39 results about "Molecular recombination" patented technology

The present invention relates to a high efficiency method of expressing multispecific antibodies in eukaryotic cells. The invention is further drawn to a method of producing immunoglobulin heavy and light chain libraries, particularly using the trimolecular recombination method, for expression in eukaryotic cells. The invention further provides methods of selecting and screening for multispecific antibodies, and antigen-binding fragments thereof. The invention also provides kits for producing, screening and selecting multispecific antibodies. Finally, the invention provides multispecific antibodies, and antigen-binding fragments thereof, produced by the methods provided herein.

The invention relates to a mammal cell high-efficiency expression vector pSNEO. The vector is constructed on the basis of neomycin resistance screening gene NEO by combined strategy of weakening screening gene expression and reinforcing target gene expression. The screening gene weakening comprises the following two sides: introducing deletion mutant into the promoter to implement transcription weakening of the screening gene, and introducing a hairpin structure sequence before the translation initial site of the screening gene to implement the translation weakening of the screening gene. The expression reinforcement of the target gene is implemented by adding a transcription control element WPRE sequence between polyclone site and polyA tail of the target gene expression frame. The pSNEO vector can conveniently and quickly complete the construction of the stable high-expression cell strain of the target gene, and provides a new tool for preparing the high-polymer recombinant protein.

The invention relates to a pSGS vector and application of the vector to development of a high-expression cell strain. The construction of the vector comprises the following steps: based on a neomycin resistance screening gene NEO, through combined strategy of weakening screening gene expression and reinforcing target gene expression, constructing an expression vector pSNEO, and in view of the amplification function of a GS system, through designing a primer, replacing the hairpin structure sequence and the NEO gene of the vector pSNEO with a hairpin structure sequence and a GS gene to obtain a universal vector pSGS for screening a high-expression and stable-transfection cell strain. Through the pSGS vector constructured by the invention, the construction of a stable and high-expression cell strain of the target gene can be finished conveniently and quickly, and a new tool is provided for preparation of macromolecular recombinant protein.

The invention discloses a composition which comprises an enzyme. The enzyme has the activity of glucosaccharase, and provides a degrading function in cellulose degradation. The enzyme is a protein shown in (a) or (b), wherein (a), the amino acid sequence of the is shown as SEQ ID NO:1; (b), one or several amino acid are displaced, removed out or added into the amino acid sequence in the (a), and the protein is derived from the (a) and has the activity of the glucosaccharase. DNA molecules of the enzyme are coded. Carriers are recombined and comprise the DNA molecules and adjusting sequences connected with the DNA molecules and used for conducting expressions. Host cells comprise the DNA molecules or the recombined carriers. Compared with the beta-glucosaccharase (with the amino acid sequence of SEQ ID NO:1), a beta-glucosaccharase mutant with the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7 has the beneficial effects that the heat stability is improved; and the hydrolysis rate of compound enzyme liquid can be improved in enzyme system compounding.

The inventive method of producing a eukaryotic viral vector comprises contacting a eukaryotic cell, which comprises a unique enzyme that nicks or cleaves a DNA molecule, with a recombinant phage vector, or contacting a eukaryotic cell, which does not comprise a unique enzyme that nicks or cleaves a DNA molecule, simultaneously or sequentially, in either order, with (i) a unique enzyme that nicks or cleaves a DNA molecule, and (ii) a recombinant phage vector. The recombinant phage vector comprises the DNA molecule comprising (a) a eukaryotic viral vector genome comprising a coding sequence, (b) a phage packaging site that is not contained within the eukaryotic viral vector genome, and (c) a promoter that is operably linked to the coding sequence.

Alternatively, the DNA molecule is not present within the recombinant phage vector. The eukaryotic cell is contacted with the first DNA molecule and a recombinant phage vector. The first DNA molecule comprises a replication deficient eukaryotic viral vector genome comprising at least one adenoviral inverted terminal repeat and a packaging signal. The recombinant phage vector comprises a second DNA molecule and a phage packaging site, wherein the second DNA molecule complements in trans the replication deficient eukaryotic viral vector genome.

