Zebrafish Poly-immunoglobulin Receptor (zplgr) Cloning,

Polymeric Immunoglobulin Receptor (pIgR), whose gene has already been cloned in several mammals, is a transmembrane protein which mediates the transmembrane transport of polymeric Immunoglobulin (including IgA and IgM) across mucosal epithelial cells. In this paper, zpIgR gene, which encodes the pIgR protein of zebrafish, was cloned by the method of molecular cloning and recombinant technics. The recombinant expression vector pET-28a(+)/zpIgR was constructed and E.coli BL21(DE3) was transformed to express the zpIgR protein. The protein was purified and used as antigen to immune animals to gain the antiserum, which was used afterwards to locate zpIgR in situ via immunohistochemical manipulations.Chosen as the subject of this research was the viscus of zebrafish, from which total RNA was isolated using Trizol method. The first strand of cDNA was obtained by reverse transcription catalyzed by M-MLV reverse transcriptase and served as the template to amplify part of the gene zpIgR. The product of amplification was treated by double endonuclease digestion and linked to pET-28a(+) expression vector. This recombinant vector was then used to transform E.coli DH5αcompetent cell and was subjected to Restriction Endonuclease Assay and DNA sequencing. The double-endonuclease-treated recombinant vector broke down into two fragments, the size of which corresponded to pET-28a(+) expression vector and PCR amplification product respectively. Moreover, DNA sequencing demonstrated a sequence completely identical to an electronically cloned gene in GenBank.The recombinant expression vector pET-28a(+)/zpIgR was then transformed into E.coli BL21(DE3) and the zpIgR protein was expressed with the induction of IPTG.. SDS-PAGE results showed that a protein with a molocular weight of 30.75 kDa was expressed within the host strain. Further studies showed the expression quantity of zpIgR protein reached the highest level after 4-hour-IPTG induction at 37℃. Inclusion bodies would form under the condition and most of the zpIgR protein was contained within them.The expressed zpIgR protein was purified with the treatment of 2M urea. Then the purified zpIgR protein was dialyzed with PBS and quantified with comasse blue. The concentration of zpIgR was adjusted for later use as an antigen. Antiserum was gained from animal immunization, then double agar diffusion experiments and Western blotting were conducted afterwards. And the titer of the antiserum was measured as 1:32 according to the results of double agar diffusion experiments.In brief, the fragment coding region of zpIgR, cloned by RT-PCR was successfully expressed in E.coli BL21(DE3) contained the recombined plasmid pET-28a(+)/zpIgR. The molecular weight of the expressed protein was identical to the anticipant one. Then the expressed zpIgR protein was purified after being washed with urea and prepared as antigen for animal immunization, and the titer of the antiserum gained was 1:32. Immunohistochemistry results indicated that zpIgR existed in intestine epithelial and muscusal cells of zebrafish.