[Prokaryotic expression and polyclonal antibody preparation of human novel gene CTRP4].
OBJECTIVE
To   further investigate the  biological function of human novel gene CTRP4 by constructing   the prokaryotic expression vector of human CTRP4,   inducing the expression of and purifying  hCTRP4-his protein in E.coli,  and preparing  polyclonal antibody against human CTRP4.


METHODS
Human CTRP4 gene was amplified by PCR,  digested with enzymes,  and subcloned into a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET-32a-hCTRP4. The pET-32a-hCTRP4 was transformed into E.coli BL21(DE3). The hCTRP4-his fusion protein was induced by IPTG,  purified by Ni-NTA purification system,  and analyzed by SDS-PAGE. The recombinant vector pcDNA3.1-myc/his(-)B-hCTRP4 expressing full-length human CTRP4 and purified prokaryotic protein hCTRP4 were used to immunize BALB/c mice to produce polyclonal antibody. The anti-serum was purified and the characteristics of the antibody were identified by ELISA,  Western blotting,  immunofluorescence cytochemistry  and immunohistochemistry.


RESULTS
The prokaryotic expression vector of pET-32a-hCTRP4 was   constructed successfully. hCTRP4-his fusion protein was expressed in E.coli BL21(DE3) after IPTG induction. The titer of the anti-serum reached 1:20 000,  and its specificity was proved by Western blotting. The results of immunofluorescence cytochemistry   and immunohistochemistry indicated that CTRP4   was       mainly localized in the cytoplasm of hepatic cells.


CONCLUSIONS
hCTRP4-his fusion protein can be successfully expressed in E.coli. A specific polyclonal antibody against human CTRP4 has been  successfully prepared.
Center for Human Disease Genomics, Peking University, Beijing, China.
Wei-feng
WF
Tan
Lan-lan
LL
Wang
Qi
Q
Li
Yang
Y
Luo
Da-xiang
DX
Na
Zhuang
Z
Ma
Lu
L
Wang
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D002460:Cell Line; D016207:Cytokines / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000906:Antibodies; C560750:C1QTNF4 protein, human; D016207:Cytokines; D011993:Recombinant Fusion Proteins