[Expression of human ciliary neurotrophic factor gene in Escherichia coli].
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.


CONCLUSIONS
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.
Huang
X
X
Institute of Medical Sciences, Nanjing Railway Medical College, Nanjing 210009 P. R. China.
Hu
X
X
Gao
Y
Y
D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D009414:Nerve Growth Factors / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*
D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins