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Construction method and application of probiotic engineering strain for treating phenylketonuria

A technology of engineering bacteria and phenylalanine deaminase, applied in microorganism-based methods, biochemical equipment and methods, applications, etc., can solve problems such as unresearched treatment of phenylketonuria

Active Publication Date: 2021-10-12
苏州优信合生技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the treatment of phenylketonuria is still under investigation

Method used

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  • Construction method and application of probiotic engineering strain for treating phenylketonuria
  • Construction method and application of probiotic engineering strain for treating phenylketonuria
  • Construction method and application of probiotic engineering strain for treating phenylketonuria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Research on the ability of phenylalanine deaminase to degrade phenylalanine

[0069] 1.1 Intracellular expression vector construction

[0070] The phenylalanine deaminase (named as TcXAL (SEQ ID NO: 1)) derived from Trichosporon cutaneum and the phenylalanine deaminase derived from Rhodotorula mucilaginosa were respectively selected. (named RmXAL (SEQ ID NO: 2)), and designed primers for vector construction. The plasmid pslpa8148 was used as the expression vector, and the constitutive promoter p32 (SEQ ID NO: 3) was used to replace the original promoter in the vector. After PCR amplification, the target gene was connected to the expression vector by Gibson method.

[0071] The details are as follows: Plasmid pslpa8148 is purified and recovered according to the instructions of the product recovery kit (Omega, D6492) after double digestion with BglII and XbalI; the p32 promoter is obtained by PCR amplification using primers p32-F and p32-R and pMG36e as a templ...

Embodiment 2

[0077] Embodiment 2 TcXAL surface display type expression vector construction

[0078] Use primers TcXAL-F2 and TcXAL-R and pUC57-TcXAL as templates to PCR amplify the TcXAL2 fragment, gel recovery and purification; use primers cnb-F and cnb-R and a plasmid containing cnb and signal peptide sp stored in the laboratory as templates , the sp-cnb fragment was obtained after amplification, and the gel was recovered and purified; the purified fragment and the p32 promoter of the enzyme-cut plasmid in Example 1 were ligated using the Gibson ligation kit (Biomed, CL116), and electrotransformed into Escherichia coli EC1000 , after sequencing verification, a positive strain containing the expression vector of TcXAL was obtained, which was named Pp32-cnb-TcXAL. Using a plasmid extraction kit (Omega, D6943), the target plasmid was extracted, and Lactobacillus reuteri CGMCC1.3264 was electroporated to obtain an engineering strain containing the above vector. Among them, the plasmid struc...

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Abstract

The invention provides a recombinant expression vector, the recombinant expression vector can be expressed in lactobacillus reuteri to generate phenylalanine deaminase, and the phenylalanine deaminase can decompose phenylalanine and metabolites thereof, which are ingested in the stomach and intestine, so that the phenylalanine and the metabolites thereof are prevented from being accumulated in a body, and the purpose of treating the phenylketonuria is achieved.

Description

technical field [0001] The invention relates to the field of biomedicine. Specifically, the present invention relates to a construction method and application of probiotic engineering strains for treating phenylketonuria. More specifically, the present invention relates to recombinant expression vectors, kits, engineering bacteria and their use in the treatment of phenylketonuria. Background technique [0002] Phenylketonuria (Phenylketonuria), referred to as PKU, is the most common disease among congenital amino acid metabolism disorders, and it is also an autosomal recessive genetic disease. A disease caused by the accumulation of amino acids and their metabolites in the body. Symptoms generally begin to appear 3-6 months after birth, and the clinical manifestations include retarded intelligence and motor development, reduced skin and hair pigment, and rat urine odor. Currently, the treatment of phenylketonuria is still under investigation. [0003] Probiotics, as the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/60C12N1/21A61K35/747A61P3/00A23L33/135C12R1/225
CPCC12N15/746C12N9/88C12Y403/01005A61K35/747A61P3/00A23L33/135A23V2002/00A23V2200/3204Y02A50/30
Inventor 孔繁智
Owner 苏州优信合生技术有限公司
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