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A food-grade Lactococcus lactis with surface-anchored human I-type metallothionein and its preparation method and application

A technology of Lactococcus lactis and metallothionein, which is applied in the field of preparation of food-grade Lactococcus lactis, can solve the problems of low expression level and introduction of resistance genes, etc., and achieve the effects of reducing protein loss, low cost, and omitting cumbersome steps

Active Publication Date: 2017-06-20
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of lactic acid bacteria to express foreign genes has low expression levels, recombinant lactic acid bacteria will introduce foreign genes, and using antibiotics as screening markers will introduce resistance genes and other problems that cannot be ignored.

Method used

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  • A food-grade Lactococcus lactis with surface-anchored human I-type metallothionein and its preparation method and application
  • A food-grade Lactococcus lactis with surface-anchored human I-type metallothionein and its preparation method and application
  • A food-grade Lactococcus lactis with surface-anchored human I-type metallothionein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Construction of Recombinant cA-hMT Protein Engineering Bacteria

[0045] The cA gene and the hMT gene were spliced ​​by overlapping PCR, a linker was introduced between the two genes, and an expression-promoting tag was introduced at the N-terminus. After double digestion with NdeI and XhoI, it was ligated with the expression vector pET21a to realize the recombinant plasmid pET21a-cA-hMT The construction of , the specific construction process is as follows figure 1 shown.

[0046] 1. Design and synthesis of target gene fragments and primers

[0047] According to the Lactococcus lactis MG1363 N-acetylmuramidase gene sequence (GenBank ID: CAL96887.1), a linker (SEQ ID No.3) was introduced at its C-terminus, thereby designing primers, wherein the forward primer is F1:

[0048] 5'-AATTC CATATG GGATCCAATACTAATTCTGGTGGCTC-3', containing restriction site NdeI (underlined part), the reverse primer is R1:

[0049] 5'-TGAACAATTAGGATCCATCTGCAGACCACCACCTTTTATTCGTAGATACT-3', co...

Embodiment 2

[0079] Expression and Identification of cA-hMT Recombinant Protein

[0080] Method: The plasmids extracted from the positive clones in the above-mentioned Example 1 and the plasmids containing molecular chaperones (dnaK, dnaJ, grpE, groES and groEL) were transformed into Escherichia coli expression strain competent cells BL21 (DE3), picked and identified by colony PCR The positive clones were inoculated into 3 mL of LB liquid medium containing 100 μg / mL Amp, 25 μg / mL chloramphenicol, and 1 mg / mL L-arabinose for expansion at 37 ° C. 600 Add 0.5mM IPTG when it is 0.5, collect the bacteria by centrifugation after 12 hours of low-temperature induction, ultrasonically lyse, add loading buffer to the supernatant and precipitate, and boil in water for 5 minutes. Concentrating gel 80V, separating gel 120V for SDS-PAGE electrophoresis, after electrophoresis, stain with Coomassie brilliant blue staining solution, and decolorize with decolorizing solution.

[0081] Result: if image 3 ...

Embodiment 3

[0084] Anchoring of recombinant hMT fusion protein to lactic acid bacteria and detection

[0085] 1. Culture of Lactococcus lactis and preparation of acid-treated Lactococcus lactis

[0086] method:

[0087]Streak a small amount of glycerol-frozen strains onto the solid medium of GM17 plates, and place them in an incubator at 30°C for static culture for 24 hours. Pick a single colony and inoculate it in 5 mL of GM17 liquid medium and culture it statically at 30°C for 12 hours. Take the overnight culture of L. lactis MG1363, inoculate it into 500 mL of fresh GM17 medium at a ratio of 1:100, and culture it statically at 30°C for 16 hours. Collect the cells by centrifugation, wash once with 250 mL of sterile water, resuspend the cells in 100 mL of acetic acid solution (pH=1), and boil in boiling water for 30 min. Wash 3 times with 250 mL of sterile PBS buffer, resuspend the bacteria with 50 mL of sterile PBS buffer, and store at 4°C.

[0088] 2. Back anchoring of cA-hMT prote...

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Abstract

The invention relates to food-grade lactococcus lactis with human metallothionein-I anchored on the surface and a preparation method and a use thereof. The preparation method comprises the following steps of 1, splicing a cA gene and an hMT gene by overlapping PCR, introducing linker between the two genes, and introducing an expression promotion label to an end N to obtain a recombinant cA-hMT gene, 2, connecting the recombinant cA-hMT gene and an expression carrier to obtain a connection product and transferring the connection product into a escherichia coli competent cell, and 3, carrying out inducible expression of the recombinant fusion protein by engineering bacteria obtained by the step 2, carrying out ultrasonication on the obtained engineering bacteria, then carrying out centrifugation and carrying out incubation of food-grade lactococcus lactis by the supernatant so that the fusion protein is anchored on the surface of the food-grade lactococcus lactis without protein purification. The prepared food-grade lactococcus lactis with human metallothionein-I anchored on the surface can enrich heavy metals. The preparation method has the advantages of high efficiency, safety, no toxicity, low cost and no protein purification.

Description

technical field [0001] The invention belongs to the technical field of gene recombination expression in the field of genetic engineering, and in particular relates to a preparation method and application of food-grade Lactococcus lactis whose surface is anchored with human type I metallothionein. Background technique [0002] Metallothionein (Metallothionein, MT) has a very conservative primary structure and similar spatial structure, widely distributed in animals, plants and microorganisms, is a class of cysteine-rich, low molecular weight (6000-7000 Daltons) ), proteins with strong ability to bind metal ions (Vasak M. Advances in metallothioneinstructure and functions. J Trace Elem Med Biol, 2005, l19:13-17.). MT has important physiological functions such as binding heavy metals, scavenging free radicals, anti-radiation, anti-aging, and regulating trace element balance (Yang F et al. 186–194.). At present, MT has become a popular research topic in the fields of medicine,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/70C12N15/62A61K35/744A61P39/02C02F3/34B01D53/84C12R1/19C12R1/01C02F101/20
CPCC07K14/825C12N15/746Y02A50/20
Inventor 王菊芳马毅李杉苏艳芳
Owner SOUTH CHINA UNIV OF TECH
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