In Vitro Expression Of Retinol Binding Protein4and Its Effects On Porcine Oocyte Maturation

Retinol Binding Proteins (RBPs), low molecular weight lipocalin, which is alsoregarded as adipokines. RBPs, transport proteins for vitamin A, are synthesized andsecreted mainly by the hepatocyte. The effects of RBP4on the growth, development,reproduction of animals have been reported. But the sources of natural RBP4islimited and the purification is very difficult. The prokaryotic expression can beeffective to improve the deficiencies.The main contents and the results of this paperare as follows:1. Prokaryotic expression vector pEASY-E1-RBP4and expression, purificationand refolding RBP4RBP4gene encoding the mature peptide region was amplified in total RNA wasextracted from porcine normal ovarian tissue, prokaryotic expression vectorpEASY-E1-RBP4was constructed, then transformed into BL21(DE3)pLysS andinducted by IPTG. The relative molecular mass of RBP4protein is21kDa, which wasespressed in the form of inclusion bodies. Unfolded the inclusion bodies with8M ureaand purificated by Ni-NTA affinity column before dialysis refolding. The porcinegranulosa cells assay showed that the protein had good biological activity. Westernblot results showed that the protein could specific bind with mouse anti-RBP4,showing good antigenicity and specificity.2. Preparation of RBP4polyclonal antibodyRefolded RBP4protein was used to immunize rabbit to obtain rabbit anti-RBP4serum. The titer was1:10000detected by indirect ELISA assay. Western-Blot resultsshowed that the target protein and porcine liver protein could be specificallyrecognized by rabbit anti-porcine polyclonal antibody.3. Effects of RBP4on porcine oocyte maturation in vitroEffects of RBP4on the first polar body release was studied. COCs were culturedin medium supplied with0,10,100ng/mL RBP4and maturation in vitro42h. Compared with the control, the first polar body release rate were significantlydecreased in treatment. Real-time PCR results showed that GDF-9and BMP-15weresignificantly down-regulated compared with the control.This study successfully constructed pEASY-E1-RBP4prokaryotic expressionvector. The strain that carried the recombinant plasmid inducted by IPTG and highlyexpressed protein.RBP4inclusion bodies protein purification, renaturation processwere established and the refolded RBP4had good biological activity. Preparation ofhigh titer had good specificity of Rabbit anti RBP4polyclonal antibody. Prokaryoticexpression RBP4stimulated porcine COCs maturation assay showed that prokaryoticexpressed RBP4significantly decreased the first polar body release rate andsignificantly down-regulated the expression of GDF-9and BMP15genes.