147 results about "Toxoplasmas" patented technology

A reagent kit based on recombinant antigen for detecting Toxoplasma is prepared through taking 542-1218 fragment (t SAG1) from the primary surface antigen gene SAG1 of Toxoplasma, subcloning it to soluble expression carrier pET32a(+), transferring it to colibacillus, configuring engineering bacterium pET32a-tSAG1/BL21, IPTG induced efficient expression, ultrasonic splitting to obtain supernatant, purifying by Ni-NTA and Sephadex-G75, and coating the microholes on ELISA plate. Its test paper can also be prepared by same way.

The invention discloses a screening method of small-molecule inhibitor capable of inhibiting proliferation of toxoplasma gondii. The method utilizes an Alphascreen experiment to primarily select 11 small-molecule inhibitors and one small-molecule accelerant, and the 12 drugs are used for clinical treatment. The 12 drugs are further screened at the cellular level, a plaque experiment proves that GSK J4HCL apparently inhibits the proliferation of toxoplasma gondii, and the value EC50 of the GSK J4HCL to toxoplasmagondii is determined to be about 4.5 muM by virtue of intracellular proliferation experiment. The AlphaScreen competitive inhibition experiment evaluates that the value IC50 of the GSK J4HCL for inhibiting the interaction of TgAtg8 to TgAtg3 is 11.01 muM. The CCK8 experiment is usedfor proving that the value IC50 of the drug for inhibiting the growth of a host cell is 34.359 muM which is far greater than the value EC50 of the drug for the insect, which illustrates that the GSKJ4HCL is a low-toxicity and high-efficiency drug for resisting the toxoplasma gondii.

A rapid detection reagent of a toxoplasma circulating antigen immune colloidal gold marker comprises a sample pad, a bonding pad, a cellulose nitrate film, a water absorbent pad and a PVC back lining, wherein, one end of the PVC back lining is sequentially adhered with the sample pad, the bonding pad, the cellulose nitrate film and the water absorbent pad, the bonding pad is coated by a colloidal gold bonder with an anti-toxoplasma polyclonal antibody, the anti-toxoplasma polyclonal antibody and a toxoplasma metaboly secrete antigen are linearly coated on the cellulose nitrate film. The toxoplasma circulating antigen immune colloidal gold marker rapid detection reagent is prepared by preparing the anti-toxoplasma polyclonal antibody, preparing the toxoplasma metaboly secrete antigen, preparing the colloidal gold, preparing a colloidal gold marker of the anti-toxoplasma polyclonal antibody; the colloidal gold marker of the anti-toxoplasma polyclonal antibody is coated on the colloidal gold bonding pad, the anti-toxoplasma polyclonal antibody and the toxoplasma metaboly secrete antigen are linearly coated in a detection region and a stagnant control region of the cellulose nitrate film and the thorough drying and other processes are carried out. The rapid detection reagent of the toxoplasma circulating antigen immune colloidal gold marker can detect the toxoplasma circulating antigens of people and various animals, which has accurate sensitivity and simple operation, and being safe and stable.

The invention provides a vaccine for preventing toxoplasma infection. The vaccine contains nucleotide sequences selected from the followings: 1) 736-2385th nucleotide sequences as shown in SEQ ID No.1, and/or 759-2411th nucleotide sequences as shown in SEQ ID No.3; 2) nucleotide sequences complementary with the nucleotide sequences as shown in 1); and 3) nucleotide sequences which perform substituting, deletion and addition modification on one or a plurality of basic groups of the nucleotide sequences as shown in 1) or 2). The vaccine has the capability of effectively preventing and controlling the infection of a toxoplasma animal model (Kunming mice), and the combination of recombinant plasmids can effectively improve the immune effect of a single-gene vaccine, therefore, plasmids pVAX-ROP5 and pVAX-GRA15 can be used as DNA (Deoxyribose Nucleic Acid) vaccine candidate antigens for preventing the toxoplasma infection; and a combined immune vaccine of a recombinant plasmid based on such two genes can be used as a cocktail composite vaccine for preventing the toxoplasma infection.

