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Preparation method of lactococcus lactis product expressing phenylalanine ammonia lyase

A technology of phenylalanine ammonia lyase and Lactococcus lactis is applied in the field of preparation of Lactococcus lactis products, and can solve the problems of high cost, limited practical clinical application, low total PAL level and the like

Active Publication Date: 2013-07-17
BEIJING TRI PRIME GENE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The obvious defect of this method is that the fermentation amount of Lactococcus lactis and the content of expression products in Lactococcus lactis are very low, resulting in a low total level of PAL obtained from the final fermentation, so the cost is extremely high, which limits its actual clinical application

Method used

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  • Preparation method of lactococcus lactis product expressing phenylalanine ammonia lyase
  • Preparation method of lactococcus lactis product expressing phenylalanine ammonia lyase
  • Preparation method of lactococcus lactis product expressing phenylalanine ammonia lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of Lactococcus lactis engineering bacteria expressing PAL by utilizing the preferred codons of Lactococcus lactis human synthetic PAL gene

[0029] Artificially synthesized literature "Food-grade expression of artificially synthesized phenylalanine deaminase gene in Lactococcus lactis" (Jia Xingyuan, Chen Xing, Su Chang, etc., Chinese Journal of Microbiology and Immunology, Volume 26, No. 1, January 2006 The sequence of the PAL gene using the preferred codons of Lactococcus lactis as shown in Figure 1 in Issue 23-26), labeled as PAL art , DNA sequencing after ligation to the T vector was verified. The PAL art There are XbaI and NcoI restriction sites at the 5' end and 3' end, respectively.

[0030] NcoI and XbaI double enzyme digestion containing PAL art Complete PAL after fragmenting the T vector art The gene fragment was then ligated with the linear plasmid obtained by double digestion of pNZ8149 with NcoI and XbaI under the action of T4DNA...

Embodiment 2

[0031] Example 2: PAL art Screening and Identification of Recombinant Expression Plasmids

[0032] Screening and identification were carried out by restriction enzyme cleavage map method and PAL enzyme activity detection method, respectively.

[0033] The restriction map method uses NcoI and XbaI double enzyme digestion, and the electrophoresis results show that the recombinant plasmid is cut into two bands of 2.8kb and 1.9kb, while the plasmid pNZ8149 is cut into two bands of about 1.9kb and 650bp, which are in line with the expected The number of bands is consistent with the size and position, and it can be confirmed that the recombinant plasmid has a 2.2kb PAL art Fragment insertion.

[0034] PAL enzyme activity detection method Take 400μl of bacterial solution, add an equal amount of 5% perchloric acid and 10mmol / L phenylalanine, adjust the pH to 8.8 with 0.1mol / L borate buffer, react at 30°C for 1 hour, and take after centrifugation at 12000rpm The supernatant was filt...

Embodiment 3

[0035] Example 3: Using the parsley PAL gene to construct Lactococcus lactis engineering bacteria expressing PAL

[0036] Extract total RNA from mechanically damaged parsley stems and leaves by conventional methods, and prepare the cDNA of PAL by RT-PCR technology, wherein the specific primers used are:

[0037] Upstream primer T1: 5′-TACGATATCAGGAACTTGGTAACAAT-3′,

[0038] Downstream primer T2: 5'-CTAAGATCTCATGCATFTCAGTTAACA-3'.

[0039] The resulting cDNA fragment was digested with EcoRV and BglII to obtain a PAL cDNA fragment with cohesive ends. Then, the expression vector pNZ8149 was digested with EcoRV and BglII, and the digested fragment was ligated with the PAL cDNA fragment under the action of T4DNA ligase to obtain the recombinant plasmid pNZ8149-PAL. Referring to the electroporation conditions of Example 1, the recombinant plasmid pNZ8149-PAL was transformed into competent Lactococcus lactis NZ3900. The screening and identification methods of the pNZ8149-PAL recom...

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Abstract

The invention belongs to the field of biological medicine, and relates to a preparation method of a lactococcus lactis product expressing phenylalanine ammonia lyase, and the method orderly comprises the following steps: 1) obtaining a gene which expresses phenylalanine ammonia lyase; 2) connecting the gene which expresses phenylalanine ammonia lyase with a expression vector which is suitable forlactococcus lactis transformation so as to form a recombinant expression vector; 3) transforming competent lactococcus lactis by the recombinant expression vector; 4) screening to obtain a lactococcus lactis strain which highly expresses phenylalanine ammonia lyase; 5) fermenting the lactococcus lactis and inducing the expression of phenylalanine ammonia lyase; 6) drying the obtained fermented strain; wherein the pH of the fermentation liquid is adjusted to be 6.0-6.5 before the induction of fermentation in step 5); the pH is adjusted to be 6.5-7.5 after the induction; the strain is continued to be cultured till the fermentation is finished. With the technical scheme of the invention, the living bacteria number of the obtained lactococcus lactis and the specific activity of phenylalanine ammonia lyase are greatly increased, wherein the former reaches up to above 1012 CFU / ml, and the later reaches up to above 80 U / g.

Description

technical field [0001] The present invention generally relates to a preparation method of Lactococcus lactis products, in particular to a preparation method of Lactococcus lactis products expressing phenylalanine ammonia-lyase. Background technique [0002] Phenylalanine (Phenylalanlne, Phe) is an essential amino acid for the human body. Except for a small part of the phenylalanine that normal people ingest from the diet and recycle in the body, most of the phenylalanine in the liver Under the action of acid hydroxylase (Phenylalanine hydroxylase, PAH), it is converted into tyrosine and then utilized. Patients with autosomal recessive genetic disease - phenylketonuria (Phenylketonuria, PKU), due to phenylalanine hydroxylase gene defect, resulting in reduced or absent PAH enzyme activity in the liver, unable to convert phenylalanine Become tyrosine, causing phenylalanine to be converted into phenylpyruvate, phenylacetic acid, phenyllactic acid, etc. through other pathways. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/01
Inventor 牛春周敏毅刘金毅程永庆
Owner BEIJING TRI PRIME GENE PHARMA CO LTD
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