胃肿瘤组织中缺失型Midkine基因的克隆和表达

目的对人胃肿瘤组织中缺失型Midkine(tMK)基因进行克隆并在大肠杆菌中表达。方法根据Genbank报道的序列,设计一对引物,通过RTPCR从胃肿瘤患者肿瘤组织中获得了tMK成熟肽DNA编码序列,与pMD18Tvector连接测序后,将该片段克隆入大肠杆菌高效表达载体pBV222中,转化大肠杆菌DH5α,在42℃进行诱导表达。结果在胃肿瘤组织中克隆到tMK表达基因,并获得高效表达tMK重组蛋白的工程菌株DH5αpBV222tMK。SDSPAGE结果表明,pBV222tMK表达的重组蛋白与预计分子量一致。结论在中国胃肿瘤患者的肿瘤组织中也有tMK的表达,经克隆重组实现了在大肠杆菌中的高效表达。

Cloning and expression of truncated Midkine cytokine from gastric carcinogenesis tissue in E. coli

OBJECTIVE: To clone the truncated Midkine from gastric carcinogenesis tissue and express in Escherichia coli. METHODS: A pair of PCR specific primers were designed according to the reported human tMK cDNA sequence in Genbank. The target DNA fragment was obtained by RT-PCR from gastric carcinoma patient's carcinogenesis tissue and cloned into pMD 18 T-vector. After sequencing, the tMK nucleotide fragment was inserted into an E. coli expression vector pBV222. The recombinant plasmid was transferred into E. coli DH5alpha and an E. coli DH5alpha expressed recombinant tMK protein, DH5alpha/pBV222-tMK, was obtained. DH5alpha/pBV222-tMK was cultured and induced with 42 degrees C. RESULTS: Truncated Midkine was cloned from gastric carcinogenesis tissue and the efficiently expressed recombinant tMK protein was obtained. SDS-PAGE indicated the molecular weight of recombinant tMK protein accorded with anticipation. CONCLUSION: The tMK was expressed in gastric carcinoma of Chinese patients' carcinogenesis tissue. The efficient expression of tMK protein was actualized in Ecoli by clone and recombination.