Recombinant Expression And Characterization Of CHP3 And CHP5, Two Kinds Of Antimicrobial Peptides From Chinese Shrimp (Fenneropenaeus Chinensis)

Antimicrobial peptides (AMPs) are widespread in plants, animals and microorganisms. They have low molecular weight, good water-solubility, resistance to heat and wide antimicrobial spectrum. They are synthesized after the recognition of an invading organism as foreign and the transduction of the signals. The expression of AMPs is an important reaction of innate immunity. To do research on AMPs helps people to perceive the function of innate immunity more deeply and more clearly. Due to their traits, AMPs are considered to have application in therapies, preventing and curing of diseases, developing new medicines, food biopreservatives and farming.Penaeidins, belonged to a unique antimicrobial peptide family, have been isolated from several species shrimp and have been divided into five groups based on their structure. Penaeidins are found to present Gram-negative antibacterial, Gram-positive antibacterial and antifungal activities. The research about Penaeidins is mainly concentrated on the functional expression of Penaeidins using bioengineering methods, the study about their structure, the mechanism and the distribution of penaeidins in the shrimp under all kinds of circumstance. Our laboratory firstly cloned two kinds of Penaeidins (Ch-penaeidin-3/5) from Chinese shrimp, Fenneropenaeus chinensis. This dissertation mainly contains two parts, one is the expression of CHP3/5 in Escherichia coli and Yeast Pichia pastoris, another is the acquisition of polyclonal antiserium to CHP3 and CHP5.1. mchp3, mchp5 were cloned into the plasmid pGEX-4T-1 and the fusion protein GST-CHP3, GST-CHP5 were expressed in E.coliBL21. The antimicrobial activities of CHP3, CHP5 were tested after GST-CHP3, GST-CHP5 were purified by affinity chromatograph and cleaved by thrombin. The results showed that the recombinant CHP3 displayed a low antimicrobial activity but the recombinant CHP5 did not.2. Using the fusion protein GST-CHP3, GST-CHP5 as antigens to stimulate the rabbit, the polyclonal antiserium to CHP3 and CHP5 were obtained. These polyclonal antiserium will be used to study the localization and distribution of CHP3.3. The plasmid pAO815 was used to express CHP3. This plasmid was designed to express foreign protein in vivo but it was difficult to decide whether CHP3 was expressed or not due to some reasons. Another two recombinant plasmids CHP5-pPIC9K and CHP5-pGAPa-A were constructed and they were introduced into Pichia pastoris KM71 too. Chp5 was secreted by the transformed cells then purified by cation ion-exchange chromatograph. The minimal growth inhibition concentration (MIC) of CHP5 was studied, which revealed that CHP5 exhibited activities against both Gram-negative and Gram-positive bacteria. The expression of CHP5 in Yeast was the fundament to illustrate its structure and was the preparation to ferment this antimicrobial candidate on a large scale.