83 results about "Echinococcus granulosus" patented technology

The invention discloses a kit for detecting echinococcus granulosus, echinococcus multilocularis and echinococcus shiquicus. Three pairs of specific amplification primers aiming at three pathogens are respectively arranged in the detection kit. By utilizing the three pairs of primers in the kit, a composite polymerase chain reaction (PCR) method can be used for detecting independent or mixed infection of the three pathogens, and whether the detected sample is infected with echinococcus can be determined according to specific strips occurring in electrophoresis of amplification products.

The invention provides a primer for detecting echinococcus granulosus in canine feces, wherein the sequence of an upstream primer is as shown in SEQ ID No. 1; and the sequence of a downstream primer is as shown in SEQ ID No. 2. The invention also provides a probe for detecting the echinococcus granulosus in the canine feces, wherein the sequence is as shown in SEQ ID No. 3. The invention further provides a kit for detecting the echinococcus granulosus in the canine feces; the kit comprises the primer for detecting the echinococcus granulosus in the canine feces and the probe for detecting the echinococcus granulosus in the canine feces, wherein the sequence of the upstream primer is as shown in SEQ ID No. 1; the sequence of the downstream primer is as shown in SEQ ID No. 2; and the sequence of the probe is as shown in SEQ ID No. 3. In the invention, by using the primer, the probe and the kit, a target gene DNA can be rapidly and specifically detected with high sensitivity; even when a specimen contains the trace amount of echinococcus DNA, the target gene DNA can be detected.

The invention discloses an indirect ELISA detection kit of sheep echinococciosis Eg95 protein antibody. The invention is characterized in that the kit comprises an echinococcus granulosus recombinantantigen Eg95 protein precoated ELISA plate, a serum diluent, an enzyme-labeled conjugate diluent, a positive control solution, a negative control solution, an enzyme-labeled conjugate, a washing solution, a substrate developing solution and a stopping solution. During the preparation process of the echinococcus granulosus recombinant antigen, a His tag sequence and a Myc tag sequence are respectively connected to the front and back of the Eg95 gene; then, EcoR I and Xho I are adopted for simultaneous digestion of a PCR product and an eukaryotic expression vector pPIC9K, the target fragment andthe vector are connected after gel extraction, recombinant plasmid pPIC9K-(His-Eg95-Myc) is constructed, the recombinant plasmid is transformed into pichia pastoris GS115 strain, and the recombinantantigen Eg95 is expressed by the induction of methanol and purified by the use of Ni-NTA. The sheep echinococciosis antibody detection kit obtained by the adoption of the dual-tag sequence and eukaryotic expression vector system has strong specificity, high sensitivity and good repeatability, and is easy to operate.

The invention discloses a kit of dog echinococcus granulosus and echinococcus multilocularis based on POCKIT Micro fluorescent PCR platform, and application of the kit. The kit comprises fluorescent quantitative PCR reaction fluid, positive comparison product and negative comparison product; the fluorescent quantitative PCR reaction fluid comprises upstream general primers of echinococcus granulosus and echinococcus multilocularis, downstream general primers of echinococcus granulosus and echinococcus multilocularis, general probes of echinococcus granulosus and echinococcus multilocularis, Taq enzyme; premixing buffer and deionized water. The nucleotide sequence of the upstream general primers of echinococcus granulosus and echinococcus multilocularis and the nucleotide sequence of the downstream general primers of echinococcus granulosus and echinococcus multilocularis are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The kit design is specific to the general primers and the probes of two hydatids; the result can be determined by detection once, thus the time is greatly saved, and detection cost is reduced.

The invention discloses an imaging model in small animal living bodies with echinococcus granulosus and a construction method thereof. The construction method comprises the steps of: feeding CF-1 male mice; carrying out in vitro culture and drug treatment on protoscolex; carrying out in vitro fluorescence imaging and fluorescence gradient analysis; pretreating mice with depilatory paste for experimental animals, injecting the abdominal cavity of each mouse with chloral hydrate anesthetic, implanting three groups of stained scoleces under Glisson capsules by injection, dividing the mice into three groups according to different groups of injected protoscolex; in a control group, injecting the livers of mice with protoscolex on which drug treatment is not carried out, collecting images every 12 hours after injection, carrying out fluorescence intensity analysis on the ROIs region, putting into a polyethylene cage after imaging, and carrying out in vitro fluorescence gradient detection in the positive control group. The construction method constructs a mouse model with echinococcus granulosus which can express luciferase, provides a platform for drug sensitive tests in living bodies with echinococcosis, and can monitor the growth and transfer of hydatid in the living model dynamically in a long term.

