Preparation Of Monoclonal Antibody Against Toxoplasma Gondii SAG1 And Preliminary Study Of Its Biosensors

Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii,which is a serious health risk for humans and can be found in the nucleated cells of almost all warm-blooded animals.Toxoplasmosis has an extremely wide distribution,and Toxoplasma gondii infections have been reported in various regions of China.Some data show that the per capita infection rate in China is around 10%to 47.3%.In recent years,Xu Bin et al.conducted antibody monitoring of Toxoplasma gondii infection in 865 sera of dogs and cats in Chongqing,and the results showed that the antibody positivity of Toxoplasma gondii infection was as high as 18.96%,indicating the seriousness of infection in dogs and cats in our city and the seriousness of surface source contamination.Toxoplasma gondii infection in pregnant women can be transmitted to the baby through the placenta and cause congenital toxoplasmosis,which can easily cause fetal malformation and can lead to miscarriage and stillbirth in severe cases.Toxoplasma gondii infection is likely to cause other important co-morbidities in people with immune deficiencies such as oncology patients and AIDS.As the only end host for Toxoplasma gondii transmission in felines,the gradual increase in pet ownership in recent years has led to an increased risk of Toxoplasma gondii transmission from pets to humans and poultry,posing a great threat to human health and the livestock industry.Currently,there are no effective therapeutic drugs or vaccines for chronic Toxoplasma gondii infections.The aim of this paper is to prepare specific and active Toxoplasma gondii SAG1 antigen and antibody,and use them as reagent materials to develop an electrochemical biosensor based on AuNPs@ZnO modified glassy carbon electrode and a MoS₂ modified micro-nano fiber optic biosensor for the rapid detection of Toxoplasma gondii antibodies,providing a new detection method for Toxoplasma gondii infection.This paper examines the following.（1）Preparation of Toxoplasma gondii SAG1 antigenThe p ET32a（+）-TG SAG1 recombinant plasmid was constructed by codon optimization and cloning into the p ET32a（+）vector according to the SAG1 gene sequence published in GenBank（accession number:S76248.1）.The recombinant plasmid was transformed into E.coli BL21（DE3）receptor cells and expression of Toxoplasma gondii SAG1 recombinant protein was induced with isopropyl-β-D-thiogalactopyranoside（IPTG）.The protein expression conditions were optimized to express Toxoplasma gondii SAG1recombinant protein under the optimal induction conditions,and the purified recombinant protein was purified by the NGC^TM protein purification system on a nickel column,and the purified recombinant protein was subjected to purity identification,concentration determination and western blot identification.（2）Preparation of monoclonal antibody against Toxoplasma gondii SAG1BALB/c mice were immunized by intraperitoneal injection using purified and dialysis-concentrated Toxoplasma gondii SAG1 recombinant protein as the immunogen.After four immunizations,blood was collected from the tails of mice for potency determination,mice eligible for cell fusion were selected,cells from spleen tissue were extracted,and splenocytes were induced to fuse with myeloma cells in vitro by PEG1 500.The supernatant of the fused cells was tested by indirect ELISA to screen positive hybridoma cells,and multiple subclonal screening was performed by multiplicative dilution to finally obtain a cell line that can stably secrete monoclonal antibodies.The purified ascites was purified using Protein G columns,and the purified Toxoplasma gondii monoclonal antibody was characterized biologically.（3）Development of an electrochemical biosensor based on AuNPs@ZnO modified glassy carbon electrode for the detection of Toxoplasma gondii antibodiesZnO was prepared by hydrothermal synthesis,ZnO and AuNPs@ZnO composites were obtained by adding chloroauric acid solution to aqueous ZnO solution drop by drop with stirring,and finally,AuNPs@ZnO composites were obtained by centrifugal drying.Characterization of the sample shape and elemental content using scanning electron microscopy（SEM）and energy dispersive X-ray spectroscopy（EDS）.The AuNPs@ZnO composite was immobilized on the surface of a glassy carbon electrode by physical adsorption,and then the Toxoplasma gondii SAG1 recombinant protein was self-assembled on the electrode surface using gold-sulfur covalent bonds,and the constructed biosensor was characterized using cyclic voltammetry（CV）.Sensitivity,specificity,clinicality and repeatability of the sensors were measured by differential pulse voltammetry.