Establishment And Application Of Indirect ELISA For Detection Of Goat PRV Based On GE Protein

Pseudorabies virus（PRV）is the pathogen of goat pseudorabies（PR）.Goats have itching,respiratory and nervous system disorders,and even sudden death when they are infected with PRV.In recent years,there have been many reports about sheep infected with PRV,which has caused some losses to the aquaculture industry in China.However,at present,there is a lack of serological methods for the detection of goat pseudorabies virus in clinic,which makes it difficult to prevent and control the disease.Therefore,based on the main antigen region of pseudorabies virus gE protein,The prokaryotic expression vector of recombinant PRV gE protein was constructed and the expression conditions were optimized and the protein was purified,an indirect ELISA method for detection of goat pseudorabies virus was established and 376 goat sera collected from national animal serum bank were tested.The result are as follows:1.The gE gene of pseudorabies virus SDPD-14 strain was successfully cloned and analyzed.The amino acid sequence analysis showed that the protein had no signal peptide,no transmembrane region,no continuous rare codon,good antigenicity and hydrophilicity.The secondary structure of the protein was predicted,including 43α-helix,12β-rotation,54 extended chains and 104 random coils.2.The recombinant PRV gE protein was successfully expressed.The optimal expression conditions were as follows:37℃,0.6 mmol/L IPTG induction for 8 h,the molecular weight of the protein was 42 ku,and the protein was successfully purified by nickel column.Western blot results showed that the protein had good immunogenicity.3.An indirect ELISA method based on pseudorabies virus gE protein was successfully established.The optimum conditions were as follows:1μg/m L recombinant gE protein was coated overnight;5%skimmed milk powder was used to seal 60min,and the serum to be tested was diluted for 60 min;enzyme-labeled secondary antibody was diluted 10000times and incubated for 60 min;chromogenic 30 min.The critical value of OD_450nmfor judging positive and negative was 0.284.The method had good sensitivity,specificity and repeatability.To sum up,the genetic evolution of gE gene of goat pseudorabies virus Shandong strain SDPD-14 was analyzed in this study.Secondly,the recombinant gE protein was expressed by prokaryotic expression technique,and an indirect ELISA method for detecting PRV gE antibody in goat serum was established,which provided an alternative detection method for breeding farms.