The DNA molecule(s) enter the eukaryotic cell, and the unique enzyme nicks or cleaves the DNA molecule in the eukaryotic cell in at least one region not contained within the eukaryotic viral vector genome, thereby inducing the production of and ultimately producing a eukaryotic viral vector.

The invention discloses a composition, containing an enzyme, wherein the enzyme has the activity of glucosaccharase and provides a degradation function in cellulose degradation, and the enzyme is the following protein (a) or (b): (a) protein with the amino acid sequence as shown in SEQ ID No: 1, (b) protein with one or more amino acids substituted, lost or added in the amino acid sequence in (a), having the activity of glucosaccharase and derived from (a). The invention also discloses a DNA molecule for encoding the enzyme, a recombinant vector containing the DNA molecule and a regulation sequence connected with the DNA molecule and used for expressing, and a host cell containing the DNA molecule or the recombinant vector. According to the beta-glucosaccharase mutant having the amino acid sequences as shown in SEQ ID No: 2, 3 and 4 provided by the invention, compared with wild beta-glucosaccharase, the thermostability is improved, and the integral hydrolysis rate of compound enzyme liquid in enzyme system compound can be improved.

The invention belongs to the fields of gene engineering and genetic engineering, and discloses a beta-galactosidase combined mutant with high transglycosylation activity. On the basis of amino acid sequences of beta-galactosidase of aspergillus candidus and aspergillus oryzae, from which signal peptides are removed, a beta-galactosidase combined mutant is obtained through fixed-site saturation mutagenesis of double sites; under the condition of keeping the oligosaccharide production amount invariable, the transglycosylation reaction rate of the mutant is improved by over 100% in comparison with a wild mutant; and meanwhile, the invention also discloses a DNA molecule of encoding the combined mutant, a recombinant expression vector containing the DNA molecule, and a host cell of expressing the DNA molecule. In addition, the invention also provides a method for preparing the beta-galactosidase with high transglycosylation activity by using the recombinant expression vector, and applications of the combined mutant, the DNA molecule, the recombinant expression vector and the host cell in preparation of the beta-galactosidase.

The invention discloses a DNA molecule, a recombinant plasmid and a recombinant bacterium for production of D-p-Hydroxyphenylglycine. The DNA molecule provided by the invention comprises a PL promoter, a D-hydantoinase gene and an N-carbamoylase gene, and expression of the D-hydantoinase gene and the N-carbamoylase gene is initiated by the PL promoter. The invention provides the DNA molecule able to stably and massively express D-hydantoinase and N-carbamoylase, and the recombinant plasmid and the recombinant bacterium based on the DNA molecule. At the same time, the invention also provides a method for converting DL-p-hydroxyphenylhydantoin into D-p-Hydroxyphenylglycine by the recombinant bacterium or fermented recombinant bacterium. The method only generates D-p-Hydroxyphenylglycine, has no need of splitting and separating an enantiomer, and the conversion rate is high. The whole production process has the advantages of mild conditions, simple operation, and environmental friendliness.

The present invention pertains to the technical field of collagens, in particular to a method for separation, purification and recombination of the collagens. The present invention takes the shark scalps as the raw materials, the soaking, the salt precipitation and the chromatography in the phosphate buffer solutions with the different concentrations are carried out sequentially for extraction and purification of the collagens. The obtained collagens can better maintain the spatial structural features of the collagens which exists naturally, the collagens can be prepared into the biomedical engineering materials with the good anti-leakage capacity and long degradation time after the molecular recombination, which can be used as various medical surgical materials with the higher requirements for reconstruction of veins and arteries and so on. The method of the present invention has rich sources, low price, simple process and no environmental pollution.

