[Cloning and expression of the extracellular domain of calcium-activated chloride channel in mouse airway goblet cells].
OBJECTIVE
To clone and express the extracellular domain of murine calcium-activated chloride channel (mCLCA3) in airway goblet cell of mouse.


METHODS
According to the gene sequence of mCLCA3 the PCR primers for N-terminal, middle and C-terminal extracellular domains were designed. Using recombinant plasmid pcDNA3.1(-)/mCLCA3 as template, the DNAs coding for the three extracellular domains were amplified. And then the DNAs encoding N-terminal and C-terminal extracellular domains were inserted into expression vector pRSET-A, while the middle extracellular domain DNA was inserted into pGEX-T1. E.coli. BL21(DE3) were transformed with the three recombinant plasmids, respectively, and were induced with IPTG for expression.


RESULTS
DNA sequencing showed that the cloned DNAs encoding extracellular domains were identical with those in GenBank (GenBank accession No. NM-017474 ). The 3 domains were expressed in E.coli and most of the expressed products existed in the form of inclusion body.


CONCLUSIONS
The expression of three extracellular domains of mCLCA3 lays the foundation for further preparing anti-mCLCA3 antibody and exploring the mechanism of modulation of mCLCA3.
Song
Li-qiang
LQ
Department of Respiratory Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. songlq@fmmu.edu.cn
Li
Yan
Y
Qi
Hao-wen
HW
Wang
Ji-cun
JC
D000818:Animals; D018118:Chloride Channels / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D020397:Goblet Cells / Q000737:chemistry; D051379:Mice; D009088:Mucoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic
D018118:Chloride Channels; C116464:Clca3a1 protein, mouse; D009088:Mucoproteins; D011994:Recombinant Proteins