Cloning,Expression And And Functional Analysis Of BPI-Like In Golden Pompano(Trachinotus Ovatus)

Bactericidal/permeability increasing protein(BPI-Like)is an important member of antibacterial peptide family and plays a key role in the immune system of organism.Studies on BPI-Like in mammals mainly focus on its immune function,while in bony fish,the study on the immune-related function of BPI-Like is not clear enough.In this study,we cloned the full-length cDNA sequence of BPI-Like in Trachinolus ovatus(TroBPI-Like),and analyzed its structural characteristics.Moreover,the antimicrobial biological function of TroBPI-Like was explored in vivo and in vitro experiments.The main results of this study are shown as follows:1.The cDNA sequence of TroBPI-Like of golden pompano was cloned.Sequence analysis showed that the predicted amino acid sequence of TroBPI-Like was similar to other mammals and fish species.Moreover,it was observed that TroBPI-Like has have the highest similarity with Oplegnathus fasciatus.The results of qRT-PCR showed that TroBPI-Like was ubiquitously expressed in eleven tissues(liver,head kidney,muscle,spleen,skin,blood,stomach,intestine,gill,heart,and brain)in healthy golden pompano,and more highly expressed in immune organs(liver,spleen and head kidney).After challenged by Edvardsiella tarda,TroBPI-Like was significantly upregulated in liver,spleen,and head kidney,the liver tissue is increased by 7 times,the spleen tissue is increased by 26 times,and the head kidney tissue is increased by 14 times.2.The prokaryotic expression vectors of TroBPI-Like were constructed.Then,the fusion protein rTroBPI-Like was successfully induced and expressed.However,the fusion protein rTroBPI-Like was insoluble.After renaturation,we analyzed its antibacterial function and the result showed that rTroBPI-Like has strong antibacterial property.The polyclonal antibody of rTroBPI-Like was also prepared.The effective dilution of the polyclonal antibody was 1:25600 by ELISA,,western blot analysis showed that the polyclonal antibody of rTroBPI-Like is specificity.This result will provide the foundation for further functional analysis on protein level of TroBPI-Like.3.The eukaryotic expression vector of TroBPI-Like(pTroBPI-Like)was successfully constructed to study its antibacterial function in vivo.The results demonstrated that overexpression of TroBPI-Like significantly reduced E.tarda colonization of tissues,the highest inhibition rate in the liver was 11.76 times in the control group,in the spleen was 8.0 times,and in the head kidney was 5.9]times.In contrast,knockdown of TroBPI-Like expression resulted in the quantity of E.tarda increasing in fish tissues,and the expression level of TroBPI-Like significantly decreased in immune tissues of T.ovatus,the highest increase was 9.87 times,3.8 times,and 8.5 times of the control group,respectively..Take together,TroBPI-Like may take part in the antibacterial immune response of T.ovatus and the result will conduce to treat drug-resistant pathogens which can be used by the pharmaceutical industry,breeding industry and agriculture.