Study On Cloning,Expression,Purification Of Mouse Periostin;Preparation Anti-Periostin Antibodies,Specificity Study And The Effects Of Periostin On PDL Cells

Periostin, also called osteoblast-specific factor 2 (OSF-2), is a disulfide linked 90 kDa bone adhesion protein secreted by osteoblasts and osteoblast-like cell lines and the protein is an attachment agent for osteoblasts. Recent research showed that Periostin is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Now the Periostin cDNA were cloned in mouse and human, and computer analysis of the deduced amino acid sequence revealed a complex protein structure with four repeats of a characteristic domain which was similar to faaciclin I, a homophilic cell-cell adhesion molecule expressed in the central nervous system of insects. But the biological functions and the underlying molecular mechanism of Periostin effects are still poorly characterized.In our study, we used the molecular biological techniques to clone and sequence the mouse Periostin fragment, and express in E. coli and purify. Then an anti-mouse Periostin antibody was prepared to proceed the Western blot and immunohistochemical study.And next a variety of biological effect of Periostin on periodontal ligament cells(PDL cells) were observed. This paper includes following 4 parts: 1.Cloning and sequencing of the terminal C encoding gene of mouse Periostin In this study, total RNA was extracted from the periodontiurn of adult mouse by acid guanidin iu in tbiocyanata-phenol-chloroform method, the desired eDNA products were obtained from the total RNA by RT-PCR with the primers including Oligo(dt) and two gene specific primers respectively. These segments (about 940bp) were inserted into pRSET-B vector and the inserting plasmids were transformed into E.coli DH5 a .The positive clone were analyzed by restriction endonuclease mapping and DNA sequencing. The results showed that the restriction endonuclease map and sequence of mouse Periostin fragment were consistent with those of the published. 2.Expression of recombinant terminal C peptide of mouse Periostin in E.coli and identification of its biological activity The cDNA fragment encoding the terminal C of periostin was inserted into expression vector pRSET-B in which foreign gene is controlled by T7 promoters. The recombinant plasmid pRS-B-Periostin was transformed into E.coli BL21(DE3) and induced at IPTG to the express the encoded protein, the expressed product was purified and then refolded. After induction , a new anticipated 36000Mr protein band appeared on SDS-PAGE gel. The espressed product existed in a form of inclusion body and after being purified and refolded, the recombinant mouse Periostin with its purity more than 95% was obtained. In order to investigate the biological activity of recombinant mouse Periostin, Solid- phase binding assay and MTT method were used to observe the ability of adhesion and spreading of osteoblast on Periostin matrix. The results showed that adhesion and spreading of osteoblast were very well on Periostin and the effects of Periostin are similar to EN. 3.The preparation and specificity study on the anti-mouse Periostin antibodies We prepared a rabbit anti-mouse Periostin IgG antibody and study it抯 specificity. Complete Freund抯 adjuvant was used when immunized the rabbit with recombinant Periostin. The specific IgG antibody was further purified with sulfuric acetate. Total protein isolated from mouse liver, brain kidney and calv...