Protein Expression and Polyclonal Antibody Preparation of a Mouse Epididymis Specific Gene β-defensin15

Abstract: The epididymis is a male accessory sex organ that is responsible for sperm maturation, storage and protection.When sperm flows through epididymis,it consecutively reacts with the microenviroment,which is created by the expression and secretion of the epididymis-specific proteins,until the sperm acquire the ability of motility and fertilization.β-defensins are important innate immune molecules that provide protective barriers between host and environment.β-defensins are ubiquitously involved in innate antimicrobial immunity,anti-virus,chemotaxis,cell differentiation,tumor development and tissue remodeling.So far,53β-defensins have been identified in the mouse.It is amazing that 18 of theβ-defensins are expressed in the male reproductive systems with segmental expression patterns in epididymis.Why so manyβ-defensins are expressed in male reproductive system, especially in epididymis? The spatially varied expression pattern indicated that they play important roles in the sperm maturation,motility and fertilization.It is promising to find new male contraception targets by studying these small peptides in detail.Mouseβ-defensin 15(mBD15)is originally identified as a member of clusteredβ-defensins in mouse chromosome 8 by bioinformatics assay.Its mRNA was specifically expressed in caput epididymis,but its protein expression property and physiological function have not been reported up to now.This research successfully cloned the cDNA sequence of mBD15 and detected the mRNA expression pattern in a variety of tissues by RT-PCR.It exclusively expressed in the caput of the mouse epididymis and lowly expressed in corpus epididymis.Using Real time PCR assay,The mBD15 mRNA were detected at the third week using Real time PCR and got a high expression level at the eighth week.In order to study the function of mBD15 in vitro,the study successfully expressed and purified soluble active protein in E.coli host cells by IMPACT-TWIN system based on intein-mediated fusion expression.Circular dichroism assay proved that mBD15 protein possessed well-folded secondary structure.Their cytostatic activity against E.coli were detected by broth dilution method,although their similar effects on microbes prevalently found in the epididymis need to be confirmed.A high quality antibody is necessary for the further research on the physiological function and the expression pattern of mBD15 on the protein level.The double-copy mBD15 protein in inclusion body in E.coli was expressed.The protein was purified from inclusion body by electro-elution from SDS-PAGE gel.Then New Zealand rabbits were immunized with the two purified recombinant protein to induce immunoreaction.Consequently,the mBD15 polyclonal antisera was obtained.The sensitivity and specificity are both good.By Western blot,a 7kDa mature mBD15 protein band was exclusively detected in caput epididymis.The expression of mBD15 protein was detected in middle caput epithelial cells by immunohistochemistry assay.Also four different siRNA segments of mBD15 were predicted and shRNA-hH1 expression plasmids was successfully constructed.The screening of effective segments is going now.The research paved the way for building animal models(overexpression,knock out,in vivo RNAi)so as to do further functional research on mBD15…
Key words: β-defensins 15(mBD15); mouse; epididymis; protein expression; polyclonal antibody

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