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Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells
Published on: May 9, 2020
[Cloning and expression of truncated Midkine cytokine from gastric carcinogenesis tissue in E. coli]
Qing-ling Wang1, Ya-hong Huang, Hao Xie
1Medical School of Nanjing University, Nanjing 210093, China.
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 E. coli by clone and recombination.