The invention provides a nucleotide sequence for prevention of toxoplasma infection. The nucleotide sequence for prevention of toxoplasma infection contains nucleotide sequences selected from (1) a nucleotide sequence as shown in SEQ ID No.1 of a sequence table, (2) a nucleotide sequence complementary with the nucleotide sequence as shown in the (1), and (3) a nucleotide sequence obtained by replacing, deleting, adding and modifying one or multiple of basic groups of the nucleotide sequence as shown in the (1) or (2) and having a function of preventing toxoplasma infection. The invention also provides a vaccine which comprises the nucleotide sequence and a medically acceptable carrier. The plasmid DNA vaccine pVAX-ROP38 can be used for effectively preventing and controlling infection of toxoplasma animal models (Kunming strain mouse), namely can be used for effectively prolonging the survival time of the mouse and reducing the worm burden of brain capsules. Therefore, the plasmid pVAX-ROP38 can serve as a DNA vaccine candidate antigen for resisting toxoplasma infection.

The present invention belongs to the field of biotechnology, and relates to a surface antigen 1 (SAG1) of Toxoplasma gondii human antibody Fab fragment, encoded gene and use thereof. According to the invention, the surface antigen 1 (SAG1) of Toxoplasma gondii human antibody Fab fragment is filtered from a base through establishing a Toxoplasma gondii human immunoglobulin, ELISA, diluting the prothrombin time, sequencing analysis, etc. Through expression purifying and authenticating, the human antigen Fab fragment is authenticated to specifically identify the tachyzoite-bradyzoite recombination SAG1 of Toxoplasma gondii and have higher affinity with the tachyzoite-bradyzoite recombination SAG1 of Toxoplasma gondii, for being identified with the specificity of Toxoplasma gondii tachyzoite-bradyzoite. The human antigen Fab fragment of the invention does not contain Fc segment and does not activate the alexin or cause the histopathological damages of human immune response, etc. when the function of restricting the invasion of Toxoplasma gondii to the host cell is exerted. The surface antigen 1 (SAG1) of Toxoplasma gondii human antibody Fab fragment is safe and reliable when applied for the human body. The antigen medicine for treating toxoplasmosis or the antigen targeted medicine can be prepared.

The invention discloses an immunologic adjuvant for preventing toxoplasma infection. The immunologic adjuvant is characterized in that the immunologic adjuvant is recombinant plasmids pVAX-IL21-IL15 formed by fusing interleukins IL21 and IL15 by using pVAX1 plasmids as carriers, and the base sequence of the immunologic adjuvant is represented by SEQ ID NO:1. The immunologic adjuvant for preventing the toxoplasma infection has a function of obviously improving the immune effect of MIC8 genes, so that the immune effect of an immune prevention vaccine against toxoplasma can be obviously enhanced.

The invention firstly provides a nucleotide sequence for preventing toxoplasma infection. The nucleotide sequence is selected from one of the following nucleotide sequences: (1) a nucleotide sequence as shown in SEQ ID No.1 in a sequence table, (2) a nucleotide sequence which is complementary with the nucleotide sequence in (1) and (3) a nucleotide sequence which is obtained by substituting, deleting and modifying one or more basic groups of the nucleotide sequence as shown in (1) or (2) and has a function of preventing toxoplasma infection. The invention also provides a vaccine comprising the nucleotide sequence and a medically acceptable carrier. The plasmid DNA vaccine pVAX-ROP7 provided by the invention can be used for effectively preventing and controlling toxoplasma animal model (Kunming mouse) infection, namely effectively prolonging the survival time of a rat and reducing the polypide quantity of a brain cyst. Therefore, the plasmid pVAX-ROP7 can be used as a candidate antigen of DNA vaccine to prevent toxoplasma infection.

The invention discloses two CD8+T cell dominant epitopes of VPNSSLVEN and SQFLSLSLL based on toxoplasmagondii bradyzoite specific surface antigens, wherein six CD8+T cell epitopes with H-2 restriction and high affinity are screened according to the protein sequences of the bradyzoite specific surface antigens of SAG2C, -2C and -2X; BALB/c mice are actively immunized by the screened epitope polypeptides; determinations for a lymphopoiesis level and a cell factor content, and an immune protection evaluation for epitope vaccines are performed after the immunization. The result indicates that powerful cell immunization can be generated by inducing the mice via the epitope vaccinesm, wherein the two antigen peptides of VPNSSLVEN and SQFLSLSLL are provided to be great in the capacity of stimulating T cell proliferation, as well as capable of inducing the mice to secrete protective cell factors and immune protection, being used as the epitope vaccines effectively resisting toxoplasmagondii infection, and reducing an encystations rate in the brain tissue of a host.

