Study On Antimicrobial Activity Of Protamine And Cloning And Expression Of Its Coding Gene

Protamine is a kind of calionic antimicrobial pcptidc(CAP) found in the nuclei of sperm of different animals species, where it replaces histone-protamine during maturation of the spermatid. Studies indicate that, being a novel natural food preservative, protamine is effective, safe, with broad anti-microbial spectrum and worthy of further exploitation. Furthermore, protamine is beneficial to health because of its nutrition and functionality, it also has been applied broadly in medical treatment.To provide some reference to the application of protamine, the experiment studied some physical and chemical factors which affect protamine's antimicrobial efficacy, in addition, we have cloned coding sequence of protamine by PCR, and expressed the fusion protein of protamine in E.coli. The main results were as following:(1) Metal ions like Ca2+ Mg2+, K+% Na+ have negative effects on antimicrobial efficacy of protamine. The higher charge and concentration of metal ions, the bigger their effects on antimicrobial efficacy of protamine, and the degree hirarchy of effect was: Ca2+>Mg2+>K+>Na+.(2) Antimicrobial efficacy of protamine is evidence under the condition of neutral and alkalescence, but it is low in the acidic condition.(3) The temperature treatment show little effect on antimicrobial efficacy of protamine, which makes protamine ideal for application in food together with heat treatment to prolong shelf life of food.(4) antimicrobial action of protamine is rapid and immediate effect, it can inhibit or kill majority of microorganism during a short time.(!>) Organic substances such as protein, fat and sugar in food have a small effect on antimicrobial efficacy of protamine. among three organic substances, the effect of fat is more significant than other.(6) Being a small peptide molecule, protamine's basic structure can be destroyed and it's antimicrobial efficacy largely affected by some protein enzymes like chymotrypsin, trypsin, papain and pepsin.(7) A fragment of protamine DNA was cloned by PCR, and its sequence determined after cloning into a pUCT vector . Compared with known protamine gene sequence, there are three single nucleic acid variations in our senquence but their homology is up to 91.1%.(8) we have also constructed the fusion expression system for prolamine using pET30a as expression vector and E.coli as expression strain. When induced by IPTG, the system expressed a new approximate 25 KD protain as checked by SDS-PAGE electrophoresis. we tentatively think that it is the target fusion protein of protamine combined with E.coli protein .