34 results about "Tetrahymena thermophila" patented technology

DNA constructs comprise a first exon sequence of nucleotides encoding a first peptide or polypeptide, a second exon sequence of nucleotides encoding a second peptide or polypeptide and a third sequence of nucleotides between the first and second sequences encoding a heterologous intron, for example that of Tetrahymena thermophila nuclear pre-rRNA, between RNA splice sites and a site-specific recombination sequence, such as loxP, within the intron, the exons together encoding a product peptide or polypeptide. Such constructs are of use in methods of production of peptides or polypeptides, transcription leading to splicing out of the intron enabling translation of a single chain product peptide or polypeptide. Isolated nucleic acid constructs consisting essentially of a sequence of nucleotides encoding a self-splicing intron with a site-specific recombination sequence within the intron, for use in creation of constructs for expression of peptides or polypeptides, are also provided.

The invention discloses a tetrahymena transgenic carrier containing HSP70 promoter and GFP and preparation method and use thereof. Plasmid T-GFP is constructed by using plasmid rDNA-GFP in the methods such as enzyme cutting and PCR enzyme cutting site mutation; thermophile tetrahymena genome DNA is used for constructing plasmid T-HSP702 in the methods such as PCR augmentation and clone; Plasmids T-GFP, T-HSP702 and pD5H8 are used for construction and finally obtaining plasmid HSP702-GFP-pD5H8 by enzyme cutting and connection. The carrier contains HSP702 promoter, thereby being capable of realizing high-efficient expression of exogenous gene; the carrier contains replaceable exogenous gene gfp so that an exogenous gene expression system is realized for tetrahymena by replacing different exogenous genes; with the sieving effect of paromomycin medicine, the carrier replaces most rDNAs originally in the tetrahymena so that the exogenous genes are largely increased in the tetrahymena so as to realize genetic transformation of target genes. The method in the invention is easy and has convenient operation, and the carrier can be used for detection of environmental tributyl tin in transfection in tetrahymena.

The ciliated protozoan Tetrahymena exemplifies a recombinant system for the expression of heterologous nucleic acids, preferably on the plasma membrane surface. Integration of a heterlogous nucleic acid into the β-tubulin gene, BTU1, of a paclitaxel-sensitive T. thermophila mutant that possesses btu1-IK350M β-tubulin allele allows screening for transformants using negative selection, as transformants have restored paclitaxel resistance. Transgenic ciliated protozoa of the invention can serve as live vaccines. For example, transgenic Tetrahymena expressing Ichthyophthirius multifiliis i-antigen protein on their surface are effective vehicles for vaccination of freshwater fish against infection by I. multifiliis.

The present invention relates to a method for degrading crude oil with protozoan infusorian. The method of the invention is characterized by comprising the following steps: A. separating and purifying infusorian population from the environment; B. domesticating the infusorian population; C. after domesticating, degrading the crude oil by the infusorian population; D. drawing an infusorian population growth curve; and F. after cultivating for 7 days, measuring the crude oil content changing of the control group and the experimental group and calculating the degration rate of the raw oil. The invention firstly finds that the protozoan tetrahymena and colpoda have strong degration capacity for the crude oil. The degration rates can respectively reach up to 68.08% and 83.06% in highest. Furthermore the infusorian has the advantages of: capacity for identifying species through an optical microscope, simple and convenient separation and purifying process, and easy cultivation. As the crude oil degration organism, the infusorian can greatly promote the control of crude oil and the microorganism exploitation of the raw oil and has particular use value for restoring the crude oil pollution through the biological method.

The invention relates to a preparation method for subunit vaccines of Cryptocaryon irritans and application of the subunit vaccines of the Cryptocaryon irritans. Approval application and industrialization of the whole organism vaccines of the Cryptocaryon irritans encounter a great challenge, mostly because the parasites cannot be cultured in vitro and only breed on fish bodies, enough Cryptocaryon irritans bodies for producing the vaccines are difficult to obtain, most importantly, the normative production process and the quality standard are difficult to make, and thus, people are forced to conduct research in a deeper level to develop genetic engineering vaccines. Candidate genes of surface proteins C1 are screened from a transcriptome database of the Cryptocaryon irritans, and recombination proteins are expressed on tetrahymena, and accordingly the vaccines are prepared for immunizing seawater fishes; and by detecting the immunogenicity and protectiveness of the vaccines, effective protective antigenic genes of the Cryptocaryon irritans are screened.

The invention relates to an organic arsenic feed additive discharge pre-alarming monitoring method in a water environment system, belonging to the environmental monitoring technical field, comprising the following steps: preparing tetrahymena culture medium; preparing arsanilic acid standard sample solution/roxarsone standard sample solution; collecting the actual water sample and separating and extracting the organic arsenic feed additive compound; inoculating the tetrahymena into the prepared culture medium and then subpackaged the inoculated culture medium into the sample culture tubes; respectively adding the organic arsenic feed additive standard solution into the sample culture tubes and respectively measuring the corresponding tetrahymena cell density using absorptometry; making the actual water sample intervening the corresponding tetrahymena biological model and analyzing and determining the corresponding tetrahymena cell density; based on the comparison of the tetrahymena cell density and the contrast of the corresponding tetrahymena cell density after the actual water sample intervention, to obtain the concentration range of the pollutant presence, thereby pre-alarming the discharge condition of the organic arsenic feed additive in the corresponding actual water environment.

