[Cloning, prokaryotic expression of CML66 and preparation of its polyclonal antibody].
OBJECTIVE
To clone and express CML66 cDNA and to prepare rabbit anti-CML66 antibody.


METHODS
cDNA isolated from the testis using RT-PCR was cloned into pGEMT. After sequencing, the cDNA was inserted into prokaryotic expression vector pET32b(+). The recombinant vector was transformed into BL-21(DE3) through electroporation. 6xHis-tagged CML66 expression was then induced by IPTG. The protein was purified through Ni(2+) affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified protein was injected into rabbits to prepare polyclonal antibody.


RESULTS
The cloned cDNA sequence was identical with that previously reported. The target protein was successfully purified. And rabbit's anti-serum with high titer was obtained.


CONCLUSIONS
We have cloned CML66 successfully, expressed and purified the protein in E.coli.Furthermore,rabbit polyclonal antibody has been obtained.
You
Qiang
Q
Shanghai Institute of Immunology, Shanghai Second Medical University, Shanghai 200025, China. youqiang@etang.com
Cheng
Lin-ling
LL
Wang
Ying
Y
Wang
Shu-jun
SJ
Zhang
Hui-zhen
HZ
Ge
Hai-liang
HL
D000906:Antibodies / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008970:Molecular Weight
D000906:Antibodies; D000951:Antigens, Neoplasm; D018076:DNA, Complementary; C431278:NUDCD1 protein, human