Preparation And Identification Of Monoclonal Antibody Against GRA7 Protein Of Toxoplasma Gondii

Toxoplasma gondii(T.gondii)is an obligate intracellularand opportunistic parasitic protozoa that can infectalmost all nucleated cells.It is considered to be one of the most common parasites in the world.It causes a disease known as toxoplasmosis which is a cosmopolitan zoonosis and has been listed by the World Health Organization(WHO)and the Food and Agriculture Organization of the United Nations(FAO)as one of the most watched food-borne parasites in the world.As an opportunistic protozoa,Toxoplasma gondii will not only bring huge economic losses to the animal husbandry industry and food industry,but also pose an enormous risk to public health problemsand pose a serious threat to public health safety in the world.At present,there is still no drug that can cure toxoplasmosis.Therefore,rapid and accurate diagnosis of Toxoplasma gondii infection is the focus of toxoplasmosis prevention and treatment.The main diagnostic methods for toxoplasmosis include etiological detection,serological detection,molecular biological detection and imaging techniques.Among these methods,etiological detection is complicated in operation and requires high professional requirements for operators;molecular biological detection and imaging techniqueshave high requirements for instruments and the cost of detection is high;serological detection is the most common method for the diagnosis of toxoplasmosis because of its low cost and simple operation.Among them,enzyme-linked immunosorbent assay(ELISA)is widely used for in vitro diagnosis(IVD)because of its ability to detect antigens and antibodies.At present,commercial indirect ELISA kits have been used to detect humans,pigs,cats,and dogs,but indirect ELISA assays have low sensitivity and are prone to missed detection errors.At present,commercial indirect ELISA kits have been used to detect Toxoplasma gondii infections in humans,pigs,cats,and dogs,but the indirect ELISA method has low sensitivity and is easily missed and misdetected.The double-sandwich ELISA based on monoclonal antibody has high sensitivity and specificity,but there is no relevant kit.In this study,a recombination prokaryotic expression vector pET32a-GRA7 was constructed.After transformed into E.coli BL21(DE3),the GRA7 fusion protein with a molecular weight of approximately 46 kDa was induced by IPTG.It was identified by SDS-PAGE and Western-blot that the purified rGRA7 protein had immunogenicity.Mice were immunized with purified protein and monoclonal antibody were prepared using hybridoma technology.One hybridomacell line named 6C3 was screened by indirect GRA7-ELISA.The immunoglobulin subclass of the 6C3 monoclonal antibody was identified as IgG2a.The titer of cell culture supernatant and ascites was1:10~4respectively measured by indirect GRA7-ELISA.The purified antibody could identify recombinant rGRA7 protein by Western-blot.The light chain and the heavy chain of the monoclonal antibody could be observed by SDS-PAGE.