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Recombinant expression vector for expressing LL-37 polypeptide, recombinant lactococcus lactis, antiviral drug, construction method and application

A Lactococcus lactis, LL-37 technology, applied in the field of genetic engineering, can solve the problems of low expression efficiency of LL-37, limited application of LL-37, and high production cost, so as to avoid low expression efficiency and solve the uncertainty of metabolism in vivo Sexuality, the effect of solving tediousness

Active Publication Date: 2020-08-07
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional preparation method of LL-37 is artificial synthesis, but the cost of artificial synthesis is high, which greatly limits the application of LL-37
The prior art discloses the use of genetic engineering methods to prepare LL-37 by constructing recombinant expression vectors containing LL-37 coding genes and recombinant bacteria, but the current methods still have low LL-37 expression efficiency and high production costs. question

Method used

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  • Recombinant expression vector for expressing LL-37 polypeptide, recombinant lactococcus lactis, antiviral drug, construction method and application
  • Recombinant expression vector for expressing LL-37 polypeptide, recombinant lactococcus lactis, antiviral drug, construction method and application
  • Recombinant expression vector for expressing LL-37 polypeptide, recombinant lactococcus lactis, antiviral drug, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of PNZ8149-7×LL-37 expression strain

[0041] 1. The complete sequence of SphI-SPusp45-LEISS-DDDDK-7×LL-37-XbaI was chemically synthesized to obtain the pUC57-7×LL-37 vector;

[0042] 2. Construction of PNZ8149-7×LL-37 vector

[0043] 1) Use the Tiangen plasmid mini-extraction kit to extract the pNZ8149, pUC57-7×LL-37 plasmids;

[0044] 2) Enzyme digestion: PNZ8149, pUC57-7×LL-37 enzyme digestion system is shown in Table 1, the total system is 50 μl, 37 ° C enzyme digestion for 3 hours;

[0045] Table 1 PNZ8149, pUC57-7×LL-37 enzyme digestion system

[0046]

[0047] 3) Preparation of 1% agarose gel electrophoresis, electrophoresis at 120V for 30 minutes, and recovery by cutting the gel;

[0048] 4) Enzyme linkage: the enzyme linkage system is shown in Table 2, the total system is 20 μl, insert fragment:vector = 7:1, overnight at 16°C;

[0049] Table 2 enzyme-linked system

[0050]

[0051] 5) Purification and recovery of the ligated pr...

Embodiment 2

[0052] Example 2 PNZ8149-7×LL-37 Electroporation of Lactococcus lactis NZ3900

[0053] 1. Competent preparation of Lactococcus lactis NZ3900

[0054] 1) Inoculate Lactococcus lactis MG1363 frozen at -80°C into 5ml of M17 liquid medium containing 5% glucose, and culture overnight at 30°C;

[0055] 2) Inoculate the obtained bacterial liquid at 1% in M17 liquid medium containing 2.5% Gly and 5% glucose, culture it statically at 30°C until the OD600 value of the bacterial cell is 0.3-0.4, and collect it for later use;

[0056] 3) Put the above-collected bacterial culture in an ice bath for 10 minutes, centrifuge at 5000 rpm at 4°C for 5 minutes; collect the bacterial cells;

[0057] 4) The precipitate was washed twice with 1 / 10 volume of ice-cold 10% sucrose and 10% glycerol mixed solution, centrifuged at 8000 rpm at 4°C for 5 min, and the precipitate was collected;

[0058] 5) Finally, the precipitate was resuspended in a mixed solution of 1 / 100 volume 10% sucrose and 10% glyce...

Embodiment 3

[0068] Example 3 Silver staining and Western detection of the protein expression ability of the PNZ8149-7×LL-37 expression strain

[0069] 1. Induced secretory expression of PNZ8149-7×LL-37 in Lactococcus lactis

[0070] 1) Introduce the strain into M17 liquid medium, culture it overnight at 30°C, transfer it to a new M17 broth medium the next day, and add the inducer nisin when the OD600 value reaches 0.4-0.6 Nisin continued to culture for 5h;

[0071] 2) Centrifuge at 10,000 rpm for 20 minutes after the culture, take the supernatant, filter the supernatant with a 0.22 μm filter membrane, add N-lauroyl sarcosinate to a final concentration of 0.1%, and keep at room temperature for 15 minutes;

[0072] 3) Add trichloroacetic acid to a final concentration of 7.5%, mix well, and place on ice for 2 hours;

[0073] 4) Centrifuge at 10000rpm for 10min, discard the supernatant, add 2ml tetrahydrofuran, and centrifuge at 10000rpm for 10min;

[0074] 5) Centrifuge at 10000rpm for 10...

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Abstract

The invention provides a recombinant expression vector for expressing LL-37 polypeptide, recombinant lactococcus lactis, an antiviral drug, a construction method and application, and belongs to the technical field of gene engineering. A skeleton plasmid for constructing the recombinant expression vector is PNZ8149; a fusion gene is inserted into the recombinant expression vector; the nucleotide sequence of the fusion gene is shown as SEQ ID NO: 1. The recombinant expression vector disclosed by the invention can be efficiently expressed in lactococcus lactis, and the LL-37 obtained by expression can effectively inhibit SARS coronavirus and SARS-CoV2 coronavirus. The invention also provides recombinant lactococcus lactis containing the recombinant expression vector in the scheme. According to the recombinant lactococcus lactis disclosed by the invention, an antiviral polypeptide drug LL-37 can be input into a human body in an oral form, the antiviral effect is good, and the production cost is low.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant expression vector expressing LL-37 polypeptide, recombinant Lactococcus lactis, antiviral drugs, construction methods and applications. Background technique [0002] LL-37 is an antimicrobial peptide that also has antiviral activity. The traditional preparation method of LL-37 is artificial synthesis method, but the high cost of artificial synthesis method greatly limits the application of LL-37. The prior art discloses the use of genetic engineering methods to prepare LL-37 by constructing recombinant expression vectors containing LL-37 coding genes and recombinant bacteria, but the current methods still have low LL-37 expression efficiency and high production costs. question. Contents of the invention [0003] The object of the present invention is to provide a recombinant expression vector expressing LL-37 polypeptide, recombinant Lactococcus lact...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/62C12N1/21A61K35/744A61P31/04A61P31/12A61P31/14C12R1/01
CPCA61K35/744A61P31/04A61P31/12A61P31/14C07K14/47C07K2319/02C07K2319/50C12N15/74
Inventor 金万洙张瀚林蒋笑笑郑爱华
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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