The invention discloses a truncated echinococcus granulosus EG95 protein tEG96 recombinant Pichia pastoris high-density fermentation process. The recombinant engineering yeast strain monoclonal EG95-1 employed by the invention is established and identified by the inventor, and is collected in China General Microbiological Culture Collection Center on February 16th, 2012 with a collection number of CGMCC No. 5764. The fermentation method provided by the invention comprises the steps that: a fermentation working seed solution is transferred into a BSM liquid culture medium; the pH value of the culture medium is regulated to 5.5, and enrichment is started; an aqueous solution containing glycerin and PTM 1 is supplemented during the enrichment process; and a solution containing a methanol solution of PTM 1 is separately added into the culture system in stages, such that protein induced expression is carried out.

The invention discloses a modification method of echinococcus granulosus EG95 protein, methods for preparing recombinant expression plasmids and recombinant engineering yeast strains by using the modified echinococcus granulosus EG95 protein, and a recombinant engineering yeast strain induction expression method. The modification method of the echinococcus granulosus major immunogenic protein EG95 comprises the steps that: on the basis of a complete EG95 protein, 23 amino acids are removed from an N- terminal, and 22 amino acids are truncated from a C- terminal. A modified tEG95 gene sequence is shown as SEQ ID No.1, and the protein sequence is shown as SEQ ID No.2. An yeast monoclonal strain EG95-1 is established and screened by using the method provided by the invention, and is collected in China General Microbiological Culture Collection Center on February 16th, 2012, with a collection number of CGMCC No.5764.

The invention discloses a preparation method of sheep echinococcosis infection-resistant gene engineering subunit vaccine. The preparation method comprises the following steps: S1, searching and downloading EG95 gene sequence as shown in SEQ ID NO.2 as well as EG95 amino acid sequence as shown in SEQ ID NO.3 of echinococcus granulosus from NCBI and performing amino acid sequence modification to obtain the modified EG95 amino acid sequence; S2, performing tandem expression on the gene sequences of a plurality of modified EG95 amino sequences through flexible linker to form recombinant multi-EG95 gene sequence; S3, cloning the recombinant multi-EG95 gene sequence to a pET28b plasmid vector, converting to Escherichia coli BL21 (DE3), and performing induction expression by a tag added fusion expression mode to obtain recombinant protein; and S4, performing protein purification on the recombinant protein to obtain the sheep echinococcosis infection-resistant gene engineering subunit vaccine. According to the preparation method, the production cost of echinotype antigen can be greatly reduced, the production process is greatly simplified and various advantages of safety, high efficiency,low cost and the like are achieved.

The invention discloses a production method of colloidal gold immunochromatography test paper strip for detecting canine Echinococcus granulosus infection. The production method comprises the following steps: cloning, expressing and purifying an EdiagA864 recombinant protein, preparing and purifying a monoclonal antibody and a rabbit polyclonal antibody, sintering a colloidal gold solution by using a trisodium citrate reducing technology, labeling the monoclonal antibody with colloidal gold to produce a colloidal gold pad, and assembling the test paper strip, and using Echinococcus granulosus EdiagA864 polyclonal antibody as adetection line and a goat anti-mouse antibody as a quality inspection line. The test paper strip has the characteristics of fastness and convenience in operation, no special devices, clear detection result, rapid judgment, and suitableness for clinic and onsite rapid diagnosis and large-scale epidemiology investigation of pastoral areas. The test paper strip has high specificity and sensitivity; and the method uses Echinococcus granulosus EdiagA864 monoclonal antibody and polyclonal antibody sandwich, the monoclonal antibody guarantees the specificity of the method, and the polyclonal antibody improves the sensitivity of the method. Compared with traditional arecoline catharsis methods, the test paper strip has the advantages of guaranteeing of the safety of detection personnel, and avoiding of egg infection.