The invention relates to a method for evaluating the biological security of solubilizing auxiliary materials. In the method, tetrahymena thermophile is taken as a model, a microcalorimetry is combined, the influence on the growth metabolism of the tetrahymena thermophile from the solubilizing auxiliary materials is inspected, and the biological security of the solubilizing auxiliary materials is evaluated. By carrying out information analysis on a thermal activity spectrum with integral characteristics and adopting characteristic parameters that reflect different growth metabolism stages, the invention inspects the influence on the growth metabolism of the tetrahymena thermophile from the solubilizing auxiliary materials totally, objectively and quantitively, evaluates the biological security of the solubilizing auxiliary materials, is simple in operation and is beneficial to the detection normalization.

The present invention provides a method for identifying nanogold in tetrahymena thermophila in situ by transmission electron microscope and energy spectrum, which is characterized by culturing the tetrahymena thermophila in a gold-containing liquid culture medium for a period of time, cutting into ultrathin sections of 50-70 nm thick with an ultra microtome after the steps of fixation, dehydration, infiltration and embedding, then dredging and drying, dyeing by uranyl acetate and lead citrate, watching under the transmission electron microscope, and confirming by the energy spectrum after watching particles. The invention can eliminate the interference of other impurity nanoparticles, achieve in-situ observation of nanogold particles in tetrahymena thermophila, and provide an effective analysis method for understanding the cycle metabolic process of nano-particles in vivo and evaluating the biotoxicity of the nano-particles.

The invention discloses a Tetrahymena expression vector for introducing exogenous genes in one step, a construction method and application. Using the plasmids HSP702-GFP and THX-HGFP, the intermediate vector TH-HGFP was constructed by PCR and enzyme-cut ligation; using the plasmids TH-HGFP, PET23a-GST and pCDNA3.1-FLAG, by PCR and enzyme-cut ligation, etc. Construct intermediate vectors TH-HIS-HGFP, TH-GST-HGFP, and TH-FLAG-HGFP; use the above intermediate vectors and plasmid pD5H8 to construct Tetrahymena expression vectors: pD5H8-HGFP, pD5H8 -HIS-HGFP, pD5H8-GST-HGFP, pD5H8-FLAG-HGFP. This series of vectors all contain the green fluorescent screening marker HGFP, which can be used for high-throughput screening of recombinants; this series of vectors can replace the cassette structure HGFP, both sides of which contain reverse I-SceI rare enzyme cutting sites, and introduce foreign genes In this case, the foreign gene fragment with the I-SceI linker is connected with the series of vectors treated with I-SceI in one step, and the Tetrahymena expression vector containing the foreign gene can be obtained. The operation of the invention is convenient and fast, and the high-efficiency expression of the exogenous gene can be realized after the Tetrahymena expression vector introduced with the exogenous gene is transfected into the Tetrahymena.

The present invention relates to heat-inducible promoters of the heat shock protein family of the ciliate Tetrahymena thermophila, especially a promoter having the nucleotide sequence of Seq. ID No. 1 or promoter-effective fragments thereof.

Furthermore the use of heat-inducible promoters of the present invention for the expression of homologous and/or heterologous proteins in the ciliate Tetrahymena thermophila is claimed.

The invention discloses a Tetrahymena expression vector of chitinases and its application in expressing chitinases. The invention provides an expression cassette, which includes an HSP70-2gene promoter as shown in the 9th-1126th nucleotides from the 5' terminal of sequence 1, an encoding gene of chitinases in sequence 2 and an HSP70-1 gene termination signal as shown in the 3835th-4312th nucleotides from the 5' terminal of sequence 1. The invention also provides a recombinant plasmid containing the expression cassette. When transferred to a Tetrahymena through electroporation, the recombinantplasmid can, under the drug screening effect of paromomycin, replace most of the original rDNA in the Tetrahymena, and multiply an exogenous gene in the Tetrahymena massively, thus realizing genetic transformation of a target gene. Under heat shock conditions, the HSP70-2 strong promoter can start the high level expression of CBD3 gene, and the expressed chitinases have bioactivity (able to hydrolyze chitin). In conclusion, the expression vector of the invention has wide application prospects.

Tetrahymena is used as a host cell in a biological assay for identification of inhibitors of tubulin glutamylases.

