[Construction of the prokaryotic expression vector and expression of human CIDE-3 gene].
OBJECTIVE
To construct a prokaryotic expression vector for human CIDE-3 gene, and to express the gene in E.coli BL21(DE3).


METHODS
The total RNA was extracted from human hepatocellular carcinoma cell line HepG2. CIDE-3 gene fragment was amplified by RT-PCR and was cloned into the pET28a(+) vector. The recombinant vector was identified by restriction endonuclease digestion analysis and DNA sequencing, and then it was transformed into E.coli BL21(DE3) through IPTG induction to express the target protein bearing His tag.


RESULTS
A 516 bp of CIDE-3 gene fragment was obtained. After E.coli BL21(DE3) was transformed with recombinant vector pET28a(+)-CIDE-3 and through IPTG induction, the recombinant protein with relative molecular masse about 23,000 was obtained. SDS-PAGE analysis showed that the expressed product was mainly inclusion bodies, accounting for 32% of the total bacterial proteins.


CONCLUSIONS
Recombinant expression vector pET28a(+)-CIDE-3 is constructed successfully. The expressed CIDE-3 protein will be helpful to our further research.
Yao
Li
L
Department of Pathology, Fourth Military Medical University, Xi'an 710032, China.
Li
Qing
Q
Li
Peng
P
Zhang
Jing
J
Ye
Jing
J
Chen
Guang-sheng
GS
Wang
Shu-fang
SF
D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C472088:CIDEC protein, human; D011506:Proteins; D011993:Recombinant Fusion Proteins