Prokaryotic Expression And Polyclonal Antibody Preparation Of TRPV6 Gene In Laying Hen

TRPV6 （transient receptor potential vanilloid receptor 6） is one member that belongs to the TRP （transient receptor potential） super-family of cation channel. The TRPV6 gene was originally cloned from rat duodenum, and distributed in calcium-transporting tissues such as intestine, kidney, ovary and bone of mammals, all of which are characterized by high Ca2+ transport requirements. TRPV6 is known as highly Ca2+-selective channels, and serves as an important rate-limiting step in facilitating the entry of Ca2+ into the epithelial cells. Considering that TRPV6 played a very important role in Ca2+ metabolism, there were two aims of this study:one was to find a way to obtain functional TRPV6 protein, the other was to provide polyclonal antibody for investigating the disease caused by calcium metabolic disturbance.To obtain the Gallus gallus TRPV6 active domain genes （GenBank submission number, XM₄16530）, the total RNA was isolated from ovarium by RT-PCR method. The objective fragment was cloned into pMD18-T vector and E.coli was transformed with the recombinant plasmid. The plasmid inserted with DNA was verified by restriction enzyme digestion and DNA sequencing. Results showed that the amplification was 375 bp and had highly similarity comparing to the sequence reported in the GenBank, the gene and amino acid sequence similarity were respectively 99% and 100%. The open reading frame （ORF） from 1801 to 2176, which encoding 124 amino acid and had no signal peptide. The TRPV6 gene was inserted to the His-tagged expression plasmid pET-32a（+）. Then the prokaryotic expression vector pET-32a（+）-TRPV6 was constructed and transformed into BL21 （DE3）, expressed with IPTG inducement. The SDS-PAGE result showed that the cloned recombinant protein expressed in the form of inclusion bodies in BL21 （DE3） with molecular weight of 35 kD. Western-blot analysis showed that the recombinant protein could react with anti-his antibody and had satisfied immunobioligical activity. The recombination proteins were purified by using Ni2+-NTA chelating column and detected by SDS-PAGE. The purified fusion protein was used as antigen and inoculated into New Zealand adult rabbits. The antiserum was extracted after four injections of antigen and purified by caprylic acid-ammonium sulphate precipitation. The results of indirect ELISA showed the titer of antiserum generated was 1:105. Western-blot analysis proved that rabbit polyclonal antiserum could specifically react with purified protein. Immunocytochemistry showed that osteoblast immunostaining identified as positive. Immunohistochemical showed that rabbit polyclonal antiserum could specifically react with natural TRPV6 protein in tissue. Extracted natural TRPV6 protein from the chicken long bones and identified by Western-blot, the result confirmed prepared antiserum could combine with natural protein and had satisfied immunobioligical activity. These findings strongly indicates that prepared rabbit against chicken polyclonal antiserum can be used for identifying natural TRPV6 protein in immunocytochemistry, immunohistochemistry and Western-blot.