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A kind of cold tolerance lactase gene and its expression vector and protein

A lactase and tolerance technology, applied in glycosylase, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2021-04-20
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the efficient production of recombinant proteins with a gene size exceeding 1.5kb using the Pichia pastoris expression system

Method used

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  • A kind of cold tolerance lactase gene and its expression vector and protein
  • A kind of cold tolerance lactase gene and its expression vector and protein
  • A kind of cold tolerance lactase gene and its expression vector and protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] This example provides an optimized artificially synthesized cold-tolerant lactase gene with a 6×His tag at the C-terminal, the specific sequence of which is shown in SEQ ID No.1 in the sequence listing, and the protein corresponding to the gene The sequence is shown as SEQ ID No.2 in the sequence listing. The optimized DNA sequences were compared by NCBI, and there was no obvious similarity.

[0064] Design primers, specific sequences are shown in SEQ ID No.5 (forward primer) and SEQ ID No.6 (reverse primer) in the sequence listing, utilize the primer amplification of SEQ ID No.5 and SEQ ID No.6 The target gene can also be subjected to related enzyme cutting and ligation operations.

[0065] The present invention synthesizes the DNA sequence shown in SEQ ID No.1 according to the sequence characteristics of the lactase gene itself and yeast codon bias, the natural DNA of lactase before optimization, and other types of enzymes after optimizing only according to the yeast...

Embodiment 2

[0067] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0068] S1: Construction of expression vector and transformation: The sequence characteristics of the gene itself and the DNA sequence synthesized by yeast codon preference in Example 1, that is, the DNA in SEQ ID No.1, were connected to the constitutive secretory expression vector pGAPZαA of Pichia pastoris to obtain Recombinant vector pGAPZαA-cold-tolerant lactase, the vector is constructed as figure 1 as shown, figure 1 It is a schematic diagram of the construction of the eukaryotic expression vector pGAPZαA-cold-tolerant lactase in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0069] (1) Digest the plasmid containing the synthetic cold-tolerant lactase gene (SEQ ID No.1) with Xho I and Xba I to obtain the target fragment. The reaction system is as follows (the endonuclease and buffer used were purchas...

Embodiment 3

[0104] In this example, the enzyme activity of the purified soluble cold-tolerant lactase was detected, and the specific steps and results were as follows:

[0105] With p-nitrophenyl-β-D-glucopyranoside as substrate, p-nitrophenol as standard substance, to the embodiment

[0106] (1) Drawing of standard curve: take 5 μmol / L p-nitrobenzene, dilute it with 20mM sodium dihydrogen phosphate solution of pH 6.0 to 800, 400, 200, 100, 50, 25 and 0nmol / L respectively . Take 100 μl of each of the above dilutions, add them to 96-well microplates, repeat each concentration three times, place them in a full-wavelength microplate reader at room temperature, select the absorbance value at 400 nm, and measure the p-nitro group at each dilution concentration. The absorbance value of benzene, and draw a standard curve, the resulting standard curve equation is: Y=0.0011X+0.0066, the correlation system r=0.9996, the detection result table of the standard is shown in Table 6.

[0107] The detect...

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Abstract

The present invention relates to an artificially synthesized gene encoding cold tolerance lactase, the gene at least contains a DNA piece of one of the following nucleotide sequences: 1) the nucleotide sequence of SEQ ID NO.1 in the sequence table; 2) the nucleotide sequence with The nucleotide sequence shown in SEQ ID NO.1 has more than 90% homology and encodes a nucleotide sequence of the same biological function protein; or 3) hybridizes with the nucleotide sequence shown in SEQ ID NO.1 and encodes Nucleotide sequences of proteins with the same biological function. According to the gene sequence of the present invention, the recombinant vector is further constructed and transformed into yeast, which can realize the constitutive secretory expression of the recombinant lactase under high-density fermentation conditions, and can obtain a purity higher than 95% through simple nickel affinity purification. And active protein with high protein concentration, the active protein has a strong activity of hydrolyzing lactose at low temperature, and will provide efficient enzyme products for converting lactose in dairy products.

Description

technical field [0001] The invention belongs to the technical field of biomolecular cloning, and relates to a cold tolerance lactase gene and its expression vector and protein. Background technique [0002] Lactose intolerance is non-infectious diarrhea caused by the inability to fully digest and decompose lactose in breast milk or cow's milk due to the low secretion of lactase, also known as lactase deficiency. The sugar in breast milk and cow’s milk is mainly lactose. The lactase in the small intestine, especially the tip of the villi on the surface of the jejunal mucosa, is less secreted or the activity is not high, so the lactose in the milk cannot be completely digested and decomposed. Part of the lactose is fermented by the colonic flora into lactic acid , hydrogen, methane and carbon dioxide. Lactic acid stimulates the intestinal wall, increases intestinal peristalsis and causes diarrhea, and occasionally may induce intestinal spasm and colic. Lactase deficiency is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/24C12N15/81
CPCC12N9/2402C12N15/815C12Y302/01108
Inventor 李洪波岳芬芳董海丽
Owner HUAIHUA UNIV
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