Cloning, Prokaryotic Expression Of Goat PTHrP, And Its Antibody Preparation

Calcium is an important constant mineral element in animal. Parathyroid hormone related protein (PTHrP) originated from malignant tumor related to hypercalcemia. During lactating period, PTHrP regulated blood calcium and bone calcium dynamic balance under regulation, and it has direct effect on dynamic balance of Calcium.Dairy goat was used as experimental model in this study. Total RNA was isolated from goat mammary gland tissues. After reverse transcription we got the fist strand of cDNA. Then we cloned the complete CDs sequences of PTHrP gene by PCR reaction. Then PTHrP was cloned into the prokaryotic expression vector pET-32a and expressed in E.coli BL21 (DE3) host cells. After selected optimal induce condition, fusion protein was overexpressed and purified. Rabbit was intradermal injected after emulsification with adjuvant. Serum was collected. Antibody titer and specificity was detected after 35 day. Western blot was performed to assay the tissue expression profile. These will provide a foundation for further investigation of PTHrP associated molecular mechanism in Calcium regulation.(1) Cloning of PTHrP geneThe PTHrP gene was cloned from breast of dairy goat by application of RT-PCR was analyzed by sequencing and bioinformatics. The results showed that the cloned PTHrP gene fragment contained complete CDs sequence with a commom characteristic of PTH. The gene encoded a protein containing 177 amino acids. Its relative molecular weight was 20.3 kD. The isoelectric point was 10.00. The molecular formula was C895H1453N271O265S2. The PTHrP protein might be located in nucleus. It had 18 probably protein kinase phosphorylation sites. N region might be its signal peptide anchor.(2) Prokaryotic expression of goat PTHrPProkaryotic expression vector was constructed. Fusion protein was expressed in E.coli BL21 (DE3) successfully with optimal expression condition (2 mmol·L-1 IPTG for 8h at 37℃) and purified.(3) PTHrP polyclonal antibody preparationPolyclonal antibody was obtained by intradermal multi-points injection. The antibody titer was 1:512 000 which was determined by indirect ELISA and it could specifically recognize PTHrP. Expression profile analysis displayed PTHrP widely expression in goat tissues.