The Rat Epididymis, A Beta-defensins Expression Methods And Antibacterial Activity Of The Monkey Epididymis-specific Expressed Genes In The Rnase 9 Expression And Function Of Exploration

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. Bin1b, a rat epididymis specificβ-defensin, plays an important role in the initiation of sperm motility. So far, 43,52 and 39β-defensins have been identified in the rat, mouse and human respectively. It is amazing that most of theβ-defensins are clustered in the male reproductive systems with segmental expression patterns. One of the aims of my research is to find an efficient and economical approach for the production ofβ-defensins. And the recombinantβ-defensins may contribute to both the physiological study and the development of antimicrobial peptides. The ratβ-defensin Bin1b was expressed in the RTS(Roche), pichia pastoris, insect cells, E.coli and Chlorella ellipsoidea expression systems. We found that the IMPACT-TWIN system based on intein-mediated fusion expression in E.coli host cells was effective. In the same system, RBD1 (a rat non-epididymis specificβ-defensin )and HBD2 (a humanβ-defensin absent in epididymis) were also expressed to evaluate the approach. The three recombinant proteins were proved active in the antimicrobial activity assays against E.coli K12D31 and Candida albicans SC5314. To my knowledge, this is the first report about the recombinant expression and antimicrobial activity of RBD1. In addition, RBD1 and rBin1b exhibited inhibition of HIV1 infection. The advances in the recombinant expression ofβ-defensins made the researches on novel defensins possible.We also expressed rhesus monkey epididymis specific ESC461 (RNase9) in the similar expression systems. ESC461 (RNase9) was produced successfully in the inclusion bodies of E.coli cells. ESC461 (RNase9) was renatured by diluting into renaturation buffer and keep stable in the solutions with high concentration of salt than in the solutions with low concentration of salt. There was no detectible RNase activity but antibacterial activity was observed. Like other members of the RNaseA super-family, ESC461 (RNase9) can bind to heparin beads. The affinity was similar with that of bovine RNaseA. Because the PI of ESC461 (RNase9) was lower than 7.0, it was controversial according to the well known antimicrobial mechanism of RNases. Many heparin binding peptides were also found antimicrobial with different mechanism from that of the cationic proteins. We supposed that ESC461 (RNase9) had the same mechanism with the one adopted by the heparin binding peptides in killing bacteria. The nature of heparin binding activity and antimicrobial activity may help a lot in the function search of ESC461 (RNase9). In the results of ScanProsite, we also found some functional domains and motifs that would give significant hints on the functional study of the novel gene.