The invention relates to the field of molecular biology, and provides specific primers for detecting Toxoplasma gondii. The sequence of an upstream primer in the specific primers is as shown in SEQ IDNo. 1; the sequence of a downstream primer in the specific primers is as shown in SEQ ID No. 2. The invention also provides probes for detecting Toxoplasma gondii. The sequences of the probes are asshown in SEQ ID No. 3-6. The invention further provides a kit for detecting Toxoplasma gondii. The kit comprises the primers and the probes for detecting Toxoplasma gondii. The invention further provides a chip for detecting Toxoplasma gondii. The chip comprises the probes for detecting Toxoplasma gondii. According to the invention, gene chip detection is carried out on Toxoplasma gondii by usingthe primers and the probes, and differential diagnosis can be specifically carried out on Toxoplasma gondii infection conditions.

The invention discloses a primer, a probe, a kit and a detection method for detecting pneumocystis carinii and toxoplasma gondii, and particularly relates to a kit and a detection method for double fluorescent quantitative PCR detection of pneumocystis carinii and toxoplasma gondii. According to the invention, the primer pair and the probe are designed by utilizing conserved regions of pneumocystis carinii and toxoplasma gondii, the primer pair and the probe which can specifically amplify pneumocystis carinii and toxoplasma gondii are screened out, the sensitivity is high, the two pairs of primers and the two probes do not interfere with each other, and a sample only containing 1 copy can be accurately detected; by utilizing the primer pair, the probe and the detection method, the contentsof the pneumocystis carinii and the toxoplasma gondii can be absolutely quantified; the kit is simple in structure, safe in detection, rapid and convenient, can be used for detecting the pneumocystiscarinii and the toxoplasma gondii and tracking and monitoring the treatment effect of medicines of a patient, and is beneficial to carrying out epidemiological investigation and preventing and controlling propagation of the pneumocystis carinii and the toxoplasma gondii.

The invention discloses a kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of the kit. The kit consists of a detachable 96-pore polymerase chain reaction (PCR) tube, PCR reaction liquid, TaqDNA polymerase, standard and control, wherein the PCR reaction liquid contains a PCR buffer solution, dNTPs, MgCl2, KCl, gelatin and various detection primers; the various detection primers are respectively specific primers for eperythrozoon, leptospira and toxoplasma. The kit is applicable to detecting whether blood is infected by one or more pathogens in the eperythrozoon, leptospira and toxoplasma. The kit disclosed by the invention is rapid in detection speed, high in detection sensitivity, good in specificity, and simple and convenient to operate; and the kit can simultaneously identify and detect one or more specimen in mixed infection of the eperythrozoon, leptospira and toxoplasma, thus greatly improving detection accuracy and reduce false positive rate.

The invention relates to a kit for absolutely quantitatively detecting Toxoplasma gondii based on digital PCR and a detection method thereof; the kit is composed of a microdrop digital PCR kit and a plurality of microdrop generator sheets; the detection method is characterized by comprising: (1) extracting total DNA of a test sample; (2) using the total DNA as a template to carry out PCR amplification; (3) placing the PCR production and microdrop reader to read signals, analyzing experimental data to obtain an absolute content of Toxoplasma gondii in the sample. The kit and method can provide direct quantitation in Toxoplasma gondii detection, have no need for standard curves, have the advantages, such as good operational convenience, high speed and efficiency, good specific sensitivity and low cost, etiological diagnosis on Toxoplasma gondii can be effectively carried out, detection rate is increased, the common problems of existing Toxoplasma gondii detection kits, such as low sensitivity and high misdiagnosis rate, are solved, and the kit and method are suitable for clinical investigations and large-scale disease detection and supervision, related preventive and control measures can be collected in time, and economic loss is decreased.

The invention provides a nucleotide sequence for preventing infection of toxoplasmas. The nucleotide sequence contains any one group of the following nucleotide sequences: (1) a nucleotide sequence of profilin; (2) nucleotide sequences of profilin, rop16, rop18, MIC6 and CDPK3; (3) nucleotide sequences of profilin and IL15; (4) nucleotide sequences of rop16, rop18, MIC6 and CDPK3; (5) complementary sequences of the nucleotide sequences from (1) to (4); and (6) nucleotide sequences prepared through carrying out substitution, deletion, addition and modification on nucleotide sequences from (1) to (5). By virtue of a pVAX-profilin vaccine and a combined vaccine, the generation of brain cysts of the toxoplasmas can be effectively inhibited, and a relatively good immune effect is achieved.

