Prokaryotic Expression Of VP2 Gene Fragment Of Porcine Parvovirus Type 1,the Preparation Of Polyclonal Antibody And Development Of Indirect ELISA

Porcine parvovirus(PPV)is a self-replicating virus belonging to the genus Parvovirus and the family Parvoviridae.It is one of the major causative virus in a reproductive failure syndromes in swine characterized by infertility,mummified fetuses,early embryonic death,and stillbirths,and brings great loss to the domestic pig raising industry and wordwide.The virus was firstly founded by Mary and Mahnel in 1966,and it spread to many countries and regions.PPV was firstly founded in China in 1980 s.The classical Porcine parvovirus disease was caused by PPV type 1.Since then,precautions,controls and eliminations of such a disease became major challenge in China.Therefore,accurate and timely examination of the antibody of the virus is the key to prevent such a disease from spreading.In this program,we developed the method of antibody indirect ELISA of PPV1,laying a firm base for the up-coming development of a rapid diagnostic kit.This experiment has successfully cloned VP2 gene of PPV type 1 and linked it to the express vector pET-30a(+).Then expressed it in the system of BL21(DE3)E.coli.Soluble analysis showed that the recombinant VP2 protein was existed in inclusion body,which was proved to have good antigenicity through Western bolt after refolding.The recombinant VP2 protein was subsequently purified by Ni-NTA chelating affinity chromatography.After four times subcutaneously immune in New Zealand white rabbits to prepare polyclonal antibodies of PPV1.The results of the indirect ELISA and Western blot showed that the polyclonal antibodies with better specificity,and the antibody titer reach 1:25 600.The antiserum was purified by ammonium sulfate and Protein A resin affinity chromatography.We have developed the method of antibody indirect ELISA of PPV1 after optimizing reaction conditions,the results showed that the method no cross react with antibodies of NA-PRRSV,PCV2,PRV and CSFV.By comparing with the national and outside detection PPV1 antibody similar detection kit,the results suggested that the detection limit of this method for positive reference serum can reach 1: 800 dilution.The sensitivity of this kit was 94.3%(132/140),which was higher to the sensitivity of the kit of Wuhan Keqian Company,and it was lower than 97.1%(136/140)of that of INGEZIM PPV.The specificity of this method was 96%(96/100),which was 94%(94/100)higher than that of Wuhan Keqian kit,and was lower than 97%(97/100)specificity of INGEZIM PPV kit.In summary,the indirect ELISA method in this study has good clinical specificity and sensitivity,and it has a greater development and utilization value.