The invention discloses a site-specific recombination-based tetrahymena thermophile expression carrier and construction and application thereof. The carrier is constructed by comprising the following steps: with plasmid pNeo4 as a skeleton, and combined serially connected HA tag sequence, restriction enzyme digestion site and tetrahymena thermophile ACTIN1 gene transcription termination sequence as modules, digesting and inserting module synthesis and digestion sites into 5'-terminal polyclonal digestion site of pNeo4, so as to obtain an expression carrier pNeo4-3HA. In use, the carrier is amplified to obtain recombinant homologous arms, namely target gene coding region C-terminal sequence and 3' flanking sequence thereof, which are subjected to enzyme-cut and connection into the 5' upstream and 3' downstream polyclonal digestion site of pNeo4-3HA. The obtained construction body is subjected to transformation of tetrahymena thermophile and resistance screening to obtain an expression mutant cell line. The carrier realizes the purpose that the C-terminal expresses the endogenous gene of a fusion tag under the regulation of self starters, the operation is simple and rapid, the immunofluorescence localization sensitivity can be enhanced through 3 HA tags, and the site-specific recombination-based tetrahymena thermophile expression carrier can also be used for protein affinity purification and protein interaction studies.

A Stylonychia lemnae pure culture system and a preparation method and a culture method thereof relate to a pure culture system and a preparation method and a culture method thereof. The invention aimsto solve the problem that there is no effective culture system for aseptic pure culture of Stylonychia lemnae at present. The culture system of the invention is composed of amino acid, nucleic acid composition, vitamins, inorganic component, organic acid and glucose. By using a Tetrahymena thermophila pure medium as the reference and through optimization of composition ratio thereof, aseptic pureculture of Stylonychia lemnae is realized. By optimizing the culture condition and establishing an efficient economic cell pure culture system, enlarged pure culture theoretical basis and technical support will be provided for the molecular biology study of Stylonychia lemnae. The invention is applied in the field of Stylonychia culture.

Tetrahymena is used as a host cell in a biological assay for identification of modulators of tubulin ligases.

The invention discloses a reagent for rapidly fixing tetrahymena thermophila for cell counting. The reagent is an aqueous solution of ethanol. The reagent has the beneficial effects that the time forfixing tetrahymena thermophila cells is short, the tetrahymena thermophila cells can be completely fixed within 5 minutes, and particularly, the reagent is colorless and can do no contamination to instruments. A 75% ethanol solution is a common sterilization reagent and is safe for human bodies.

The invention provides a biological toxicity detection method for coal chemical industry wastewater and belongs to the technical field of biological toxicity detection, aiming at solving the problemsthat an existing water quality biological toxicity detection method is not applicable to biological toxicity detection of the coal chemical industry wastewater. The biological toxicity detection method adopts tetrahymena as a toxicity testing specie; the tetrahymena commonly exists in the coal chemical industry wastewater and has certain adaptability on biological toxicity substances and the sensitivity of the tetrahymena is not too high, so that the biological toxicity detection device for the coal chemical industry wastewater, provided by the invention, is applicable to detection of the coalchemical industry wastewater. On the other hand, compared with green algae, daphnia magna or zebra fish, the tetrahymena has the advantages of easiness for culturing and short reproductive period. Therefore, compared with an existing water quality biological toxicity detection method based on the green algae, the daphnia magna or the zebra fish, the biological toxicity detection method for the coal chemical industry wastewater has the advantages that a detection period is relatively short and the device is easily popularized and generalized in the field of the biological toxicity detection ofthe coal chemical industry wastewater.

Methods and compositions are provided for studying and targeting RNA structures. In particular, antibodies and antibody fragments that contain an Fab were produced. Phage display libraries that were engineered to produce RNA-specific antibody and antibody fragments were made. Antibodies, or fragments thereof, that have at least one Fab that specifically binds tRNA_i^Met, the P4-P6 domain of Tetrahymena Group I intron, the glycine riboswitch from Vibrio Cholerae, or the glycine riboswitch from Fusobacterium Nucleatum were identified. The production of these RNA-specific antibodies and antibody fragments can be used to study the structure of the RNA as well as to treat diseases with known RNA targets. For example, an Fab specific to tRNA_i^Met was produced that can be used to treat cancer in subjects where tRNA_i^Met is overexpressed.

The invention relates to a method for expressing duck-sourced avian influenza virus NS1 protein and application of the method. The method comprises the following main steps: (1), constructing a tetrahymena expression vector pD5H8-NS1; (2), constructing and screening a tetrahymena recombinant cell strain; and (3), performing induced expression and identification on the NS1 protein. The method for expressing the duck-sourced avian influenza virus NS1 protein is applied to preparation of an avian influenza virus identifying diagnostic kit and preparation of a vaccine; a tetrahymena expression system is selected as an expression system of the duck-sourced avian influenza virus NS1 protein, and can efficiently and stably express the duck-sourced avian influenza virus NS1 protein; a transgenic expression vector pD5H8 used by the selected tetrahymena cell expression system contains a high-efficiency metallothionein gene MTT5 promoter, so that an important guarantee is provided for efficient expression of a foreign gene in tetrahymenon.