| Eggshells of the poultry are formed in the evolutionary process of birds, which is a kind ofself-protection structure for theirs offspring. Furthermore, it has an important significance at thechick hatching. OC-116synthesized and secreted by the uterus is the major protein of eggshellmatrix protein, is located mainly in the egg white and shell membrane and barrier layer in theeggshell. The studies showed the intermediate peptide of the OC-116protein tend to form β-sheet,which indirectly controls the direction of crystal nucleation and growth of calcium and phosphorusinteract with calcite hydroxyapatite crystal protein and so on. At present, physical structure andfunction of proteins about OC-116are not known.According to Gallus OC-116gene, the full-length encoding sequence of OC-116was clonedand constructed into corresponding eukaryotic and prokaryotic expression plasmid in theexperiment. OC-116Z, the middle section of OC-116was induced, expressed and purificated toobtain recombinant proteins and polyclonal antibody; In addition, we further research thephysicochemical property of the OC-116Z protein, such as stability, polymerization conditionsetc., laying the foundation of the follow-up study of OC-116structure and function.The main results/points are listed as follows:1. Reference to the original encoding gene sequence of OC-116of chicken (G. gallus), threegene fragments were cloned using three pairs of primers which were designed based according toits functional areas; By sequencing, digesting and connecting them, eventually we get thefull-length encoding gene of OC-116; Then we also construct the corresponding prokaryoticexpression vector. By induced expression, it was fond that OC-116N, the N-terminal of OC-116was almost unexpressed; OC-116Z, the middle peptide of OC-116was a soluble fusion protein;but OC-116C, the C terminal of OC-116was partially soluble.2. OC-116Z expression in prokaryotic cells and protein purification system established: Byoptimizing，Rosetta strain was chosen as the expression host, and by screening of expressionconditions, an optimal expression strategy by inducing at20℃for13hours with0.2mM IPTGand by pET-28b vector was achieved. By optimizing its purification process, the final selection ofWash Buffer was20mM,40mM imidazole, and the Elution Buffer was100mM. The high purityprotein was obtained through Ni-NTA column and the concentration is3mg/ml.3. Preparation and detection of the antibody: The purified OC-116Z was send to Geneticsand Developmental Biology at the Chinese Academy of Sciences, and polyclonal antibody wasprepared by immune rabbits, then detected its potency and specificity by Western-blot using1:1000, the dilution ratio of antibody. The experimental result shows a single band which indicategood antibody specificity. 4. Identification of OC-116Z stability and domain (major functional areas): OC-116Z proteinwas stable at room temperature; enzyme digestion by chymotrypsin showed that there ware tworelatively stable domain in the OC-116Z.5. Identification of OC-116Z polymer properties: based on analyses of the size exclusionchromatography(SEC) and the glutaraldehyde crosslinking assay, OC-116Z displayed as apolymer and showed containing of trimer and tetramer unit.6. Using the hanging drop method to culture crystal of OC-116Z protein in vitro, thecrystallization conditions screened ware0.02M Calcium chloride dehydrate(pH=4.6),0.1MSodium acetate trihydrate,30%v/v (+/-)-2-Methyl-2,4-pentanediol, and14%(v/v)2-propanol(pH=4.6),0.07M Sodium acetate,0.14M Calcium chloride,30%(v/v) Glycerol.In summary, the full-length sequence of OC-116was cloned and constructed eukaryoticexpression plasmid in this study; Through purifying, finally OC-116Z with high purification wasobtained and the antibody was also made; Furthermore, We studied physical properties of majorfunctional areas of OC-116and conditions for protein crystallization in vitro too; this laid thefoundation for the study of protein structure and biological research of OC-116. |
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