The invention provides a semi-nested PCR method for detecting toxoplasma gondii, which determines whether a sample is toxoplasma gondii infection positive or negative by arranging a PCR detection kit, extracting the DNA of a test sample and carrying out the semi-nested PCR amplification and the analysis of the amplified products. The method of the invention can rapidly and accurately detect the toxoplasma gondii in the test sample, and can also be used for the molecular epidemiological investigation and the efficacy monitoring of toxoplasmosis. The template DNA preparation steps of the method are simple, the cost is low, however, the conventional method needs to be processed by lysozyme, protease K, sodium dodecyl sulfate, cetyltrimethyl ammonium bromide and other reagents, the time is long and the cost is high. The method is established on the molecular biology, which can exclude the interferences of bacteria and impurity particles, thus greatly improving diagnostic accuracy and reducing false positive rate.

The present invention is toxoplasma metabolic secretion antigen vaccine and its preparation process. The toxoplasma metabolic secretion antigen vaccine is prepared with toxoplasma metabolic secretion antigen. The preparation process includes the following steps: inoculating toxoplasma tachyzoite to abdominal cavity of mouse, killing the mouse and soaking in alcohol for surface sterilizing; collecting polypide; obtaining abdominal cavity liquid via centrifuging and collecting supernatant; inoculating toxoplasma tachyzoite to vero cell to culture and collecting the cultured liquid after cell falling off via centrifuging and collecting supernatant; thrice freezing and thawing abdominal cavity liquid and cultured liquid; mixing, measuring protein content of the mixed liquid; mixing the mixed liquid with M206 adjuvant, emulsifying and packing as the composite toxoplasma vaccine with antigen protein content of 10 mg/ml. The vaccine is for inducing humoral immunity and cellular immunity.

The invention discloses an anti-toxoplasma composition drug and a screening method thereof, and belongs to the field of medicine. According to the anti-toxoplasma composition drug and the screening method thereof, mouse test toxoplasmosis animal models are established, the ten drugs of a sulfadiazine sodium injection, a compound sulfamethoxazole, a lincomycin hydrochloride injection, a sulfamonomethoxine sodium injection, a florfenicol injection, acetylspiramycin, pyrimethamine, roxithromycin, artemisinin and radix sophorae flavescentis are selected out, two compositions which have the best anti-toxoplasma effect are screened out, one composition comprises the sulfamonomethoxine, the pyrimethamine and TMP, and the other composition comprises the compound sulfamethoxazole and the acetylspiramycin; animal models in mice and pigs are established, it is verified that the two compositions are both effective on the treatment of artificially infected toxplasmosis in pigs, and the treatment effect on the pigs is in accordance with that of the mouse models. By means of the anti-toxoplasma composition drug and the screening method thereof, it is proved that the the feasible method is achieved by utilizing the mouse test toxoplasmosis models to evaluate the efficacy of drugs on treating toxplasmosis in pigs, and meanwhile two anti-toxoplasma drug compositions with good effects are provided.

The invention provides a nucleotide sequence for preventing toxoplasma infection. The nucleotide sequences selected from the followings are contained: 1) 724-1470th nucleotide sequences as shown in SEQ ID No.3, and/or 724-2472nd nucleotide sequences as shown in SEQ ID No.1; 2) nucleotide sequences complementary with the nucleotide sequences as shown in 1); and 3) nucleotide sequences which perform substituting, deletion and addition modification on one or a plurality of basic groups of the nucleotide sequences as shown in 1) or 2). The pVAX-CDPK (Calcium Dependent Protein Kinase)1 can effectively prevent and control the infection of a toxoplasma animal model (Kunming mice), and pVAX-IL21-IL15 not only can be independently used for effectively preventing and controlling the toxoplasma infection, but also can be used as a cytokines adjuvant for effectively improving the immune effect of a vaccine pVAX-CDPK1.

The invention discloses a toxoplasma wx2 gene deletion strain, a construction method and application. The wx2 gene of a toxoplasma RH strain is knocked out through CRISPR/Cas9 technology to successfully obtain the wx2 gene deletion strain with pyrimethamine drug resistance. The specific method is that CRISPR plasmid Psag1-Cas9-U6-sgwx2 and drug-resistant fragment wx2-DHFR are constructed, targetedshearing of the CRISPR plasmid and homologous recombination of the drug-resistant fragment are utilized, the identification of a monoclonal strain and a monoclonal deletion strain is carried out through drug screening, and the wx2 gene deletion strain of the toxoplasma RH strain can be successfully obtained. The toxoplasma wx2 gene deletion strain lays a solid foundation for the study of molecular mechanism of growth of the toxoplasma, and provides a theoretical basis for development of the toxoplasma vaccines and study of the toxoplasma drug target molecules.