Engineering probiotics for treating phenylketonuria as well as construction method and application thereof
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A probiotic and engineering technology, applied in the field of medicine, can solve the problems of limiting the degradation efficiency of phenylalanine and achieve the effect of reducing intake
Active Publication Date: 2022-01-25
SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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However, the existing E. coli engineering bacteria for the treatment of phenylketonuria mainly colonize the large intestine. In addition, the existing E. ...
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Embodiment 1
[0065] Construction of the project probiotics LA205
[0066] Lactobacillus acidophilus NCFM strain departure, integrated protein secretion and processing domains prtP gene phenylalanine lyase PAL gene, comprising the steps on the genome:
[0067] Gene Synthesis Examples and sequenced by Shanghai Sangon Biological Engineering Limited of the present embodiment. Molecular Biology comprises preparing competent cells, transformation and the like refer to "Molecular Cloning, A Laboratory Manual" (Third Edition) performed.
[0068] The method of designing a promoter-containing SL (DNA sequence NO3), protein secretion and processing domain gene prtP (DNA sequence NO1) and phenylalanine ammonia lyase gene PAL (DNA sequence NO2) gene fragment SPAL, whole gene synthesis DNA fragments and cloned into cloning vector pUC57, pUC57-SPAL vector is obtained.
[0069] Lactobacillus acidophilus NCFM genome template, L-F and L-R as primers, PCR amplified upstream homology arm L-arm. In NCFM genome tem...
Embodiment 2
[0085] Construction of the project probiotics LA1314
[0086] Lactobacillus acidophilus NCFM strain departure, phenylalanine lyase gene integration and phenylalanine PAL pheP transporter gene on the genome, comprising the steps of:
[0087] Gene Synthesis Examples and sequenced by Shanghai Sangon Biological Engineering Co. present embodiment; Molecular Biology comprises preparing competent cells, transformation and the like refer to "Molecular Cloning, A Laboratory Manual" (Third Edition) performed.
[0088] The method of designing a promoter-containing SL (DNA sequence NO3), phenylalanine lyase gene PAL (DNA sequence NO1) and phenylalanine transporter gene pheP (DNA sequence NO2) gene fragment PALP, whole gene synthesis DNA fragments and cloned into cloning vector pUC57, pUC57-PALP obtained vector.
[0089] Lactobacillus acidophilus NCFM genome template, L-F and L-R as primers, PCR amplified upstream homology arm L-arm. In NCFM genome template, R-F and R-R as primers, PCR amplifi...
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Abstract
The invention relates to the fields of medicine and biotechnology, in particular to engineering probiotics for treating phenylketonuria as well as a construction method and application thereof. The recombinant expression vector comprises a recombinant expression vector I and a recombinant expression vector II; the recombinant expression vector I is an expression vector containing a phenylalanine ammonia lyase gene PAL nucleotide sequence, a protein secretion processing domain coding gene prtP nucleotide sequence and a lactobacillus acidophilus constitutive promoter SL; the recombinant expression vector II is an expression vector containing a phenylalanine ammonia lyase gene PAL nucleotide sequence, a phenylalanine transfer pump gene pheP and a lactobacillus acidophilus constitutive promoter SL; the engineering probiotics capable of secreting phenylalanine ammonia lyase to the outside of the bacteria in gastrointestinal tracts are constructed for the first time, phenylalanine in digested food in the gastrointestinal tracts is effectively degraded, the phenylalanine intake of patients is reduced, and the engineering probiotics are used for treating PKU.
Description
Technical field [0001] The present invention relates to the field of pharmaceutical fields and biotechnology, and therefore specifically involved engineering probiotics and construction methods and applications thereof. Background technique [0002] The information disclosed in the background is disclosed only to increase the understanding of the overall background of the present invention, and is not necessarily considered to admit or imply that the information is intended to be a prior art known to those skilled in the art. [0003] Phenylketone urine PKU is a congenital phenylalanine metabolic disorder. At present, the only clinical therapy for phenyltrinone urine patients is a strategy of low-phenylalanine PA diet, feeding low PA milk powder in infancy, Follow the low protein diet during the childhood and adults, control phenylalanine intake; but strict low-protein dietary treatment strategies have great side effects on adolescent development and adults, affecting patient phy...
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