| Toxoplasma gondii,a kind of opportunistic pathogenic protozoa residing in the nucleated cells of human and other vertebrate animals.In recent years,the integrated infection of Toxoplasma gondii along with other pathogens could cause critical problems in animal husbandry and human health.Thus,the study of the technology in the immunological detection will contribute to the rapid diagnosis of Toxoplasma gondii.ELISA is the most commonly used method for diagnosising toxoplasmosis.Choosing the appropriate antigen is a prerequisite for the development of ELISA kits.Toxoplasma gondii dense granule protein is a kind of target antigen that closely related to the growth and development of parasites residing in cells.In our previus studies,the recombinant protein of Toxoplasma gondii dense granule protein GRA8 was expressed.It has been proved that GRA8 was a supreior diagnostic antigens for chicken toxoplasmosis by ELISA and Western blot.Given that the recombinant protein GRA8 was low expressed in Escherichia coli BL21 cells,the prokaryotic expression system of truncated sGRA8 gene was reconstructed by cutting the signal peptide and hydrophobic region whith related molecular biology technology.Western blot analysis showed that the recombinant sGRA8 protein was of high immunogenicity.With its high sensitivity and specificity,the infection of Toxoplasma gondii in chicken can be detected in 130 days by sGRA8 protein.In this study,the sGRA8-ELISA method was applied to the detection of Toxoplasma gondii antibody in rat,pig,cattle,sheep and human for the standardization and preliminary application of Toxoplasma gondii infection.On the basis of our previous studies,we explore and optimize the dilution ratio of the primary or second anibody for further investigation.In this study,the sGRA8 protein was expressed by the prokaryotic expression vector BL21-pCold,and purified by His affinity chromatography,got the soluble purified coating antigen that could be used for the Toxoplasma gondii ELISA antibody detection.10 SD rats were infected with Toxoplasma gondii tachyzoites,while 10 SD rats were not.Serum was collected in 3-109 days after infection,all the 20 rats were verificated by PCR;the rat serum was tested by sGRA8-ELISA method in comparision with the imported commercialization kit purchased from BioTSZ.The results show that both detection methods have a certain effect;Four and six positive sera were detected by sGRA8-ELISA and the BioTSZ kit respectively;from 7 to 75 days after infection,the positive detection rate by sGRA8-ELISA reached 75%-100%and the BioTSZ's kit up to 67%;The positive-detection proportion of BioTSZ kit show a downward trend from 75 days after infection and could not be used at 109 days since infection,while the positive rate of sGRA8-ELISA was 67%(4/6)at 109 days.The sensitivity of the sGRA8-ELISA method was 80%(74/92),while the BioTSZ kit was 51%(47/92).There were no significant differences in the detection of negative sera by both detection methods,and the specificity was 100%(60/60).The results showed that sGRA8-ELISA was superior to that of BioTSZ kit.Through the sGRA8 protein polyclonal antibody detection,the titer reached 1012.The results showed that sGRA8-ELISA kit had good effects on the detection of swine,cattle and sheep Toxoplasma gondii antibody.The test results between the sGRA8-ELISA and Nanjing Angle Co.,Ltd ELISA kits have a high coincidence rate.Using sGRA8-ELISA method,809 pig sera from 16 pig farms were tested and the overall positive rate was 6.43%(52/809).The positive and negative sera were screened by the antibody test kit of Toxoplasma gondii antibody from Zhuhai Haitai Biotechnology Co.,Ltd.,and 38 positive sera and 52 negative sera were re-tested by sGRA8-ELISA.The results showed that the coincidence rate of the positive samples was 84%(32/38)and that of the negative samples was 94%(49/52).In summary,sGRA8-ELISA method is remarkable in detecting toxoplasma antibodiesof rat,pig,cattle,sheep and human,it provides a prerequisite for development of commercialization kits. |
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