48 results about "Bimolecular fluorescence complementation" patented technology

The invention relates to the field of plant antiviral genetic engineering, and provides a plant antiviral new target. On the basis of tobacco vein banding mosaic virus infection cloning, photosystem II oxygen-evolving complex protein PsbO1 is screened through immunoprecipitation and mass spectrography, and a PbsO1 gene is obtained through reverse transcription PCR amplification. Immunoprecipitation and bimolecular fluorescence complementation prove that the PsbO1 can interact with the tobacco vein banding mosaic virus 6K2. Reduction in expression of the PsbO1 gene enables an infected plant to generate resistance to the tobacco vein banding mosaic virus and potato virus Y.

The invention relates to the field of genetic engineering and provides construction methods and applications of two PVX (potato virus X) over-expression vectors and one BiFC (bimolecular fluorescence complementation) vector. A genome of a PVX 1985 isolate is cloned to a downstream 35S promoter through genetic recombination, obtained infectious clone can infect solanaceae crops such as tobacco, tomatoes, potatoes and the like through agrobacterium infiltration. Infectious clone is reconstructed, and the over-expression vectors capable of effectively expressing one or two types of heterologous protein in a host plant body as well as the BiFC vector capable of identifying interactions between protein are obtained.

The invention provides a protein interaction detection method with low false positive rate, relates to a detection system and belongs to the field of a molecular biological technology. According to the method, two generally needed bimolecular fluorescence complementation (BiFC) detection systems based on single expression plasmid vectors are built in a dual expression plasmid vector system by a BiFC technology based on fluorescent protein, so the false positive rate of the BiFC detection method in research on protein-protein interaction can be reduced obviously, and quantitative analysis can be realized. The method also can be applied to screening research on unknown protein-protein interaction based on a gene library and detection on protein-protein interaction in living animals.

The invention discloses a method for screening interacting protein based on the bimolecular fluorescence complementation (BiFC) technique. The method comprises the steps of 1, dividing YGFP into an N end (sequence 1, 1-157) and a C end (sequence 1, 158-238); connecting a coding gene of target protein A to the 3' terminal of an N-end coding gene through a connecting sequence (connecting peptide as shown in coding sequence 2), so as to form a fusion gene fragment A; connecting a coding gene of target protein B to the 5' terminal or 3' terminal of a C-end coding gene through the connecting sequence, so as to form a fusion gene fragment B; and 2, importing the fusion gene fragments A and B into receptor yeast cells, conducting culture to obtain transgenic yeast cells, and detecting whether the transgenic yeast cells generate green fluorescence, wherein an interaction or candidate interaction relation exists between the target protein A and the target protein B if yes, and no interaction or candidate interaction relation exists between the target protein A and the target protein B if not. The BiFC technique has high sensitivity and specificity, and is suitable for high-throughput protein-protein interaction screening in yeast.

The invention discloses imaging and tracking of interaction between proteins in living cells by utilizing bimolecular fluorescence complementation technology based on a self-linkage label, and revealsan interaction relation between the proteins and explores that a dynamic behavior of an interaction compound is already become a hot spot for proteomics research. The interaction between proteins forms a basis of cellular activity. An existing method for researching the interaction of the proteins do not simultaneously have the characteristics of living cells, high temporal-spatial resolution, single molecules and the like, and therefore, a new method for making and tracking the interaction between the proteins needs to be developed. The invention discloses a method for novel marking the interaction compound of the proteins by adopting the bimolecular fluorescence complementation based on the self-linkage label. The self-linkage label is split into two parts at the proper site, and two proteins having the interaction effect are fused respectively, and due to the interaction of the two proteins, the split self-linkage labels are pulled spatially and closely so as to form a complete self-linkage label; by adding dyes, the interaction compound can give out fluorescent light. The novel marking method utilizing the bimolecular fluorescence complementation based on the self-linkage label is widely applied to cell biology, and single molecular horizontal detection and tracking on the interaction of the pair of proteins in the cells in a high temporal-spatial resolution manner can berealized.

The invention provides a novel expression vector for bimolecular fluorescence complementation (BiFC) research. Expression cassettes of 2 segments of a fluorescent protein are integrated onto the vector; and gene sequences of 2 target proteins can be simultaneously cloned to the vector by one-step reaction (Golden Gate cloning or closed-loop Gibson splicing process) and respectively form fusion expression with the 2 segments of the fluorescent protein. Therefore, when the vector is used for BiFC research, only one vector is needed and introduced into cells, thereby greatly facilitating the operation of the plant BiFC experiment. The vector is compatible with the two novel cloning methods of Golden Gate cloning or closed-loop Gibson splicing. The vector is a high-copy plant expression vector containing T-DNA sequence, and can be simultaneously used for instantaneous and stable expression of plants.

