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  • Title: [Expression of human ciliary neurotrophic factor gene in Escherichia coli].
    Author: Huang X, Hu X, Gao Y.
    Journal: Zhonghua Yi Xue Yi Chuan Xue Za Zhi; 1998 Apr 10; 15(2):69-71. PubMed ID: 9531641.
    Abstract:
    OBJECTIVE: To express biologically active human ciliary neurotrophic factor(hCNTF) gene in Escherichia coli. METHODS: Total RNA was extracted from human peripheral nerves and cDNA was synthesized by superscript reverse-transcriptase, a polymerase chain reaction(PCR) was conducted to obtain a full length cDNA fragment encode for hCNTF gene. After recovery from gel and purification, the PCR product was cloned into the pGEM-5Zf(+) vector and DNA sequence analysis was performed to verify hCNTF gene. The hCNTF gene was then placed under control of T7 promoter in the expression vector pET-11d and transformed into Escherichia coli strain BL21(DE3). Cultures of this transformat were induced by IPTG for the expression of recombinant protein. The bioactivity of recombinant protein was evaluated by its ability to support the survival of embryonic chick dorsal root ganglion neurons in culture. RESULTS: The human CNTF gene was cloned and biologically active hCNTF was expressed efficiently. Based on densitometry of stained gel,the recombinant hCNTF accounted for more than 25% of the total bacterial protein. CONCLUSION: The cloning and expression at high level of hCNTF gene in E.coli provides a basis for understanding the structure-activity relationship of CNTF and its potential clinical application.
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