Research On The Purification,Heterologous Expression And Anti-inflammatory Mechanism Of Ovocalyxin-36 From Eggshell

OCX-36 is a major matrix protein in the eggshell,but at present there is no relevant research on this protein in China.This project was focused on OCX-36protein:A method for extraction and co-purification of OCX-36 and other eggshell matrix proteins was established.The gene sequence function of OCX-36 were analyzed by bioinformatics.OCX-36 gene was expressed in prokaryotic and eukaryotic expression systems,respectively.Finally,the antiinflammatory activi ty of OCX-36 was studied,and its possible mechanism was discussed.The main findings are as following:(1)The extraction,isolation and purification of eggshell matrix protein Ovocalyxin-36 was studied.First of all,we improved the shell-membrane separation method,and the eggshell matrix protein was extracted by PB solution which is neutral and mild.Then extracts were enriched and separated by serial CM-DEAE anion exchange chromatographies to prepare three kinds of egg shell matrix proteins.This co-purification method was with good yield,high purity,environmental protection,high efficiency and rapid synthesis and low cost.Ovocleidin-17and Ovocalyxin-116 were purified from CM ion exchange chromatography with purity of 80.15%and 73.22%respectively.Ovocalyxin-36 with purity of 96.82%was obtained from DEAE ion exchange column and maintained higher activity.(2)The bioinformatics tools were used to predict and analyze the gene sequence of OCX-36 and Homology modeling was constructed.The theoretical molecular weight of OCX-36 is 48.79 k Da,isoelectric point is 5.61,belongs to hydrophobic protein,and no transmembrane area,one signal peptide contained.18phosphorylation sites are Ser(15),Thr(2),Tyr(1),4 N-glycosylation sites are located at 31,96,260 and 369,respectively,and 2 O-glycosylation sites are at254,255 sites,respectively.OCX-36 is a precursor protein,the mature protein will take off 1-21 peptide then secretion to the extracellular.Functional predictive analysis showed that OCX-36 may play an important role in signal transduction,transcriptional regulation and binding and co-factor biosynthesis during the process of transport.The main participant biological process are immune system and the response to stimulus.Homology modeling of OCX-36 protein,and evaluation of the model was higher credibility.The three-dimensional structure showed that the maximum length of the three-dimensional structure was 128.3 nm,which composed of three highly distorted?-sheets and five?-helices.There was a lipid-binding pocket structure lays at the concave surface of the center,which was the core of its biological activity.(3)The expression of OCX-36 gene in prokaryotic system was studied.The OCX-36 gene was obtained from the layer chicken and full-length CDS sequence was amplified,the codon was optimized.p ET28b-OCX-36 was constructed after codon optimization and transformed into Escherichia coli C41,BL21 and T7E strains respectively,optimum temperature is 37?.The results showed that the protein could be expressed in all three kinds of host bacteria,and the expressed protein was in two forms:soluble and inclusion body.After inclusion and dissolution,the inclusion bodies were purified by affinity chromatography column,SDS-polyacrylamide gel electrophoresis showed that the molecular weight of the purified protein was close to the theoretical molecular weight of OCX-36 protein.The activity test showed that the expression protein was structurally correct and had good activity.The results provide an empirical reference for the successful expression of animal genes in prokaryotic expression system.(4)The expression of OCX-36 gene in Bac-to-Bac/Ac MNPV baculovirus system was studied.The vector p Fast Bac1-OCX-36 was constructed and sequencing correct,the expression vector was transformed into Bacmid-OCX-36 in the insect cell line,P2 recombinant baculovirus can achieved higher expression levels.The optimal expression conditions were 2×10~6 cells/m L,adherent culture,MOI value of1,cultured 48 h.Add a small amount of dextran to the commercial serum-free medium and replace the peptone with the soy protein hydrolyzate to give a better yield.The recombinant OCX-36 protein with high activity was successfully expressed in the Bac-to-Bac/Ac MNPV baculovirus system.To achieve the efficient expression of heterologous genes and formation of large-scale production of OCX-36 protein technology.(5)The in vitro anti-inflammatory activity of recombinant OCX-36 protein was studied.CKK-8 test showed that rOCX-36 protein was non-toxic to RAW 264.7 cells.Flow cytometry showed that rOCX-36 inhibited LPS-activated macrophage apoptosis significantly,with early apoptotic rate decreased by 187%,late apoptotic rate decreased by 122%.Further studies have s hown that the inhibitory effect of rOCX-36 on i NOS,TNF-?,IL-1?,IL-6 is regulated by regulating its m RNA transcription level.(6)The anti-inflammatory mechanism of recombinant OCX-36 protein was studied.The effect of rOCX-36 protein on NF-?B and MAPK signal pathways in LPS-activated macrophages RAW 264.7 cells was studied.It was found that rOCX-36 protein significantly inhibited phosphorylation of p38,JNK and ERK proteins and was dose-dependent.It was indicating that rOCX-36 achieve anti-inflammatory effect is through the inhibition of MAPK signaling pathway activation,especially ERK1/2 MAPK pathway.