TH14170U - Vectors containing genes for delivering proteins outside the cells of the Lactobacillus casei bacteria. - Google Patents

Abstract

The vector containing the gene for delivering an extracellular protein from the bacterium Lactobacillus casei is a 6,224 base pair DNA ring, composed of the following components: Genes involved in extracellular protein synthesis include promoter genes, signal peptide genes, and green fluorescent protein (GFPuv) genes, ampicillin (APR) and erythromycin (EryR) resistance genes, the replication A1 (RepA1) protein gene, the origin of replication (Ori), and 14 enzyme-specific multiple cloning sites: AatII, NotI, SacII, EcoRI, HindIII, KptiI, AgeI, NdeI, XhoI, BamHI, SaII, SpeI, PstI, and XbaI. These vectors can synthesize the target proteins for extracellular secretion in Lactobacillus casei.

Claims (3)

1. The vector containing genes for extracellular protein delivery from Lactobacillus casei is a DNA ring with the following components: a. Genes for extracellular protein synthesis, consisting of: a promoter, which is the lactate dehydrogenase promoter (LdhProl) gene of Lactobacillus casei, located at nucleotide sequence 4858-5102 of the vector. This site is where the RNA polymerase enzyme binds and moves to the structural gene to synthesize RNA. a signal peptide gene, which is a cell wall-associated hydrolase (Cwh) gene responsible for delivering target proteins across the cell membrane and out of the cell. The vector contains the following genes: a. The green fluorescent protein (GFPuv) gene, located at nucleotide sequences 5103-5238 of the vector, which synthesizes the green fluorescent protein (GFPuv) used to track the target protein (protein of interest). b. The erythromycin resistance gene (EryR), located at nucleotide sequences 97-828 of the vector, is used for selection in Lactobacillus casei. c. The replication A1 gene (RepA1), located at nucleotide sequences 1377-2225 of the vector, is a gene segment that synthesizes the replication A1 protein, which aids in protein replication. D. The starting point for replication in Lactobacillus casei (ORI) is at nucleotide position 2365-2468 of the vector, which is the position that allows the vector to replication in Lactobacillus casei. E. The starting point for replication in E. coli (ORI) is at nucleotide position 3972, which is the position that allows the vector to replication in E. coli. F. The ampicillin (APR) resistance gene is located at nucleotide position 4691-3834 of the vector, which is used for selection in Escherichia coli. G. A multiple cloning site is a location on the vector where a gene sequence can be cut by a restriction enzyme (restriction enzyme site). This area is used for inserting the gene of interest at a specific cut site. This vector has multiple cloning sites specific to 14 enzymes as follows: - Specific site for AatII enzyme is at nucleotide position 4822. - Specific sites for NotI enzyme are at nucleotide positions 4848 and 5991. - Specific site for SacII enzyme is at nucleotide position 4852. - Specific sites for EcoRI enzyme are at nucleotide positions 4858, 5973, 6224, and 2185. - Specific sites for HindIII enzyme are at nucleotide positions 5104, 1030, and 2652. - Specific sites for KpnI enzyme are at nucleotide positions 5239,12. - Specific site for AgeI enzyme is at nucleotide position 5242. - Specific site for NdeI enzyme is at nucleotide position... 5483 and 6011 - XhoI-specific loci are located at nucleotide position 5675. BamHI-specific loci are located at nucleotide position 5774, 1000. SalI-specific loci are located at nucleotide positions 5857, 6005, 24, 987, and 1012. SpeI-specific loci are located at nucleotide position 5979. PstI-specific loci are located at nucleotide positions 5999 and 1018. XbaI-specific loci are located at nucleotide position 1006. 2. A vector containing a gene for the extracellular protein delivery of Lactobacillus casei, as per claim 1, with a size of 6,224 base pairs. 3. A vector containing a gene for the delivery of an extracellular protein of Lactobacillus casei, as per claim 1 or 2, which can accommodate a gene or DNA fragment ranging in size from 100 to 1888 base pairs.