Development And Preliminary Of Colloidal Gold Immunochromatographic Strips And Fluorescence Immunochromatographic Strips For The Detection Of Toxoplasma Infection In Cat

Toxoplasma gondii is an obligate intracellular protozoan parasite.According to statistics,one third of the person in the world has been infected with Toxoplasma gondii.Among them,cats are the hosts of Toxoplasma gondii.In this study,three proteins of SAG1,GRA1-GRA7 and GRA6-GRA2 were expressed in prokaryotic cells.The highly sensitive proteins was selected by ELISA.It is generated as Colloidal Gold Immunochromatographic Strips and Fluorescence Immunochromatographic Strips for cat Toxoplasma infection.In this study,the recombinant plasmids p ET28a-GRA1-GRA7,p ET28a-SAG1 and p ET28a-GRA6-GRA2 were optimized and synthesized by molecular biology methods according to the preference of Escherichia coli and convenience of protein purification.The plasmids were transformed into ER2566 competent cells for expression.In order to obtain high expression of recombinant protein,the expression conditions were optimized.The size of GRA1-GRA7 protein is about 50 ku.Under the conditions of IPTG(1 mmol/L),high expression of protein can be obtained at 25°C;The size of SAG1 protein is about 56 ku.Under the conditions of IPTG(0.2 mmol/L)at 25℃,the protein can be highly expressed.The size of GRA6-GRA2 protein is about 64 ku.Under the conditions of IPTG(1 mmol/L)at 25℃,the protein can be highly expressed.The expression products were purified by His chromatography column,and Western Blot.The expression product were identified with good antigen specificity.ELISA method was used for detection and comparison,and it was determined the GRA6-GRA2 with highest sensitivity.The establishment of Colloidal Gold Immunochromatographic Strips method for cat Toxoplasma infection.In this study,GRA6-GRA2 protein was used as the labeled antigen.Staphylococcus protein A(SPA)was used as the detection line(T),and the rabbit anti-GRA6-GRA2 antibody was used as the quality control line(C).They were used to prepare the Colloidal Gold Immunochromatographic Strips of cat Toxoplasma antibodies.In order to optimize the p H value,add 12μL 0.2mol/L K2CO3 to 1m L of colloidal gold;The labeled amount of GRA6-GRA2 protein was 3.75μg/m L.The sensitivity of the test strip is 1:256;the positive sera of feline calicivirus,feline parvovirus,and feline herpes virus are tested without cross-reactivity;when the test is repeated,the repeatability is good.This test paper tests 289 clinical samples of serum.The results of the indirect hemagglutination kit showed that the positive detection rate of the test strip was 2.42%,the positive detection rate of the indirect hemagglutination kit was 2.07%,and the coincidence rate of the two was 99.5%.Establishment of Fluorescence Immunochromatographic Strips for Cat Toxoplasma Infection.In this study,purified GRA6-GRA2 protein was used as the labeled antigen.After optimization,100μL of fluorescent microspheres should be added to 90μg of GRA6-GRA2 antigen;1mg/m LSPA+1mg/m L rabbit anti-GRA6-GRA2 antibody is the best coating ratio;The results show that the best detection time is 5 minutes after loading the sample.The sensitivity of the test strip is 1:1024;the positive sera of feline calicivirus,feline parvovirus,and feline herpes virus are tested without cross-reactivity;When the test is repeated,the repeatability is good.The test strips and the Toxoplasma antibody indirect hemagglutination method were used to compare 289 clinical sera,the positive rates were2.42% and 2.07%,and the coincidence rate was 99.5%.