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Lactic acid bacterium double-gene expression box and construction method and application of lactic acid bacterium double-gene expression box

A construction method and expression cassette technology, applied in the field of genetic engineering, can solve the problems of lowering cholesterol, not having, not being able to degrade bile salts, etc., and achieve the effect of efficient co-expression

Inactive Publication Date: 2017-03-22
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although L. casei is able to tolerate the organism's defense mechanisms, including enzymes in the oral cavity, low pH in gastric juice, and bile acids in the small intestine, etc., the L. casei genome does not contain a bile salt hydrolase gene. Therefore, L. casei Can't degrade bile salts, so it doesn't have the effect of lowering cholesterol

Method used

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  • Lactic acid bacterium double-gene expression box and construction method and application of lactic acid bacterium double-gene expression box
  • Lactic acid bacterium double-gene expression box and construction method and application of lactic acid bacterium double-gene expression box
  • Lactic acid bacterium double-gene expression box and construction method and application of lactic acid bacterium double-gene expression box

Examples

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Embodiment 1

[0033] This example is the construction of the double-gene expression cassette and its expression vector for lactic acid bacteria.

[0034] The construction method of the lactic acid bacteria double-gene expression cassette and expression vector provided by the present embodiment comprises the steps:

[0035] Step 1, using the Lactobacillus casei LC2W genome as a template, using primers Pldh-F and primers Pldh-R to amplify to obtain a first fragment, the first fragment includes a first promoter, and the first promoter is a pldh promoter ;

[0036] Step 2, using the pIB184 plasmid as a template, amplifying with primers P23+ter-F and primers P23+ter-R to obtain a second fragment, which includes a second promoter and a second terminator, the second The promoter is the p23 promoter;

[0037] Step 3, using the pMG36e plasmid as a template, using primer PMG-F and primer PMG-R to amplify to obtain a third fragment, the third fragment includes the first terminator;

[0038] Step 4,...

Embodiment 2

[0044] This example is the verification of the expression vector obtained in Example 1.

[0045] The expression vector obtained in Example 1 was transformed into Escherichia coli Top10, and the transformed Escherichia coli was cultured on a plate at 37°C for 24 hours to obtain Escherichia coli colonies; a single colony on the plate was taken for liquid culture, and primers Pldh-F, P23+ ter-R carries out PCR amplification and obtains plasmid vector (that is, the expression vector that embodiment 1 obtains), and amplification result is as follows image 3 shown.

[0046] image 3 It is the electrophoresis diagram of the PCR verification of the expression plasmid in Example 2. Band M in the figure is DNA Marker DL2000, and bands 1, 2, 3, 4, 5, and 6 are the PCR verification bands of bacterial solution.

[0047] From image 3 It can be seen that the plasmid obtained by bacterial liquid PCR has a clearly visible band at about 950bp, which is in line with expectations.

[0048] ...

Embodiment 3

[0051] This example is an investigation of the transformation efficiency of the pMG-pldh-p23 expression vector in lactic acid bacteria.

[0052] In the present embodiment, the plasmid transformation adopts the electroporation method, and the specific steps are as follows:

[0053] Step 1: Add the plasmid obtained in Example 2 to the competent cells of Lactococcus lactis NZ9000, Lactobacillus casei LC2W and Lactobacillus plantarum WCFS1, mix gently, transfer to an electric shock cup pre-cooled on ice, and adjust the electroporation parameters (Lactococcus lactis NZ9000 and Lactobacillus casei LC2W are 2.0KV, Lactobacillus plantarum WCFS1 is 2.0KV, 4.0ms), electric shock electric conversion cup, then quickly add 1ml of pre-cooled recovery medium to the electric transformation cup, mix well Transfer to a 1.5ml centrifuge tube, and place in an anaerobic incubator for static recovery culture (cultivation conditions are: Lactococcus lactis NZ9000 is 30°C, 2h; Lactobacillus casei LC2...

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Abstract

The invention provides a lactic acid bacterium double-gene expression box which is used for conducting expression on an exogenous gene in the lactic acid bacterium after being inserted in the exogenous gene. The lactic acid bacterium double-gene expression box comprises a first expression box and a second expression box. The first expression box comprises a pldh promoter and a first terminator; and the second expression box comprises a p23 promoter and a second terminator. The invention further provides an expression carrier comprising the lactic acid bacterium double-gene expression box and a construction method of the expression carrier. By means of the expression carrier comprising the lactic acid bacterium double-gene expression box, coexpression of two objective exogenous genes in the lactic acid bacterium can be achieved, lactobacillus casei can express two kinds of bile salt hydrolase and show bile salt degradation activity.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a double-gene expression cassette for lactic acid bacteria and a construction method and application of the double-gene expression cassette. Background technique [0002] Lactic acid bacteria (such as Lactobacillus plantarum, Lactobacillus dryum and Streptococcus thermophilus, etc.) are recognized as safe-grade microorganisms, and many of them belong to probiotics, which can improve the intestinal flora, so they are widely used in In the production of food, health care products and medicines. At the same time, since the functional characteristics of natural strains can no longer meet the needs of the society, the current hot spot of lactic acid bacteria research is to carry out genetic engineering on them, so that various functional proteins can be expressed in the obtained recombinant lactic acid bacteria. [0003] At present, the expression vectors used in lactic acid bacteria ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/67
CPCC12N15/67C12N15/74C12N2800/60
Inventor 艾连中熊智强王光强夏永军张汇
Owner UNIV OF SHANGHAI FOR SCI & TECH
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