Expression of CIRP from the Testis of BALB/C Mouse in E.coli and Preparation of Its Polyclonal Antibody

Abstract: Cold inducible RNA-binding protein(CIRP)is the first cold shock protein found in mammalian cells and overexpressed upon a temperature downshift.This discovery have been divulsing the prologue of investigating CIRP and its biological functions.At present,some scientists had confirmed that CIRP not only plays an essential role in cold-induced growth suppression,but also might be involved in other physiological processes such as human and animal reproductive development,neural development and regulation,embryonic development,tumorigenesis and animal hibernation,and so on.Thus, establishing scientific model of laboratory animals and preparing the antibody of CIRP will be the practicing foundation of furtherly studying the biological functions of CIRP and exploiting the applying value of it.In the present study,the eDNA of CIRP was obtained from the testis of BALB/C mouse treated by cold(4℃)rather than that at room temperature(25~28℃)in the first time by RT-PCR and used to construct pGEM-T-CIRP plasmid.The positive clones were screened by PCR and enzymic excision, then sequenced.The result indicated that the 531-bp coding sequence of CIRP was successfully cloned from the testis tissue of BALB/C mouse and 100%consistency with the one of Mus musculus CIRP (GenBank accession no.NM_007705;XM_986308).The coding sequence of CIRP was obtained by digesting pGEM-T-CIRP plasmid with BamHⅠand SalⅠand inserted into the BamHⅠand SalⅠsites of the expression vector pQE-30 Xa.To obtain recombinant CIRP,the competent E.coli XL-1 blue was transformed with this expression plasmid.The recombinant CIRP was induced by IPTG,detected by SDS-PAGE and Western-blot using the monoclonal antibody against histidine;Meanwhile,optimizing IPTG concentration,analyzing the content of this recombinant protein in the total proteins of bacteria by the Bandscan software and identifying the expression pattern of this recombinant protein.The results showed that recombinant CIRP whose molecular weight is about 21.89kDa was unsolubly expressed.Its expression was higher after 6h by the induction of 0.2mM IPTG at 33℃and approximately accounted to 18%of the total protein of bacteria.Recombinant CIRP was purified by electroelution in dialysis bags and used to immunize the rabbits.The polyclonal antibody against CIRP with the higher valence(between 1:12800 and 1:25600)was examined by the indirect ELISA.The datas revealed that the recombinant protein had good antigenicity.Finally,CIRPs in the recombinant bacterium and the testis,liver of BALB/C mouse treated under 4℃for eight hours were detected by Western-blot using the polyclonal antibody prepared.The results indicated that a specific band whose size is 21.89kDa and two specific bands whose sizes are both 18kDa were clear and confirmed that the prepared polyclonal antibody could specifically bind the recombinant CIRP and the natural CIRP,be utilized to furtherly evolve the applying and functional research on CIRP as a biological reagent.Therefore,this study have established the experimental model for investigating the expression of CIRP in the living animal,provided the technological support for launching the functional research on CIRP,and settled the material basis for the application of recombinant CIRP…
Key words: CIRP; prokaryotic expression; polyclonal antibody

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