The invention discloses a real-time quantitative PCR (Polymerase Chain Reaction) method for detecting echinococcus multilocularis and echinococcus granulosus by types based on an MGB (Minor Groove Binder) probe and a detection kit. The real-time quantitative PCR method comprises the following steps: designing primers, designing the MGB probe and selecting PCR amplification conditions; the detection kit contains an outer primer pair sequence of a PCR primer pair and an MGB type detection probe sequence. The method disclosed by the invention can be used for pertinently detecting samples containing the echinococcus multilocularis and / or the echinococcus granulosus by types and the accuracy is high; mitochondrion DNAs (Deoxyribonucleic Acid) of the echinococcus multilocularis and the echinococcus granulosus can be accurately detected by types; the sensitivity is high so that the echinococcus multilocularis and the echinococcus granulosus can be rapidly identified by types in a large batch; the operation is simple and convenient, and trace insect source mitochondrion DNAs in the samples can be amplified and the level of detecting by types is realized.

The invention discloses a loop-mediated isotheral amplification (LAMP) detection kit for detecting whether echinococcus granulosus pathogen exists in dog excrement or not and application of the kit. The kit comprises four specific primers, wherein the four specific primers comprise an upstream external primer SEQ No. 1, a downstream external primer SEQ No. 2, an upstream internal primer SEQ No. 3 and a downstream internal primer SEQ No. 4. The kit also comprises LAMP reaction liquid, a primer mixture, Bst DNA polymerase, a standard positive template and a color developing agent, so that the kit is convenient to use. The kit can quickly detect whether the echinococcus granulosus pathogen exists in the dog excrement or not, has high sensitivity and high specificity, and is easy and convenient to operate.

The invention discloses a liver echinococcus gene segment screening method, an amplification primer and a kit.The screening method comprises the following steps: eliminating an influence of a human genome and a close genetic relationship tapeworm group genome from whole genomes of echinococcus granulosus and echinococcus multilocularis; and screening to obtain a third echinococcus granulosus gene segment, a third echinococcus multilocularis gene segment and a common gene segment, and designing three types of amplification primers by using three types of the gene segments respectively. A primer pair group for detecting echinococcosis of human tissues is obtained by further screening and a kit and a use method of the kit are provided based on the primer pair group. False positive results caused by human genes or close genetic relationship tapeworm genes existing in to-be-detected tissue DNA is avoided from the source, the to-be-detected DNA aiming at the primer during design is a human tissue sample, the false negative results in clinical detection are remarkably reduced, specific primers have higher accuracy and higher specificity, and clinical use effects of the primer pair and the kit are obviously enhanced.

The invention discloses a vaccine for blocking transmission of echinococcosis pathogeny echinococcus granulosus from a source. The vaccine provided by the invention is a CTB-EgM123 vaccine or a complete set of vaccine consisting of the CTB-EgM123 vaccine and a subunit vaccine of an EgM123 protein recombinant mycobacterium smegmatis vaccine. An active ingredient of the CTB-EgM123 vaccine is a fusion protein formed by fusion of a cholera toxin B subunit and an echinococcus granulosus EgM123 protein; the active ingredient of the EgM123 protein recombinant mycobacterium smegmatis vaccine can express the recombinant mycobacterium smegmatis of the echinococcus granulosus EgM123 protein. The vaccine provided by the invention can stimulate the immune response in a host, improves the immune level of the host, enhances the intestinal mucosal immune response, and plays an important protective role in resisting infection of echinococcosis.

