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Recombinant lactococcus lactis with laccase excreting capacity, and construction method and application thereof

A technology of Lactococcus lactis and laccase, which is applied in the field of genetic engineering of enzymes, can solve the problems of low copy number, low expression amount, and difficulty in realizing exocrine expression in a lactic acid bacteria expression system, and achieves good thermal stability and acid and alkali resistance. , the effect of increasing the yield

Active Publication Date: 2020-08-25
INNER MONGOLIA UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although a variety of genes have been expressed heterologously in food-grade lactic acid bacteria, due to the shortcomings of the lactic acid bacteria expression system, such as low copy number, low expression level, and difficulty in exocrine expression, it is particularly important for genes to be efficiently secreted and expressed in lactic acid bacteria. difficulty

Method used

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  • Recombinant lactococcus lactis with laccase excreting capacity, and construction method and application thereof
  • Recombinant lactococcus lactis with laccase excreting capacity, and construction method and application thereof
  • Recombinant lactococcus lactis with laccase excreting capacity, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of recombinant Lactococcus lactis with ability to secrete laccase

[0048] 1.1 Design and synthesis of CotA laccase gene and construction of pUC57-Simple-S-CotA recombinant cloning vector

[0049] The mature peptide of Bacillus subtilis CotA laccase was obtained through GenBank (accession number: FJ663050.1). Designed to add Usp45 secretion signal peptide and His tag to the CotA laccase sequence, according to the selection of restriction sites on the lactic acid bacteria expression vector pMG36e, add Xma I restriction sites and Hind III restriction sites to the N-terminus and C-terminus of the protein sequence respectively location. The designed sequence is optimized according to the codon preference of Lactococcus lactis, and the optimized sequence is shown in SEQ ID NO.3. The optimized CotA laccase sequence containing Usp45 secretion signal peptide was named S-CotA. The optimized CotA laccase sequence was entrusted to Hangzhou Baosai Biotechn...

Embodiment 2

[0090] Example 2 PCR amplification of CotA laccase gene

[0091] 2.1 Extraction of pUC57-Simple-S-CotA recombinant plasmid

[0092] Construct the pUC57-Simple-S-CotA recombinant cloning vector and transform it in E.coli DH5α, activate the recombinant strain on the LB solid medium containing 100 μg / mL ampicillin (Ampr), use the plasmid small extraction kit (EasyPureHiPure Plasmid MiniPrep Kit) to extract the pUC57-Simple-S-CotA recombinant plasmid, the specific operation steps were the same as 1.2.2 using the EasyPure HiPure Plasmid MiniPrep Kit to extract the pMG36e-S-CotA recombinant plasmid.

[0093] 2.2 Primer design for CotA laccase gene

[0094] According to the codon-optimized base sequence of the laccase gene in Bacillus subtilis (no Usp45 signal peptide) and the selection of restriction sites on the Escherichia coli expression vector pET-30a(+), primers were designed by using Primer 5.0 software. Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the synt...

Embodiment 3

[0106] Example 3 Identification of pMG36e-S-CotA recombinant expression vector

[0107] 3.1 PCR identification of positive clones

[0108] 1) Primer design

[0109] The primers were designed and identified according to the sequence on the pMG36e vector. The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The synthetic primers are shown in Table 4.

[0110] Table 4 Primer sequences

[0111]

[0112]

[0113] 2) PCR reaction system

[0114] The PCR reaction system and reaction conditions are shown in Table 5 and Table 6. After the PCR was finished, the PCR results were detected by 1% agarose gel electrophoresis.

[0115] Table 5 PCR reaction system

[0116]

[0117] Table 6 PCR reaction conditions

[0118]

[0119] 3) PCR identification results

[0120] The result is as image 3 As shown, the size of the amplified band is about 1900bp, which is consistent with the size of the expected band (1942bp), preliminarily proving that the recomb...

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Abstract

The invention provides recombinant lactococcus lactis with laccase excreting capacity, a construction method and application of the recombinant lactococcus lactis. According to the invention, a bacillus subtilis CotA laccase gene sequence is obtained through GenBank; codon optimization is carried out on the CotA laccase gene containing a Usp45 signal peptide according to the codon preference of lactococcus lactis NZ9000; then the codon-optimized CotA laccase gene is connected with a pMG36e vector; and the codon-optimized CotA laccase gene is electrically transferred into lactococcus lactis NZ9000 to construct the recombinant lactococcus lactis with laccase excreting capacity. The recombinant lactococcus lactis is high in extracellular laccase yield, can improve the ensiling efficiency andimprove the ensiling straw quality, and is an ensiling lactic acid bacterium strain with potential application prospects.

Description

technical field [0001] The invention belongs to the technical field of gene engineering of enzymes, and in particular relates to a recombinant Lactococcus lactis capable of secreting laccase and its construction method and application. Background technique [0002] Silage is a method in which green feed is formed by anaerobic fermentation of lactic acid bacteria in a closed environment, which is palatable, nutritious, storage-resistant, and can maintain various nutritional characteristics of green feed all year round. Silage is an important material basis for the development of animal husbandry. With the rapid development of animal husbandry in my country, the problem of shortage of forage grass for herbivorous livestock is becoming more and more serious. Improving the utilization rate of feed is the key to solving the shortage of feed. Lactic acid bacteria determine the quality of silage. However, the number of lactic acid bacteria attached to natural pastures is relatively...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/53A23K30/18C12R1/01
CPCC12N9/0061C12Y110/03002C12N15/746A23K30/18C12N2800/22C07K2319/02C07K2319/21A23V2400/231
Inventor 李冠华汪露露孔祥婕
Owner INNER MONGOLIA UNIVERSITY
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