Prokaryotic Expression Of Main Epitope Domain Of Pseudorabies Virus Glycoprotein E And Development An Indirect ELISA For The Detection Of Viral Glycoprotein E Antibodies

In this research, gE gene of Pseudorabies Virus Strain SL was amplified and bioinformatics analysis, then selective subcloned its main antigen into the prokaryotic expression vectors, prokaryotic expression and the initial establishment of the ELISA deteetion methods.1. Cloning and bioinformatics analysis of gE geneBased on the sequence of gE gene of Pseudorabies Virus registered in GeBank (registerNo.NC₀06151), using the Pseudorabies Virus Strain SL as template to amplify gE gene. Then the amplified product was cloned into pMD19 T-Simple Vector, the recombinant plasmid was named pMD-gE. Enzyme digestion and sequencing showed that the sequence is 1964 bp including a 1734bp ORF which codes 578 amino acids and shared 97.5%～100.0% nueleotide sequences identity and 94.8%～99.8% the amino acids sequence homology among 11 virus stains. The results showed that the gE gene was highly conserved between different PRV stains. Bioinformatics analysis showed that, G, C appeared frequently in the gE gene codon, accounting for 40% and 30%, the four most frequent codon is GGG, GAC, CTG and CGC, and 96.89% of GC3S content, showed the gE gene had apparent preference to G and C; secondary structure is mainly the corner and random coil; by hydrophobicity analysis and transmembrane prediction, gE protein has two main hydrophobic regions, the first hydrophobic region (3～25 amino acid residue locations) for the signal peptide, the second hydrophobic region (429～452 amino acid residues locations) a transmem-brane, N terminal in the outer membrane, C terminal in the inner membrane; antigenic peptide Prediction, gE gene antigenic peptide mostly in the N terminal 300 amino acids.2. Construction of gE main antigen gene fragment prokaryotie expression plasmid and its activity detectionA pair of primers was designed to amplify the main antigen of gE gene (mgE). Using the pMD-gE plasmid as template, the mgE gene fragment was amplified by PCR and cloned into the pMD19 T-simple vector and named pMD-mgE. Then the recombinant plasmid pMD-mgE was sequenced, the sequence of mgE was identical with gE gene which cloned on former stage. The pMD-mgE plasmid was digested with EcoR I and Sal I to obtain mgE gene fragment, then cloned into the pET32a(+), after identification the reeombination prokaryotic expression plasmid was named pET-mgE and transformed into E.coli Rosetta TM(DE3). Induced by IPTG, the fusion protein was expressed and its molecular weight 42Ku was determined by SDS-PAGE. Detectde by western blot, the pET-mgE possesses specific reaction-genicity.3. mgE protein purification and the initial establishment of ELISA detectionThe mgE recombinant protein which expressed in form of inclusion body is purified as coating antigen, and optimizing conditions in antigen-coated,reaction time and choice of substrate, and finally test the effect of this ELISA through the specificity experiment, the duplicated experiment, blocks examination and contrasting with the standard reagent kit. We establish initially the detection method of porcine pseudorabies virus by indirect ELISA. The research results shows that optimum concentration of the recombinant antigen coated is 8.3ug/mL, the most conditions suitable for coating is 4℃overnight, serum' dilution is 1:40, serum and enzyme-labeled second antibody reaction time is respectively 90min and 60min at 37℃, TMB solution as substrate colored 37℃10min. After statistis analysis, the definite masculine and feminine elements's marginal value is 0.32. And through some testing experiments show that the ELISA has deteetive specificity and good duplication, high sensitivity; the ELISA method that established is compared with American IDEXX Co. PRV antibody kit, the relative specifieity and the sensitivity respectively are 95.7% and 90.9%, the coincidence rate achieves 93.8%, the test result non-significance difference.This research cloned and expressed an antigen concentration fragmentof PRV gE gene, and the initial establishment of the indirect ELISA detection method. From some aspects as the bio-information, molecular biology, immunogenicity and so on, the research providing the refer for more detailed study on PRV.