The invention discloses an echinococcus granulosus developmental-stage secretory protein expression gene chip. The echinococcus granulosus developmental-stage secretory protein expression gene chip is characterized in that sequences of isolated cDNA expression sequence labels of echinococcus granulosus protoscolex, vesicle, oncosphere and adult developmental stage secretory proteins are combined with vectors; and the cDNA expression sequence labels are shown in the formulas of SEQ ID No.1 to SEQ ID No.30. The echinococcus granulosus developmental-stage secretory protein expression gene chip has commercial values and scientific research values. Through the novel cDNA expression sequence labels, a genetic map is drawn. The echinococcus granulosus developmental-stage secretory protein expression gene chip has an important effect of research of secretory protein gene expression. The expression sequence labels can realize screening of targets of a cystic echinococcosis-resistant drug and through the expression sequence labels, the optimal echinococcosis-resistant drug molecules are selected out. The echinococcus granulosus developmental-stage secretory protein expression gene chip can be used for screening of a cystic echinococcosis diagnostic marker and can be used for research and development of an echinococcosis diagnostic kit having high specificity. The echinococcus granulosus developmental-stage secretory protein expression gene chip can be used for screening of cystic echinococcosis protective antigens and can be used for research and development of echinococcosis vaccines suitable for the animal husbandry and pet dogs.

The invention discloses a kit for assaying echinococcus in lesion tissue or dog feces by multiplex recombinase-aid amplification (RAA) and multiple PCR. Meanwhile, the kit is also used for assaying echinococcus granulosus G1 and echinococcus multilocularis. The kit comprises primers as follows: echinococcus granulosus specific primers shown in SEQ ID NO.1 and SEQ ID NO.2 and echinococcus multilocularis specific primers shown in SEQ ID NO.3 and SEQ ID NO.4. In addition, the invention further discloses a multiplex RAA and multiplex PCR assay method for the echinococcus granulosus G1 and echinococcus multilocularis. Various verifications prove that the kit has characteristics of rapidness, sensitivity and high specificity, complex steps for repeated assay are eliminated, two genes can be assayed simultaneously in one reaction, functions of pathogens are made clear, the workload is reduced greatly, the assay time is shortened, the assay cost is reduced, and the kit has a greater development trend.

The present invention provides an echinococcus granulosus imago diagnosis protein gene, further relates to a monoclonal antibody and a polyclonal antibody prepared by using the diagnosis gene protein so as to establish the canine echinococcus granulosus coproantigen detection method, further provides a protein gene for canine echinococcus granulosus ELISA detection and applications of the protein gene in establishment of the canine echinococcus granulosus double antibody sandwich ELISA method, and belongs to the technical field of canine disease detection.

The invention discloses a genetic engineering subunit vaccine capable of resisting goat hydatidosis infection. The genetic engineering subunit vaccine comprises a recombinant protein of which the encoding gene sequence is disclosed by SEQ ID NO:1. An EG (Echinococcus granulosus) 95 protein is optimized and is used for preparing the genetic engineering subunit vaccine capable of resisting the goathydatidosis infection, the antigenicity, the immunogenicity and the function are similar to the antigenicity, the immunogenicity and the function of a natural protein, an expression level of the obtained vaccine is high, the immunogenicity of the obtained vaccine is high, a good immune effect can be provided only by a small quantity, and the obtained vaccine does not have pathogenicity for goats.Meanwhile, the Eg 95 protein subjected to expression optimization by CHO cells and the like is adopted, a glycosylation level is high, solubility is high, an expression quantity (which can be 2-3 g / L) is high, and the genetic engineering subunit vaccine can be prepared in a bioreactor through large-scale serum-free suspension culture, and the production cost of the vaccine is greatly lowered.

The invention discloses a sheep echinococcosis ELISA antibody detection kit and a preparation method and application thereof. In the detection kit, an elisa plate is pre-coated with an echinococcus granulosus cocktail antigen; the echinococcus granulosus cocktail antigen is composed of an EPC1 recombinant antigen, an EgAgB1 recombinant antigen, an EgAgB2 recombinant antigen and an EgAgB4 recombinant antigen. The echinococcus granulosus cocktail antigen is composed of an EPC1 recombinant antigen, an EgAgB1 recombinant antigen, an EgAgB2 recombinant antigen and an EgAgB4 recombinant antigen. Thedetection kit provided by the invention can be used for rapidly detecting the echinococcosis antibody in the sheep serum, is simple to operate, short in consumed time, high in sensitivity and good inspecificity, and is convenient for simultaneously detecting a large number of samples. The sheep echinococcosis ELISA antibody detection kit provided by the invention has important application valuein echinococcosis antibody level detection and epidemiological investigation.

