11 results about "Bacterial artificial chromosome" patented technology

The invention belongs to the technical field of genetic engineering, and particularly relates to a universal bacterial artificial chromosome recombinant virus transfer vector and a construction method thereof, the transfer vector comprises a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as a basic skeleton and pUC19-BAC as a functional sequence; according to the universal bacterial artificial chromosome recombinant virus transfer vector and the construction method thereof, a drug screening target gene reading frame and an EGFP fluorescent protein gene reading frame are connected through an IRES double-expression connection sequence, and double screening can be carried out; in the BAC construction process, screening genes and functional sequences are respectively constructed to a high-copy T vector, so that sufficient plasmid DNA can be conveniently copied and prepared. And through a proper restriction enzyme cutting site, introduction of various homologous arms is facilitated, the alignment is wide, simultaneous operation of various genomic sequences is facilitated, and the research efficiency is improved.

The disclosure provides for methods of constructing synthetic chromosomes including the steps of providing host cells with an endogenous chromosome, transforming a cloning vector and a cloning cassette into the host cells, excising target genomic nucleic acids from the endogenous chromosome, recombining the excised target genomic nucleic acids with the cloning cassette via homologous recombination to form heterologous vectors comprising cloned sequences, extracting the heterologous vectors containing the cloned sequences from the host cells, digesting the heterologous vectors with a restriction endonuclease to release the cloned sequences from the heterologous vectors to provide released cloned sequences, and introducing the released cloned sequences, a centromere cassette, and a yeast artificial chromosomes or bacterial artificial chromosomes into a second host cell, such that the released cloned sequences, the centromere cassette, and the YAC or BAC recombine with one another via homologous recombination to produce the synthetic chromosome.

The invention relates to a hog cholera virus full-length infectious clone and a construction method thereof, the gene sequence of the hog cholera virus full-length infectious clone is as shown in SEQ ID NO.1, the used carrier is pBAC-smCSFV, through segmented PCR amplification and overlapping PCR connection, the genome sequence of a CSFV Shimen strain, a CMV promoter sequence, an RZ sequence and a BGH sequence are integrated into a bacterial artificial chromosome carrier, and the hog cholera virus full-length infectious clone is obtained. The complete hog cholera virus full-length infectious clone is obtained. According to the invention, a single-copy bacterial artificial chromosome vector and a human cytomegalovirus early promoter capable of starting RNA synthesis in eukaryotic cells are combined, and the construction method of the CSFV full-length infectious clone, which is simple and convenient to operate, efficient and stable, is provided. According to the method, the filial generation virus can be efficiently saved and obtained, and the filial generation virus rCSFV and the female parent wild type virus wtCSFV have the same growth characteristics and can be used for subsequent foundation and application research.

The invention discloses a method for constructing high-yield engineering bacteria of an oritavancin precursor cloireomycin. A high-yield chassis strain capable of being used for efficiently synthesizing glycopeptide antibiotics is constructed by knocking out an NRPS gene responsible for ristomycin biosynthesis in TNS106, inserting an integration site and then inserting a pJLL10 plasmid with a double-copy bbr gene. A bacterial artificial chromosome (BAC) plasmid containing a glycopeptide antibiotic biosynthetic gene cluster is introduced into the high-yield chassis strain to obtain the glycopeptide antibiotic high-yield engineering strain. The shake flask fermentation yield of the cloirusin high-producing strain obtained by the method disclosed by the invention can reach 1440mg / L, and the cloirusin high-producing strain is the strain with the highest shake flask fermentation yield in a public report.

The present invention relates to the use of a bacterial artificial chromosome (BAC) for the preparation of a vaccine wherein the BAC comprises:-an inducible bacterial ori sequence for amplifying the BAC to more than 10 copies per bacterial cell, and-a viral expression cassette comprising cDNA of an attenuated RNA viral genome, and comprising a cis-regulatory element for transcribing the viral cDNA in mammalian cells and for processing the transcribed RNA into infectious viral RNA.

