13 results about "In vitro recombination" patented technology

The invention discloses radix tetrastigme flavone synthase and application thereof. The radix tetrastigme flavone synthase comprises an amino acid sequence as shown in SEQ ID No. 2. The key enzyme-flavone synthase gene (ThFNS) related to synthesis of apigenin in radix tetrastigme is cloned for the first time, in-vitro recombinant protein expression and enzymatic reaction system verification show that flavone synthase can catalyze naringenin to generate apigenin, and technical support is provided for follow-up metabolic engineering modification for an apigenin biosynthetic pathway.

This application relates to the field of maize technology, specifically to the ZmPAR1 gene and its applications. A gene encoding a nucleoside kinase was discovered in the maize genome. This nucleoside kinase was obtained through in vitro recombinant expression of the encoded gene. Testing revealed that this nucleoside kinase exhibits binding activity to various nucleoside substrates and can catalyze the synthesis of monophosphate nucleotides from nucleosides. This gene influences maize plant height, leaf angle, ear height, leaf length, and leaf width. Knocking down or overexpressing this gene can yield new maize materials, providing technical support for the breeding of new maize materials and the construction of germplasm resources.

The invention relates to a recombinant hybrid protein REC2z with a structure as defined in the description, recombinant DNA sequences encoding this protein, vectors containing the recombinant DNA sequences, and eukaryotic and prokaryotic expression systems using the in vitro recombinant DNA sequences described below. The invention also relates to the use of the recombinant hybrid protein REC2z as a component of bioink for bioprinting and also as a component of biomaterials. In addition, the invention relates to the use of recombinant DNA sequences, expression vectors and expression systems to produce the recombinant hybrid protein REC2z. The invention finds applications in bioprinting, medicine and cell and tissue engineering.

The invention discloses a method for synthesizing and assembling large-fragment DNA, and relates to the technical field of biology, the method comprises the following steps: S01, sequence splitting: splitting target large-fragment DNA into N small-fragment DNA with the length of 3000-5000bp, designing a 25-40bp overlapping region between adjacent fragments, and respectively introducing 30bp complementary sequences homologous with a linearized vector into the head fragment and the tail fragment; s02, in-vitro recombination: mixing the small-fragment DNA and a linearized vector according to the molar ratio of 1: 1 (inter-fragment) and 3: 1 (total fragment: vector), and adding recombinase to carry out Gibson homologous recombination; and S03, rolling circle amplification: taking a recombinant product in the step S02 as a template, adding a primer Primer-F and Phi29 DNA polymerase, and amplifying at 30 DEG C for 5-6 hours. And adding a specific reverse primer to carry out second-round amplification by taking a first-round rolling amplification product as a template, and cyclizing through enzyme digestion and connection. According to the method, the large-fragment plasmid is enriched through two rounds of phi29 polymerase amplification, the conversion efficiency is improved, a complex template can be unwound through the strong strand displacement activity of the phi29 polymerase, the single extension reaches 100 kb, the fidelity is 1000 times higher than that of Taq enzyme, and preparation of the high-quality large-fragment plasmid is ensured.

This application discloses an in vitro reconstructed vaginal mucosa model, its construction method, and its application. The construction method includes an improved culture medium: HVU medium is added to immortalized vaginal epithelial cell line VK2 / E6E7, which is then seeded into Transwell chambers and cultured. The HVU medium on the surface of the culture in the Transwell chambers is aspirated, and the Transwell chambers are raised to the air-liquid interface for air-liquid surface culture. The culture medium is then replaced with HVA medium to obtain the vaginal mucosa model. This method improves the stability of cells during in vitro culture and constructs an in vitro reconstructed vaginal mucosa model with a tissue structure highly similar to human vaginal mucosa (possessing a complete stratified squamous epithelial structure and normal intercellular connections), thereby enhancing the model's physiological functional simulation.

The application discloses a method for preparing recombinant plant lactobacillus over-expressing GAPDH and application thereof, and comprises the following steps: designing specific primers according to the sequence of a GAPDH gene, amplifying a gene fragment containing FLAG; inserting the gene fragment into a constitutive shuttle expression vector pMG36e to construct an in-vitro recombinant expression plasmid; transforming the recombinant expression plasmid into MC1061F competent cells by a heat shock transformation method and then amplifying the recombinant expression plasmid; transforming the amplified recombinant expression plasmid into wild-type competent plant lactobacillus by an electroporation method to prepare the recombinant plant lactobacillus over-expressing GAPDH. E. coli The prepared recombinant plant lactobacillus over-expressing GAPDH is used for improving the speed and capacity of biofilm formation and enhancing the intestinal mucosal adhesion and colonization capacity. In the experiments for determining the biofilm amount and the adhesion of the enterocyte of lateolabrax japonicus, it is proved that the over-expression of GAPDH can enhance the adhesion and colonization capacity of the plant lactobacillus LP-PO23.

The present invention discloses a multi-enzyme cascade catalytic reaction system for synthesizing trehalose-6-phosphate and its application, belonging to the field of biocatalysis technology. The present invention constructs a multi-enzyme cascade catalytic reaction system containing maltose phosphorylase, polyphosphate glucokinase, trehalose-6-phosphate phosphorylase, and polyphosphate kinase through in vitro recombinant expression of the enzymes. The multi-enzyme cascade catalytic reaction system catalyzes the synthesis of trehalose-6-phosphate from the raw material maltose. The method of the present invention has a high raw material utilization rate, and the conversion efficiency of maltose to the target product trehalose-6-phosphate reaches 90%, which is the highest level currently reported domestically and internationally, laying the foundation for the industrial synthesis of trehalose-6-phosphate.

