Expression And Characterization Of Newborn Larvae' Desoxyribonuclease â…¡ Of Trichinella Spiralis

At present, our country is one of countries that are the most severelythreaten by trichinelliasis. In our country, trichinelliasis is classified as firstof all three major classzoonotic parasitosis (Trichinelliasis, cysticercosisand hydatidosis), which must be examined in the meat products. Studyingon how to control trichinelliasis is of great significance for improvement ofour meat quality and safty.That the problem of search for the specific,sensitive antigen todiagnose and protecte is still unsolved. New born trichinellasapiralis'antigen has more protective ability than adult's and muscularlarva's which is problely relative to expression of specific-stage gene in thenew born stage. Specific stage expression gene in the new born stage isisolated and expressed in order to screen protective antigen and earlierperiod diagnostic antigen and mass production. People have focus onscreening specific stage expression gene in the new born stage andavailable antigen.New born stage is formation of cyst stage, so we can isolate andevaluate new born specific gene. We can further studying on moleculemechanism of invasion of Trichinella spiralis,and adjustment of muscle cell.The function of muscular larva's 43kDa excretion glycoprotein in host thatcan induce epithelia to nurse cell has provided theorecial foundation onTrichinella spiralis'immunity therapy..1 Cloning and analysis of Trichinella spiralis new born gene : RetrieveN5 gene which was screened by our laboratory with interproscan, the resultshowed that N5 protein possesses DNaseⅡconservative structure domainwhich belongs to DNaseⅡfamily. N5 protein which has 338 amino acid infull length. From 1 to 18 is hydrophobic signal peptide sequence.Transmembrane leader sequence locates in from 67 to 87 amino acid.Maturation protein is about 35.3kDa in molecular mass and dissolvableprotein, which isoelectric point is 9.30. By comparison, we found that twogene of Trichinella spiralis in the new born stage share great homology. Tosum up, N5 gene belongs to DNaseⅡ. 2 Studying on characterization of N5 protein: Establish expression vectorpET28a-N5 which removes the sequence which encodes signal peptide.Transform recombination expression vector into expression bacteriumBL21(DE3)and optimize the expression conditions. Schizolysis inducedbacterium with hypersound and extract inclusion body. Dissolve inclusionat TE buffer and PBS buffer respectively at 4℃. Analyze it by digestingλDNA. The result demonstrates dissolved inclusion body in PBS has somenuclease properties. It has some other properties: 1 the optimal pH is Ph5.0;2 independent of divalent cation; 3 insensitive to EDTA. Due to thischaracter, we think N5 encode DNaseⅡ.3 Studying on enzymatic active site: Comparing with some other DNase Ⅱ,N5 has conservative structure domains. We make some conclusions: 1DNaseⅡpossesses signal peptide which is beneficial to secretion; 2 Sixconservative C residue forms disulphide bands which maintain higherstructure. Among the conservative domain, each has H residue and H is theamino acid residue of endonuclease;3 DNaseⅡof other spices and ofTrichinella spiralis share one conservative domain DHSKW, but in thisdomain the H residue has been changed into S or K in Trichinella spiralis; 4Another conservative domain is JFWLVH, the H in this domain wassupposed to the conservative H amino acid residue, at least in trichinellaspiralis. Substitute H in the domain JFWLVH for S or K. Replace critical...