Analysis Of Recombinant Expression And Function Analysis Of Antheraea Pernyi Malectin

Antheraea pernyi is an important economic insect in China,but it is easily infected by pathogenic microorganisms in the four stages from larva to adult,which has caused huge economic losses to the Antheraea pernyi industry,Therefore,it is crucial to study the immune mechanism of Antheraea pernyi.The Antheraea pernyi rely on the natural immune system to resist the invasion of exogenous microorganisms,where pattern recognition receptors are essential for insect humoral immunity,they can recognize pathogenic pattern related molecules,such as lipopolysaccharides on the surface of bacteria,peptidoglycans,etc.Their interaction activates host defense mechanisms that protect the Antheraea pernyi itself from pathogenic microorganisms.Malectin is a newly discovered endoplasmic ER-resident lectin protein,participating in quality control of glycoproteins,and has immune-related effects.We cloned the Antheraea pernyi Malectin(Ap MLEC),quantified its expression,and conducted a preliminary analysis of its function.The results are as follows.The Ap MLEC gene was cloned by PCR and analyzed by bioinformatics: the gene contained 798 bases,encoding 266 amino acids.The predicted molecular weight of the fulllength Ap MLEC protein is 29.9 k Da,the isoelectric point is 5.12,the amino acids at positions1-21 are signal peptides,the four aromatic residues mediating carbohydrate interactions are Y77,Y99,Y109 and F110,the amino acids at positions 244-263 are transmembrane domains,and the amino acids at positions 26-188 are in the Malectin-domain.Submit the Ap MLEC sequence information to the NCBI-Gen Bank database under the login number OL519584.The gene was connected to the p ET-28 a prokaryotic expression vector.A recombinant protein with a molecular weight of approximately 33 k Da and His-tag was obtained,which purified by proximal expression system and affinity chromatography.Exploring the ability of the protein to bind polysaccharides,agglutinate bacteria and bacteriostatic,the results confirmed that Ap MLEC was able to bind to maltose,isomaltose,lipopolysaccharides and peptidoglycans,agglutinates bacteria and inhibits the growth inhibition activities of E.coli and S.aereus;and the minimum bacteriostatic concentration was 250 μg/m L.In order to identify the role of Ap MLEC in the replication and proliferation of Antheraea pernyi nuclear polyhedron viruses,the purified Ap MLEC and Ap NPV/egfp recombinant viruses were added to Tn-High Five cells,and the antiviral effect of this protein was identified by the expression of recombinant viruses with egfp fluorescent labels.In order to identify the effect of endogenous Ap MLEC on Antheraea pernyi nuclear polyhedron viruses,this study cloned the Ap MLEC gene and the Ap MLEC-mutant gene defective gene,connected the genes to the transfer vector p Ap Bac Dual-egfp,respectively.Both expression vectors were transfected with cells and Antheraea pernyi to explore their antiviral effects.The results showed that endogenous expression of Ap MLEC can inhibit Antheraea pernyi nuclear polyhedron viruse replication expression.Preliminary functional analysis of Ap MLEC shows that it may play an important role in the natural immunity of Antheraea pernyi,laying a foundation for future research on the immune system of Antheraea pernyi.