This invention provides a method for seamless editing of a double-copy target gene in a bacterial artificial chromosome. The method includes: first, constructing a bacterial artificial chromosome containing a double-copy target gene and introducing it into *E. coli* SW102 to obtain a first recombinant bacterium; second, constructing a target fragment including a chloramphenicol resistance gene, a gentamicin resistance gene, and a galactokinase encoding gene, contacting this target fragment with the first recombinant bacterium, and obtaining a second recombinant bacterium through a first homologous recombination; finally, constructing a target fragment for editing the target gene and obtaining a third recombinant bacterium through a second homologous recombination, thus completing the seamless editing of the double-copy gene. The method provided by this invention can simultaneously recombine and modify a double-copy target gene in a bacterial artificial chromosome, avoiding the problems of low double-site knock-in rate and high screening difficulty associated with single galactokinase encoding gene marker screening, and achieving seamless editing of the double-copy target gene.

The invention relates to the technical field of gene detection, and provides a mycoplasma pneumoniae nucleic acid detection quality control product based on a recombinant artificial chromosome and application of the mycoplasma pneumoniae nucleic acid detection quality control product. Aiming at the problems of difficulty in integration of multi-target full-length genes, accurate drug-resistant mutation, insufficient stability and the like of an existing quality control product, a recombinant artificial chromosome carrying full-length sequences of mycoplasma pneumoniae key target genes (16S rRNA, 23S rRNA, a P1 adhesion protein gene and a community-acquired respiratory distress syndrome toxin gene toxin) is constructed; important clinical drug-resistant mutation sites (A2063G, A2064G, C2617T and A2067G) are accurately introduced into the 23S rRNA gene, and the 23S rRNA gene is stably integrated into escherichia coli engineering bacteria by using a bacterial artificial chromosome system. The quality control product exists in a low-copy form, has the advantages of multi-target coverage, accurate simulation of drug-resistant mutation, high genetic stability and no biological safety risk, and is suitable for performance analysis verification and quality control of a detection kit.

The invention discloses a polyene macrolactam antibiotic Weidel, a production method thereof and a production strain. According to the invention, a bacterial artificial chromosome library of streptomyces DSS69 genome DNA is constructed, bacterial artificial chromosomes containing polyene macrolactam compound biosynthetic gene clusters are selected from the bacterial artificial chromosome library, and are cloned and subjected to heterologous expression, antibacterial activity screening and compound separation and identification, so that the novel polyene macrolactam antibiotics are found; the compound is named as Weddelamycin, and the compound is named as Weddelamycin. An antibacterial experiment is further carried out on the Weddellamycin, the Weddellamycin has very good antibacterial activity on all tested gram-positive strains, and the MIC (minimal inhibitory concentration) value of the Weddellamycin is between 0.14 mu g / mL and 1.07 mu g / mL. Especially, the MIC values to MRSA, MRSE and enterococcus faecalis are 0.14 [mu] g / mL, 0.27 [mu] g / mL and 1.07 [mu] g / mL respectively.

The present invention relates to a process for preparing recombinant Modified Vaccina Virus Ankara (MVA) using bacterial artificial chromosome (BAC) based permanent dominant selection.

Provided here are compositions for a bacterial artificial chromosome-based construct containing a replication-competent recombinant severe acute respiratory syndrome coronavirus-2 (rSARS-CoV-2) genome with a deletion of ORF3a and ORF7b group-specific open reading frames and methods of making such compositions and uses thereof. The rSARS-Cov-2 provided herein can be attenuated and safely worked with under BLS-2+ conditions.

This invention discloses a novel antitumor macrolide compound, UCN370, its preparation method, and its applications. The structural formula of compound UCN370 is shown below. This invention successfully expressed the bacterial artificial chromosome (BAC) plasmid pUCN370, containing the UCN370 biosynthetic gene cluster, using the chassis strain *Streptomyces albus* J1074, resulting in the novel scaffold compound UCN370. The novel antitumor macrolide compound UCN370 described in this invention exhibits potent inhibitory activity against tumor cells such as HK-2 and can be used to prepare antitumor drugs.