Screening And Function Analysis Of Cellular Proteins Interacting With Chicken Annexin A2 In Chicken Follicular Membrane Cells

Annexin A2(ANXA2)is a member of the calcium-dependent membrane phospholipid-associated protein family.It is widely present in various tissues and cells of animals and is mainly involved in cell growth,proliferation,apoptosis,vesicle transport,Functions such as ion channels and cytoskeleton formation.Studies have shown that chicken follicular membrane cells can not only secrete estrogen,but also regulate the expression of ANXA2 in follicular membrane cells,and the up-regulation of ANXA2 gene expression in follicular membrane is closely related to follicular development.In order to explore the role of ANXA2 in chicken follicular membranes,this study firstly cloned chicken ANXA2 gene and analyzed biological information.By constructing chicken ANXA2 gene recombinant prokaryotic vector,the prokaryotic expression and purification of recombinant protein His-ANXA2 were carried out,and His Pull-down and mass spectrometry techniques were used to screen the cellular proteins that interact with chicken ANXA2 from chicken follicular membrane cells.1 Cloning and bioinformatics analysis of chicken ANXA2 geneThe CDS region of the ANXA2 gene was amplified from chicken follicular membrane cells by RT-PCR,and subcloned into the prokaryotic expression vector p ET-32a(+)to construct the recombinant prokaryotic expression vector p ET-32a-ANXA2,and used bioinformatics software to analyze the chicken ANXA2 performs bioinformatics analysis.The results showed that the full length of the chicken ANXA2 gene coding region was 1020 bp,encoding a total of 339 amino acids,the theoretical p I was 6.92,and the molecular weight was about 38.6 Ku.Chicken ANXA2 protein has a rich secondary structure,dominated by ?-helix and random coils;this protein has two structural domains,which are a variable amino-terminal functional domain and a conserved carboxy-terminal core Regional functional domain,but the amino terminal position of the functional domain in chicken and quail is different from that of humans and mammals.Genetic evolution analysis found that chickens and turkeys are closely related(98.1% nucleotide homology).STRING software predicts that there are 10 kinds of proteins that interact with ANXA2 protein,which are mainly distributed in the cytoplasm and participate in fibrinolysis,immune system,signal transduction and other functions.2 Prokaryotic expression and recombinant protein purification of chicken ANXA2In this study,the successfully constructed chicken ANXA2 gene recombinant prokaryotic expression vector p ET-32a-ANXA2 was transformed into E.coli BL21(DE3),using different concentration gradients of IPTG(0.1,0.5,1.0 and 1.5mmo L/L)and different induction temperatures(25,30 and 37 ?),different induction time(2,3,4 and 5 h)to optimize the expression conditions of the recombinant protein,and analyze the expression form of the recombinant protein.The results showed that the recombinant protein His-ANXA2 induced high expression level of recombinant E.coli at a temperature of 30 ?,an IPTG concentration of 0.1 mmo L/L,and an induction time of 5 hours.The recombinant protein was expressed in both the supernatant and inclusion bodies,but mainly Inclusion body form exists.The recombinant protein in the supernatant was further purified with His Pur Ni-NTA Magnetic Beads agar balls and identified by Western blotting.The results showed that a higher purity recombinant protein was obtained from the supernatant of the induced bacterial lysate.Western blotting detected that the protein band at the protein molecular weight of about 53 Ku was consistent with the expected size of the recombinant protein band.3 Screening and functional analysis of cellular proteins interacting with ANXA2 in chicken follicular membrane cellsWith the recombinant protein His-ANXA2 as the bait protein and the tag protein His as the control,the prepared primary chicken follicular membrane cells were used to extract the whole protein,and the His-ANXA2 and His-ANXA2 and His-ANXA2 and His-ANXA2 The His-interacting proteins were identified by liquid chromatography tandem mass spectrometry LC-MS/MS to identify the cellular proteins that interact with the recombinant protein His-ANXA2 and the tag protein His.The results showed that a total of 87 proteins were identified,of which 41 proteins only interact with His-ANXA2 and 14 proteins interact with His-tag protein.The 41 proteins interacting with His-ANXA2 were analyzed by GO function annotation,KEGG signal pathway analysis and protein interaction network diagram.The results show that the identified interacting proteins are mainly distributed in the cell membrane and cytoskeleton,and are involved in biological processes such as cytoskeleton formation,response to stimulation,and translation.The molecular functions are mainly manifested in binding,catalytic activity,and ribosomal structure formation.KEGG signaling pathway analysis shows that interacting proteins are mainly involved in stress response,metabolism,translation,signal transduction,immunity and protein localization pathways.The analysis of protein interaction network diagram shows that there are complex interactions between proteins.The above experimental conclusions lay the foundation for the in-depth study of the mechanism of ANXA2 involved in chicken follicular development.