Preliminary Identification Of Chicken OC-116Post-translational Glycosaminoglycans Modification Related Enzymes

OC-116matrix is organic components playing important roles in the formation of eggshell， Nostudies have reported about features on OC-116eggshell matrix glycosaminoglycan enzymes yet，including the specific expression mechanism in the process of shell formation. Here, screening of theeukaryotic expression vector for chicken OC-116encoding gene，transfection of the uterine endothelialcells with the Eukaryotic recombinant， and preliminary identification of the chicken OC-116posttranslational glycosaminoglycan modification related enzymes were performed.1) Screening of the Eukaryotic Expression Vector for Chicken OC-116Encoding Gene:4plasmidsof OC116-GFP、 OC116-pcDNA、 OC116(M1)-pcDNA and OC116(M1)-GFP were prepared andtransfected into CHO cells respectively. After transient expression, cDNA and total protein wasextracted. Expression level of recombinant plasmids acquired by Site-directed mutagenesis with Kozaksequence(1.77times and4.13times) in pcDNA3.1/myc-His(-) A vector(1.42times and3.43times) wastested higher than wild type using Real-time PCR. The translational expression of plasmids above wasidentified higher than wild type using Western blotting.2) Plasmids of pcDNA3.1/myc-His(-)A、 OC116(M1)-pcDNA and OC116(M1-5)-pcDNA weretransfected into the chicken Uterus Epithelial cells respectively. After preparation of cDNA and totalprotein, the existence of glycosaminoglycan and its modification was detected using Western blotting.7glycosaminoglycan modification related enzymes is reespectiveiy Chondroitin synthase、 ChPF、 DSepimerase、GalT-I、GalT-II、Xyl2-O-kinase、GlcAT-I were preliminary identified in transcription levelusing Real-time PCR. This study offered foundation for the further research OC-116post-translationalglycosaminoglycan modification and the regulating of OC-116proteoglycan molecular mechanisms oneggshell quality.