EXPRESSION OF Β-DEFENSIN OF MICROPTERUS SALMOIDES IN BACULOVIRUS SYSTEM AND ITS ANTIBACTERIAL ACTIVITY ANALYSIS

Largemouth bass (Micropterus salmoides) has been widely cultured in many countries and has become an important freshwater product in Chinese aquaculture. In recent years, germplasm degradation and disease resistance are caused by inbreeding and intensive breeding. In order to control and cure diseases caused by microorganisms, antibiotics are widely used. However, the overuse of antibiotics caused the problem of antimicrobial resistance and food safety. β-defensin is an important natural immune factor, and it has efficient and broad-spectrum antimicrobial activity which plays an extremely important role in host defense system. In this study, we focused on expressing recombinant protein of β-defensin of Micropterus salmoides (MSBdefe) by baculovirus expression vector system, and preliminarily evaluated its bacteriostatic effect. First, we characterized MSBdefe and our results showed that it shared the highest amino acid sequence identity (98.14%) with Dicentrarchus labrax. Multiple sequence alignments showed that MSBdefe has six highly conserved cysteine residues which is similar to β-defensin in other species. We synthesized MSBdefe gene with codons optimized for inset cells expression. With the insect codon optimization synthesis of MSBdefe gene, it was recombined into baculovirus shuttle vector pYBDM-IM plasmid by double restriction enzyme digestion and ligationlinking methods. The recombinant plasmid MSBdefe-pYBDM-IM was transformed into the competent cell MultiBac/rSW106/asd-/inv+. We screened bacterial colonies using blue/white screening with X-Gal and IPTG and Gm/Kan/Dap/TeT/Spe antibiotics occurred by identifying a white spot and confirmed positive colonies by PCR. The positive recombinant strain MSBdefe-pYBDM-IM-AM was directly used to infect Sf9 cells. Red fluorescence was observed under fluorescence microscope for 72h after infection which confirmed successful production of to obtain recombinant baculovirus AV-MSBdefe. The high titer of AV-MSBdefe was obtained in the supernatant by through multiple rounds of infection. SDS-PAGE and Western-Blot confirmed presence of a 12.2 kD recombinant MSBdefe protein were used to detect the protein expression of Sf9 cells infected with AV-MSBdefe, and the protein with molecular weight of 12.2 kD was obtained. The recombinant protein was purified through Ni-chelating affinity chromatography. The concentration Concentration of purified protein was detected by BCA protein concentration method. Spread plate count method was used to detect the bacteriostatic activity of recombinant protein against Aeromonas hydrophila, which is the most common pathogen in freshwater fish. The results showed that when the final concentration of recombinant protein MSBdefe was 30 μg/mL, the bacteriostatic efficiency was 83.00%, and when the concentration was 15 and 7.5 μg/mL, the bacteriostatic efficiencies were 54.33% and 33.67%, respectively.β-defensin has broad application prospects. However, it is challenging to produce it through genetic engineering approach. Although the β-defensin has been successfully expressed in the prokaryotic cell system, recombinant β-defensin produced this way is not biologically active because of lacking of post-translational modification in prokaryotic cells. Eukaryotic yeast expression system offers high protein yield, lesser expression time, post translational modifications and requires simple media. But there are still limitations in the yeast expression system such as the secretion efficiency of this system is low and the excessive glycosylation problems. Baculovirus expression system is mainly used to express recombinant proteins in insect cells. As the insect cells carry out posttranslational processing of proteins similar to that of mammalian cells, the protein can be expressed and fold correctly, and are close to natural products in terms of biological activity, antigenicity and immunity. Moreover, due to the presence of polyhedron strong promoter Polh and P10, high level of recombinant protein expression can be obtained. The recombinant protein system in this study is based on Zero-background Tn7-mediated transposition in Escherichia coli. One advantage of baculovirus system is that recombinant bacmid DNA can be transfected into insect cells or injected directly into the instar larvae for virus production and protein expression. Thus, the system provides a way of efficient generation of recombinant baculovirus and a more convenient method of expressing recombinant protein in insect cell and larvae.In conclusion, the baculovirus system vector of MSBdefe was constructed, and recombinant MSBdefe was successfully expressed and purified. The recombinant MSBdefe protein had an obvious antibacterial effect on Aeromonas hydrophila. The results of this study lay a foundation for the large-scale production of fish β-defensin using insect bioreactors. Our results also show that MSBdefe is a good candidate for natural antibacterial agents that can replace or partially replace antibiotics.