The invention provides an ST2 detection kit based on a bimolecular fluorescence complementation technology and a preparation and use method thereof. The kit includes a fluorescent protein N-end segment resistant to ST2 antibody coupling and a fluorescent protein C-end segment resistant to ST2 antibody coupling. The invention also discloses the preparation method of the ST2 diagnosis kit based on the bimolecular fluorescence complementation technology. The preparation method includes preparation of the fluorescent protein N-end segment resistant to ST2 antibody coupling and preparation of the fluorescent protein C-end segment resistant to ST2 antibody coupling. Finally, the invention also discloses the use method of the kit. The kit has the advantages of being easy to operate, wide in linear range, excellent in specificity, free of cleaning, high in accuracy and the like and can provide convenience for clinical detection and usage; the kit is used for predicting unfavorable prognosis cardiovascular events happening to patients suffering from acute myocardial infarction (AMI) and monitoring the capacity of evaluating the coronary artery disease degree; by means of the kit, the clinical evaluation and risk stratification accuracy of clinicians on AMI patients or AMI high-risk groups are improved, and therefore the kit has a considerable market value.

The invention discloses a BiFC (Bimolecular Fluorescence Complementation) intracellular detection method and system for indicating NrF2 (Nuclear factor erythroid 2-related factor)-Keap1 (Kelch-like ECH associated protein 1) interaction. A recombinational expression vector constructed by Nrf2 protein which is fused with eGFP N-end 158-locus amino acid and a Keap1 protein coding gene which is fused with eGFP C-end 159-locus amino acid is provided; meanwhile after cells are transfected, a complete fluorescent protein is reconstructed through fused N-end and C-end of fluorescent protein eGFP under the expressed NrF2-Keap1 interaction. By adopting the BIFC system, cells are transfected instantly or constructed into a stable expression cell line, a specific signal generated in a cell nucleus can be observed, the signal can be displayed specifically and reflect the activity state and change of a Nrf2-Keap1 signal path in a living cell, and a foundation is laid for screening of small molecule compounds capable of influencing Nrf2-Keap1 interaction and antitumor medicaments by detecting BiFC fluorescence change.

The invention provides a NGAL diagnostic kit based on bimolecular fluorescence complementary technology. The kit comprises an anti-NGAL antibody conjugation fluorescent protein N-terminal fragment andan anti-NGAL antibody coupled fluorescent protein C-terminal fragment. The present invention also discloses a preparation method of the NGAL diagnostic kit based on the bimolecular fluorescence complementary technology. The preparation method comprises preparation of the anti-NGAL antibody coupled fluorescent protein N-terminal fragment and preparation of the anti-NGAL antibody coupled fluorescent protein C-terminal fragment. The present invention also finally discloses a use method of the kit. The kit has the advantages of fast detection, simple operation, good specificity, no cleaning, highprecision, and the like, is convenient for clinical detection, can be used for monitoring of acute kidney injury, has great significance for preventing the acute kidney injury, and has great market value.

The invention provides an IL-6 diagnostic kit based on a bimolecular fluorescence complementary technology. The kit comprises an anti-IL-6 antibody coupled fluorescin N-terminal fragment and an anti-IL-6 antibody coupled fluorescin C-terminal fragment. The invention further discloses a preparation method of the IL-6 diagnostic kit based on the bimolecular fluorescence complementary technology. Themethod comprises the steps of preparing the anti-IL-6 antibody coupled fluorescin N-terminal fragment and preparing the anti-IL-6 antibody coupled fluorescin C-terminal fragment. Finally, the invention further discloses a use method of the kit. The kit provided by the invention has the advantages of simplicity in operation, wide linear range, good specificity, free from cleaning, high accuracy and the like, is convenient in clinical detection use and can be used for improving the accuracy of sepsis diagnosis when the kit is applied to infection monitoring, thereby being extensively popular tothe market and having a great market value.

The invention provides a cTnT diagnostic kit based on a bimolecular fluorescence complementary technology. The kit comprises an anti-cTnT antibody coupled fluorescin N-terminal fragment and an anti-cTnT antibody coupled fluorescin C-terminal fragment. The invention further discloses a preparation method of the cTnT diagnostic kit based on the bimolecular fluorescence complementary technology. Themethod comprises the steps of preparing the anti-cTnT antibody coupled fluorescin N-terminal fragment and preparing the anti-cTnT antibody coupled fluorescin C-terminal fragment. Finally, the invention further discloses a use method of the kit. The kit provided by the invention has the advantages of rapidness in operation, good specificity, good precision, high accuracy and the like, is convenientin clinical detection use and can be used for improving the accuracy of acute myocardial injury diagnosis when the kit is applied to myocardial injury monitoring, thereby having a great market value.

The invention provides a KIM-1 diagnostic kit based on a bimolecular fluorescence complementary technology. The kit comprises an anti-KIM-1 antibody coupled fluorescin N-terminal fragment and an anti-KIM-1 antibody coupled fluorescin C-terminal fragment. The invention further discloses a preparation method of the KIM-1 diagnostic kit based on the bimolecular fluorescence complementary technology.The method comprises the steps of preparing the anti-KIM-1 antibody coupled fluorescin N-terminal fragment and preparing the anti-KIM-1 antibody coupled fluorescin C-terminal fragment. Finally, the invention further discloses a use method of the kit. The kit provided by the invention has the advantages of rapidness in operation, good specificity, good precision, high accuracy and the like, is convenient in clinical detection use and can be used for improving the accuracy of acute renal injury diagnosis when the kit is applied to acute renal injury monitoring, thereby having a great market value.