6 results about "In vitro transfection" patented technology

The application discloses a multifunctional multi-arm star polymer and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The functional multi-arm star polymer disclosed by the application takes an internal ring polymer as a core molecule, and a functionalized acrylate monomer is used to prepare the multi-arm star polymer through ATRP.The polymer prepared by the application has the characteristics of adjustable terminal reaction site of the core molecule, controllable and narrow molecular weight distribution of the number, length and functionalization unit of the arm molecule, excellent affinity to mRNA, and the like.The complex nanoparticles formed by the polymer and mRNA are uniformly distributed and have good stability.The in-vitro transfection result shows that the polymer can efficiently deliver mRNA in various tissue cell lines, which provides an application basis for further genetic disease, cancer gene therapy and virus vaccine development.

This invention discloses a method for screening and validating personalized neoantigens for liver cancer and its application. DNA and RNA are extracted from tumor and adjacent normal tissue samples from patients and subjected to high-throughput sequencing. Mutant peptides are screened, and the patient's HLA typing is determined. The binding affinity of the mutant peptides to HLA alleles is then evaluated, and antigen presentation ability is predicted. Candidate neoantigen peptides are comprehensively screened. The candidate neoantigens undergo coding sequence sequencing, cloning, PCR amplification, in vitro transcription, capping and tailing modifications, and encapsulation to prepare an mRNA-LNP vaccine. The obtained mRNA-LNP vaccine is then transfected into 293T cells in vitro to verify antigen expression efficiency, completing the functional quality control of the mRNA-LNP vaccine. This invention provides a highly efficient, closed-loop system for screening personalized neoantigens for liver cancer and validating vaccines, an integrated approach that directly guides mRNA vaccine construction and in vivo immunogenicity evaluation from multi-omics data screening.

The utility model discloses a micro-fluidic chip for preparing LNP, an LNP synthesis module and an automatic preparation system of mRNA vaccine and medicine. The micro-fluidic chip is provided with a bent sample introduction channel and a mixing flow channel consisting of transverse flow channels and vertical flow channels which are alternately communicated, so that a water phase and an organic phase can be automatically and fully mixed, and LNP can be prepared in a high-flux manner. According to the micro-fluidic chip, an mRNA vaccine and medicine preparation system is introduced through an LNP synthesis module, the system is also integrated with various peripheral equipment and a pipetting workstation, and complete-flow, high-throughput, rapid, integrated and automatic preparation of mRNA preparation and purification, LNP encapsulation, in-vitro transfection and cellular level characterization in mRNA vaccine and medicine research is completed for the first time; and a foundation is laid for realizing standardization of a whole mRNA preparation process.

The invention relates to the field of gene engineering, in particular to a vector and a method for transfecting non-activated T cells in vitro. The invention discloses a vector and a method for contacting a non-activated T cell and transfecting the non-activated T cell by using the vector. The surface of the vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof, wherein the antibodies or the antigen-binding fragments thereof and / or the ligands or the receptor-binding fragments thereof are specifically bound with an endocytosis receptor of the non-activated T cell; the vector can carry nucleic acid for coding the CAR, and enters and effectively transfects the non-activated T cells through the endocytosis caused by the endocytosis receptor specifically combined with the non-activated T cells, so that the technical problem that the non-activated T cells are difficult to effectively transfect in the process of preparing the CAR-T cells in vitro is effectively solved, and the CAR-T cells can be used for preparing the CAR-T cells. Further, the cost for preparing the CAR-T cells is reduced, and the preparation period is shortened.

The application discloses a kind of automatic high-throughput preparation mRNA vaccine and medicine method.The method uses following microfluidic chip and preparation system.Microfluidic chip has curved sample channel and mixed flow passage of alternating communication transverse flow channel and vertical flow channel, can be fully mixed and high-throughput preparation LNP to water phase and organic phase.The microfluidic chip is introduced mRNA vaccine and medicine preparation system by LNP synthesis module, the system is also integrated with a variety of peripheral equipment and pipetting workstation, for the first time, from mRNA vaccine and medicine research mRNA preparation and purification, LNP encapsulation, in vitro transfection, cell level characterization The whole process of high-throughput, fast integrated automatic preparation is completed, and foundation is laid for realizing the standardization of mRNA whole preparation process.

The application discloses an in vitro transcription system of a transformed cell of a circular theileria and a construction method thereof, and the in vitro transcription system comprises IVT-EGFP-mRNA, and the structure is 5'UTR-Cla1-Kozak-EGFP-PacI-3'UTR-polyA(120). The mRNA coding the EGFP gene is synthesized by in vitro transcription, and is subjected to capping, purification and agarose gel electrophoresis analysis. Finally, the expression of the target gene in the TaNM1 cell is verified by in vitro transfection test and indirect immunofluorescence technology. The result shows that the green fluorescent protein EGFP is successfully expressed by the in vitro transcription system coding the EGFP, which indicates that the system for transfecting the EGFP gene into the transformed cell TaNM1 of the circular theileria by the in vitro transcription method is successfully established, and the system provides important reference value for the design and research and development of the RNA vaccine of theileria annulata.