The present invention relates to a high efficiency method of expressing immunoglobulin molecules in eukaryotic cells. The invention is further drawn to a method of producing immunoglobulin heavy and light chain libraries, particularly using the trimolecular recombination method, for expression in eukaryotic cells. The invention further provides methods of selecting and screening for antigen-specific immunoglobulin molecules, and antigen-specific fragments thereof. The invention also provides kits for producing, screening and selecting antigen-specific immunoglobulin molecules. Finally, the invention provides immunoglobulin molecules, and antigen-specific fragments thereof, produced by the methods provided herein.

The invention relates to mineral coating for ceramic and a preparation method thereof. The mineral coating is prepared from the following raw materials according to a weight percent: 20-35% of anti-bacterial base liquid, 20-40% of potassium metasilicate, 1-5% of dispersing agent, 1-4% of diluent, 3-10% of emulsion, 1-7% of talcum powder, 1-5% of ferric oxide, 5-10% of aedelforsite and 10-15% of kaoline. According to the provided mineral coating for the ceramic and the preparation method thereof, through inorganic molecular recombination, a molecular structure of a product is changed from an original crystal structure to a net-like structure, a molecular gap is less than a water molecule, and greater than a water vapor molecule, so water of a wall body is turned into water vapor under a temperature difference effect and is freely discharged from a coating, a pressure to a wall surface caused by evaporation of the water is relieved, and a base surface is kept dry. Through the good air permeability of a coating film, the coating is guaranteed not to crack, and maintains a good appearance effect. Through adding the anti-bacterial base liquid, the sterilizing effect can be achieved, andthe safety of the coating is improved.

The invention discloses a knockout method for a stem cell gene, and relates to the technical field of molecular biology. The method specifically includes the following steps: S1, establishing a gene sequence, querying a code sequence (CDS) of a target gene in a database according to the gene sequence, obtaining a complete CDS of the gene by using a single-stranded CDNA as a template, and performing comparison to obtain a complete target gene exon sequence; S2, designing the target gene according to the S1, designing homologous recombination double exchange homologous arms, and constructing target gene knockout plasmid on the basis of knocking out template plasmid; and S3, recombining the target gene and DNA molecules homologous with a specific fragment of a target gene in cells onto a vector with a marker gene. According to the knockout method for the stem cell gene, the stem cell gene can be quickly knocked out on the basis of not introducing exogenous genes by using the knockout method for the stem cell gene provided by the invention, so that a worker can analyze the target gene function more accurately in the later stage, and the gene knockout efficiency is improved.

Novel packaging cell lines which produce recombinant retrovirus, free of detectable helper-virus are disclosed. Also disclosed are methods of making the cell lines and methods of producing recombinant retroviruses from the cell lines. Retroviruses produced by the cell lines include lentiviruses, such as HIV, capable of transfering heterologous DNA to a wide range of non-dividing cells. The packaging cells contain at least three vectors which collectively encode retroviral gag, pol, and env proteins, wherein the gag and pol genes are separated, in part, onto two or more different vectors. This is made possible by fusing Vpr or Vpx to pol proteins separated from gag so that the proteins are targeted to assembling virions. Among other advantages, the packaging cells provide the benefit of increased safety when used in human gene therapy by virtually eliminating the possibility of molecular recombination leading to production of replication-competant helper virus.

The invention discloses a shRNA molecule of knockdown mouse endogenous CYP3A. The nucleotide sequence of the shRNA molecule is as shown in SEQID No.6. The invention also discloses a recombinant expression vector pRIME-CMV-eGFP-miR-shRNA containing the shRNA molecule, a preparation method of the vector and an application of the shRNA molecule in the knockdown mouse endogenous CYP3A. Expression of a mouse endogenous CYP3A gene is effectively blocked in transgenic mouse produced by the invention. By the use of the invention, a transgenic mouse model of the knockdown mouse endogenous CYP3A is established and human CYP is expressed in the mouse model. The invention can be applied in drug research and development to make the drug effect index in mouse to be more close to that in human.

The invention relates to DNA molecules, recombinant vectors and cell cultures for use in methods for expression of bile salt-stimulated lipase (BSSL) in the methylotrophic yeast Pichia pastoris.