA of 648bp was obtained after the RT-PCR amplification. It is completely in accordance with the foregone sequence reported by GenBank. Recombinant expression vector for HMGB1 protein was reconstructed. The protein of interest was obtained and identified by western blotting. Conclusion HMGB1 cDNA was cloned. Prokaryotic expression vector was reconstructed and protein of interest was obtained and identified.