The invention relates to application of an inhibitor in preparation of drugs for treating cystic echinococcosis, and particularly discloses application of an inhibitor for preparing drugs for treatingcystic echinococcosis. The inhibitor is selected from the following inhibitors: a deubiquitinating enzyme inhibitor, an EGFR inhibitor or a combination thereof. The inhibitor can effectively kill echinococcus granulosus (particularly echinococcus granulosus larva), and then the cystic echinococcosis is treated.

The invention discloses application of tauroursodeoxycholic acid salt used as an active ingredient in preparation of a drug for treating echinococcosis. The tauroursodeoxycholic acid salt is used as the active ingredient for killing pathogen-echinococcus granulosus of the echinococcosis; an obvious echinococcosis protoscolex restraining and killing effect is achieved, and thus, the treatment of the echinococcosis is realized.

The embodiment of the invention discloses a bovine echinococcosis granulosa time-resolved fluorescence immunochromatographic assay test strip and a preparation method thereof, and belongs to the technical field of biological assay. A combination pad of the test strip is coated with a goat anti-bovine IgG antibody marked by time-resolved fluorescent microspheres. The nitrocellulose membrane is provided with a detection line (6) and a quality control line (7) which are separated from each other, the detection line is coated with a recombinant multi-epitope antigen, the quality control line is coated with a rabbit anti-goat IgG secondary antibody, and the recombinant multi-epitope antigen is formed by splicing seven peptide fragments (named as Eg-H1). The test strip disclosed by the inventionhas the advantages of simplicity and convenience in operation and good sensitivity and accuracy; overcomes the defects of incapability of field operation and low accuracy of ELISA, colloidal gold andother diagnosis methods, also has good specificity, provides a rapid diagnosis method for epidemiological investigation of bovine echinococcosis granulosa and diagnosis of the disease, and has wide application prospects.

The invention discloses an echinococcus granulosus recombinant protein, which is characterized in that four EgG1Y162-2 protein fragments are connected in series through a linker sequence 'GSGGSG' to form the recombinant protein; experiments show that the recombinant protein vaccine can promote maturation of dendritic cells. The action principle and effect of the recombinant vaccine EgG1Y162-2 (4) are disclosed for the first time, and a foundation is laid for preparing vaccines or diagnostic kits for preventing and treating echinococcosis of people or livestock.

The invention provides an echinococcus granulosus immunogenic protein EG95 constitutive expression vector and a pichia pastoris genetically engineered bacterium containing the expression vector. The applicant successfully realizes secretory expression of EG95 protein in a pichia pastoris constitutive expression system, the concentration of the protein expressed by a triangular flask shaking tablereaches 0.5g / L, and the purity of the target protein reaches 90% or above. Westernblotting tests prove that the expressed recombinant protein has good reactogenicity and specificity (no reaction withother recombinant protein antibodies) with existing sheep echinococcosis EG95 subunit vaccine immune positive serum. The engineered bacterium capable of efficiently and stably secreting and expressingthe EG95 recombinant protein is screened out from constructed p-GAPZ alpha A-EG95 recombinant plasmid transformed pichia pastoris SMD1168, no inducer is needed, the production period is remarkably shortened, the production cost is remarkably reduced, and the production complexity is remarkably reduced.

The invention relates to a typing detection method for applying a loop-mediated isothermal nucleic acid amplification technology to alveolar echinococcus / echinococcus granulosus and a detection kit. The typing detection method comprises designing an LAMP primer for distinguishing alveolar echinococcus / echinococcus granulosus, selecting LAMP amplification conditions and detecting an LAMP amplification product. The method can perform typing detection on a sample containing alveolar echinococcus / echinococcus granulosus, is high in accuracy, is high in sensitivity, and is simple and convenient tooperate; and amplification results can be directly observed through naked eyes.