（4）Development of a MoS₂-modified micro-and nano-fiber optic biosensor for the detection of Toxoplasma gondii antibodiesThe sensing area of the tapered fine-core micro-nano fiber is treated by hydroxylation and silylation to make the fiber surface with mercaptans.The defective sites formed by the absence of sulfur atoms on the surface of MoS₂ can bind to sulfhydryl groups and then immobilize the Toxoplasma gondii antigen on the fiber surface by the high hydrophobicity of MoS₂.The biosensor obtained was prepared for the detection of Toxoplasma gondii antibodies,and the sensitivity,specificity and clinicality of the sensor were evaluated.Results.（1）Preparation of Toxoplasma gondii SAG1 antigenThe constructed p ET32a（+）-TG SAG1 recombinant plasmid was identified by double digestion with Bam HI and Xho I,and a nucleic acid electrophoretic band appeared at 1020bp,which was consistent with the expected target band.The recombinant plasmid was transformed into E.coli BL21（DE3）receptor cells to successfully construct genetically engineered bacteria,and the expression conditions（temperature,time,IPTG concentration）of SAG1 recombinant protein were optimized,and the optimal induction conditions were screened:25°C,8 h,0.4 mmol/L.Purified by NGCTM protein purification system and identified by SDS-PAGE electrophoresis for purity,the results showed a clear single band near 55 k Da with high purity as expected.The concentration of purified SAG1recombinant protein was 0.4519 mg/m L as measured by the BCA Protein Concentration Quantification Kit,and a clear protein blot near 55 k Da was identified by Western blot,indicating good specificity.（2）Preparation of monoclonal antibody to Toxoplasma gondii SAG1A cell fusion experiment was performed and the cell fusion rate was 100%with a positive rate of 98.3%.Using indirect ELISA to screen positive hybridoma cells and performing three subclonal screens,two cell lines that could stably secrete monoclonal antibodies were obtained,and both were identified as Ig G1 type by subtyping and named:5-2C and 5-11A.The cell line was expanded and cultured,and the ascites monoclonal antibody was induced by intraperitoneal injection into BALB/C mice.The ascites was purified using Protein G columns and identified by SDS-PAGE,which showed significant bands at 25 k Da and 50 k Da with high purity.The BCA protein quantification kit measured its concentration to be 0.5034 mg/m L.The purified Toxoplasma gondii SAG1 monoclonal antibody was identified by Western blot,which showed a significant specific immunoblot near 55 k Da.Finally,the potency of Toxoplasma gondii SAG1 monoclonal antibody was measured by indirect ELISA at 1:512 000.（3）An electrochemical biosensor based on AuNPs@ZnO modified glassy carbon electrode was established,The performance of the sensor was measured by differential pulse voltammetry.The results showed that the linearity of the standard curve was good using the constructed AuNPs@ZnO/SAG1 recombinant protein/glass carbon electrode based biosensor for the determination of Toxoplasma gondii antibodies,with the equation of the standard curve being:y=-4.501x+51.377,linear correlation coefficient of 0.9965 and lower limit of detection of 0.0998 pg/m L（S/N=3）.The sensor was tested for repeatability and the RSD=1.6%,indicating good repeatability.The specificity of the sensor was tested using pseudorabies antibody,rabies antibody,brucellosis antibody,Toxoplasma gondii antibody and a mixture of these four antibodies,and the results showed that only the solution containing Toxoplasma gondii antibody reacted specifically with the antigen on the surface of this sensor,resulting in a decrease in peak current,indicating good specificity of the sensor.The final test was a clinical serum sample of Toxoplasma gondii,which showed significant specific binding of the clinical sample to the antigen on the sensor,indicating that the sensor can be used for clinical testing.（4）A MoS₂-modified micro-nano fiber-optic biosensor was established and used for the detection of Toxoplasma gondii antibodies.Experimental results show that the sensor has a detection saturation point of 100ng/m L and detects Toxoplasma gondii antibodies in the concentration range of 1pg/m L-100 ng/m L.The specificity of the sensor using rabies antibodies and pseudorabies antibodies showed only a slight change in wavelength,indicating good specificity.Finally,clinical serum samples of Toxoplasma gondii were tested and the results showed that positive clinical serum samples specifically bound to the Toxoplasma gondii antigen on the sensor,causing a significant change in the spectrum and a red shift in wavelength,indicating that the sensor is suitable for clinical detection.Conclusion.In this study,Toxoplasma gondii SAG1 recombinant protein was successfully expressed and Toxoplasma gondii SAG1 monoclonal antibody was prepared.A method for the detection of Toxoplasma gondii antibodies based on AuNPs@ZnO modified glassy carbon electrode electrochemical biosensor was developed,and a MoS₂ modified micro-nano fiber optic biosensor was also developed and used for the determination of Toxoplasma gondii antibodies.The electrochemical and fiber-optic biosensor developed in this paper provides a new technique and means for the detection of toxoplasmosis,which has important application value.