The application relates to the technical field of biological medicine, in particular to preparation and application of an anti-HPV6 virus L1 murine monoclonal antibody; the anti-HPV6 virus L1 murine monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1; and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2. The murine monoclonal antibody is a unique single-chain antibody, and can be specifically combined with HPV6 virus L1. Western blot experiment results show that the HRP-labeled murine monoclonal antibody recombined in vitro is combined with HPV6 virus L1 to form obvious bands, and the murine monoclonal antibody has the characteristic of targeting HPV6 virus L1.

The application discloses a banana fusarium wilt gene knockout mutant back-donation vector constructed by using an in-vitro recombination technology and application thereof, and the construction steps of the back-donation vector are as follows: 1) replacing resistance genes on an initial vector with HYG resistance genes or NEO resistance genes respectively to construct an intermediate vector; 2) amplifying a ptef1 promoter sequence, a 3GFP sequence and a terminator sequence; and 3) after double enzyme digestion of the intermediate vector into a linearized vector, in-vitro recombination is carried out on the linearized vector, the ptef1 promoter sequence, the 3GFP sequence and the terminator sequence to construct the back-donation vector. The back-donation vector constructed by the application does not need in-vivo recombination transformation of yeast, avoids the cumbersome in-vivo recombination step of yeast, is simple to operate and reduces time. The back-donation vector is higher in gene expression, the back-donation strain can express a more intense fluorescent signal, and is more favorable for subcellular localization observation.

The invention provides an anti-human PD-1 monoclonal antibody with agonistic activity or an antigen binding fragment thereof and application of the anti-human PD-1 monoclonal antibody. The anti-human PD-1 monoclonal antibody with agonistic activity or the antigen binding fragment thereof is characterized in that a mouse is immunized through in-vitro recombinant expression of protein antigens of human PD-1 Fc and his tags, spleen, bone marrow and lymph node cells of the mouse are collected, and plasma cells of a secreting target cell binding antibody are enriched by adopting a microfluidic droplet sorting system; carrying out single-cell library construction on plasma cells, and carrying out NGS sequencing to obtain antibody sequences naturally paired with light and heavy chains; the method comprises the following steps: selecting an antibody sequence to carry out mammalian cell high-throughput expression, carrying out ELISA detection, flow detection and reporter gene cell line verification, selecting a representative antibody to carry out animal in-vivo experiment verification, and carrying out screening verification to obtain a series of anti-human PD-1 monoclonal antibodies with agonistic activity or antigen binding fragments thereof. The polypeptide has high affinity and high specificity, and has good antigen-antibody binding activity and agonistic activity on the cellular level.

The present invention discloses ToNK-lysin, an antimicrobial peptide from Takifugu obscurus, whose amino acid sequence is shown in SEQ ID NO. 1. The present invention utilizes in vitro recombinant expression technology to obtain ToNK-lysin, an antimicrobial peptide from Takifugu obscurus. This recombinant protein has no cytotoxic effect on pufferfish head kidney cells and can significantly eliminate Vibrio harveyi in pufferfish. Furthermore, the ToNK-lysin recombinant protein can degrade the genomes of Escherichia coli and Vibrio harveyi and significantly inhibit their growth. As an effective immune effector molecule, ToNK-lysin has potential applications in the development of antimicrobial drugs, new immune preparations, and feed additives.

The invention discloses a recombinant lactobacillus plantarum method for overexpressing GAPDH and application of the recombinant lactobacillus plantarum, and the method comprises the following steps: designing a specific primer according to a GAPDH gene sequence, and amplifying a gene segment containing FLAG; inserting the gene segment into a constitutive shuttle expression vector pMG36e to construct an in-vitro recombinant expression plasmid; transferring the recombinant expression plasmids into E.coli MC1061F competent cells by using a heat shock conversion method, and carrying out mass amplification; and transferring the amplified recombinant expression plasmid into wild competent lactobacillus plantarum by an electric shock conversion method to prepare the recombinant lactobacillus plantarum for overexpressing the GAPDH. The constructed overexpression recombinant lactobacillus plantarum is used for improving the forming speed and capacity of a biological membrane and enhancing the intestinal adhesion colonization capacity. Experiments for measuring the biofilm amount and the intestinal epithelial cell adhesion of paralichthys olivaceus prove that the adhesion and colonization capability of the lactobacillus plantarum LP-PO23 can be enhanced by overexpression of the GAPDH.

The disclosure discloses a method for preparing a multisubunit SCF E3 ligase with a fusion protein through in vitro reconstitution, and a use of the multisubunit SCF E3 ligase. The method for preparing a multisubunit SCF E3 ligase with a fusion protein through in vitro reconstitution disclosed by the disclosure includes: subjecting an engineered SKP1-Cullin1-RBX1 fusion protein (referred to as eSCR) to a reaction with an F-box protein in a reaction system to obtain the multisubunit SCF E3 ligase, where the eSCR fusion protein has an amino acid sequence from position 10 to position 1062 of SEQ ID NO: 2. Experimental results show that different multisubunit SCF E3 ligases are successfully prepared with the eSCR fusion protein through in vitro reconstitution in the disclosure; the reconstituted multisubunit E3 ligase has biological activity.