The invention provides an echinococcosis granulosa gene engineering vaccine. The sequence of the diagnostic antigen P-29 gene of the echinococcosis granulosa Uruguay strain is retrieved from the Genbank of www.ncbi.nlm.nih.Gov, the P-29 gene is amplified through RT-PCR, with the total RNA of the echinococcosis granulosa pathogens of echinococcosis patients in Ningxia region, China as the template, and the DNA sequence of the amplified gene is consistent with that of the gene of the Uruguay strain. The cloned P-29 gene and the expression vector pET-28a are recombined through the recombinant DNA technology to transform and construct the engineered escherichia coli strain. The P-29 recombinant protein is prepared and the obtained P-29 recombinant protein immune mice are attacked by the protoscolex pathogens of echinococcosis patients in Ningxia region. Through computation in the experiments, the protective immunity of EgP-29 is 96.6%. The P-29 recombinant protein is proved to have immunology effect of the vaccines.

The invention belongs to the technical field of biotechnology, and particularly discloses an echinococcus granulosus EgTeg and EgFABP1 multi-epitope vaccine and application. Theechinococcus granulosus EgTeg and EgFABP1 multi-epitope vaccine comprises a CD8 + T cell epitope, a CD4 + T cell epitope and a B cell epitope of an EgTeg protein and an EgFABP1 protein. The invention further discloses a screening method and application of the multi-epitope vaccine, and compared with a vaccine obtained by a traditional antigen epitope screening method, after the EgTeg and EgFABP1 multi-epitope vaccine provided by the invention is inoculated, the wet weight of the echinococcus granulosus cyst in a mouse body infected by echinococcus granulosus protoscolex can be effectively reduced; and the screening time and economic cost are low, the experimental process is simplified, and the echinococcus granulosus EgTeg and EgFABP1 multi-epitope vaccine is suitable for wide screening, popularization and application of the antigen epitopes.

The invention discloses a gene engineering vaccine resistant to sheep echinococcosis infection. The vaccine is a protein rEg.P29 vaccine which is prepared by extracting RNA from a China strain of Echinococcus granulosus, obtaining Eg.P29 gene molecules through RT-PCR, building a P29 / pET28a prokaryotic expression vector and performing IPTG induced expression and recombination in Escherichia coli BL21. The vaccine is collected under CCTCCM2015425. Immunoprotection verification of an Echinococcus granulosus recombinant vaccine is performed on sheep, and immune protection efficiency reaches 94.8%. Vaccine candidate molecules are expected to be applied to preparing vaccines resistant to sheep echinococcosis infection and expected to play a great role in preventing and treating liver echinococcosis.

The invention discloses a specific detection antigen of bovine echinococcosis granulosa and application of the specific detection antigen. Specific detection antigen protein of the bovine echinococcosis granulosa is (a1) or (a2): (a1) protein of an amino acid sequence shown as SEQ ID NO.1, and (a2) protein obtained by carrying out substitution and / or deletion and / or addition of one or more amino acid residues on an amino acid sequence of the protein defined by the (a1) and with the same function. Time-resolved fluorescent microspheres are used as tracers, immunochromatographic test strips areprepared, the clinical operation is simple, the diagnosis is rapid, the sensitivity is high, and the specificity is high.

The invention relates to the technical field of biology and discloses a series of new applications of an echinococcus granulosus mitochondrion gene ND6. The echinococcus granulosus mitochondrion geneND6 is taken as molecular diagnosis marker, whether a slaughtered animal is infected with echinococcus granulosus or not is detected by PCR, high reliability, specificity and sensitivity are achieved,meanwhile, by means of direct PCR, detection efficiency can be improved, and the invention is particularly suitable for large-scale gene detection and high-throughput analysis.

The present invention provides primers for simultaneously and rapidly identifying 4 types of taenia solium larvae of cysticercus cellulosae, taenia asiatica cysticercus, cysticercus tenuicollis and echinococcus granulosus, and also provides a corresponding detection kit and a detection method. The designed specific primers for the taenia solium larvae can quickly identify 4 types of the widely prevalent taenia solium larvae in China in one PCR reaction system, overcome a problem that insect bodies are similar in forms and cannot be accurately identified by morphology, and also overcome a defect of piece weight PCR identification of a single species, besides, PCR products do not need sequencing and sequence alignment, molecular identification of the 4 types of the taenia solium larvae can be realized only according to sizes of electrophoretic fragments of the PCR products, which greatly shortens time of identification diagnosis, and the primers can be used for rapid identification diagnosis of the 4 types of taenia solium larvae and epidemiological investigation of metacestodiasis.