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Secretory expression method of bacteriocin lacticin Q

An expression method and an expression vector technology, which are applied in the field of secretion and expression of bacteriocin lacticin Q, can solve problems such as unfavorable large-scale production and application, and difficulty in separation and purification of wild bacterial bacteriocin, and achieve the goal of simplifying the separation and purification process and increasing protein production Effect

Inactive Publication Date: 2013-07-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, people mainly obtain bacteriocins by screening and cultivating wild strains, but it is difficult to separate and purify wild strains of bacteriocins, which is not conducive to large-scale production and application
Bacteriocin is a protein substance synthesized through the ribosome pathway. Many researchers have carried out research on bacteriocin genetic engineering technology and achieved many important results, but it is still necessary to further increase the yield and simplify the separation and purification process.

Method used

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  • Secretory expression method of bacteriocin lacticin Q
  • Secretory expression method of bacteriocin lacticin Q
  • Secretory expression method of bacteriocin lacticin Q

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construction of embodiment 1 SUMO-lacticin Q fusion gene

[0042] According to the amino acid sequence of lacticin Q, its gene sequence (as shown in 345-504 in the sequence table SEQ ID NO.1) was synthesized, loaded into T vector, and transformed into Escherichia coli DH5α. The primers P1 (as shown in SEQ ID NO.3 in the sequence table) and P2 (as shown in SEQ ID NO.4 in the sequence table) were designed using the software Premier5.0 to amplify the lacticin Q gene.

[0043] PCR reaction system:

[0044]

[0045] Reaction program: denaturation at 95°C for 5min; denaturation at 95°C for 30Sec; annealing at 55°C for 30Sec; extension at 72°C for 1min, 25 cycles, and finally extension at 72°C for 8min. After cutting the target band by 2% agarose gel electrophoresis, it was purified and recovered with a DNA gel extraction kit.

[0046] Since the PCR product amplified by Taq DNA polymerase contains prominent A bases at both ends, and the carrier pET SUMO has been linearize...

Embodiment 2

[0047] Example 2 Expression plasmid construction

[0048] 1) PCR amplification of SUMO-lacticin Q fusion gene

[0049] According to the SUMO-lacticin Q fusion gene sequence and the pWB980 cloning site, primers P3 (as shown in the sequence table SEQ ID NO.5) and P4 (as shown in the sequence table SEQ ID NO.6) were amplified from the plasmid pET SUMO-lacticin Q. Add the coding sequence of the SUMO-lacticin Q fusion protein gene containing the His tag (as shown in the sequence table SEQ ID NO.1), wherein primer P3 is introduced into the HindIII site, and primer P4 is introduced into the XbaI site, so that the fusion gene is cloned into the pWB980 plasmid .

[0050] PCR reaction system:

[0051]

[0052] Reaction program: denaturation at 95°C for 5min; denaturation at 95°C for 30Sec; annealing at 49.5°C for 30Sec; extension at 72°C for 1min, 20 cycles, and finally extension at 72°C for 8min. After cutting the target band by 2% agarose gel electrophoresis, it was purified and...

Embodiment 3

[0055] The acquisition of embodiment 3 recombinant lacticin Q

[0056] 1) secreted expression

[0057] A single colony of the screened positive recombinant bacteria WB600 / pWB980 / SUMO-lacticin Q was inoculated into LB medium, and shaken at 150 rpm for 12 hours at 37°C. Then it was transferred to fresh LB medium at a ratio of 4%, and was shaken at 37°C and 200rpm for 36h to secrete and express SUMO-lacticin Q in the medium, and the expression effect was analyzed by Tricine-SDS-PAGE ( figure 1 ), SUMO-lacticin Q accounted for more than 21% of the total supernatant protein.

[0058] Tricine-SDS-PAGE analysis

[0059] The configuration of stacking gel and separating gel is as follows

[0060]

[0061] After the gel is solidified, add a quarter volume of 5xloading buffer to the sample, boil for 5min, and load the sample for electrophoresis after cooling.

[0062] The voltage of the stacking gel is 60V, and the voltage of the separating gel is 120V.

[0063] After electrophor...

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Abstract

The invention provides a secretory expression method of bacteriocin lacticin Q. The method comprises the following steps of: cloning lacticin Q and an SUMO fusion gene SUMO-lacticin Q to pWB980; transferring to bacillus subtilis; expressing the SUMO fusion gene SUMO-lacticin Q in a secretory manner in a culture medium; separating and purifying the SUMO fusion gene SUMO-lacticin Q I; and digesting and releasing the lacticin Q to further purify and obtain the lacticin Q. The activity detection confirms that the obtained recombinant protein is good in antibacterial activity which is equivalent to that of natural lacticin Q. According to the method provided by the invention, not only can the separating and purifying process of the lacticin Q be simplified, but also the yield of the lacticin Q is greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for secreting and expressing bacteriocin lacticin Q. Background technique [0002] Bacteriocin is a kind of polypeptide or protein with antibacterial activity produced by some bacteria through the ribosome synthesis mechanism. It is a kind of antimicrobial peptide derived from microorganisms. Bacteriocins have broad prospects in medicine, feed antibiotic substitutes, food bio-preservation, and plant bio-control. [0003] At present, there are hundreds of bacteriocins discovered by people, but very few of them are actually used. Nisin is a bacteriocin approved for food applications. In 1953, nisin was first introduced to the market in the UK. In 1969, nisin was approved by the United Nations The Food and Agriculture Organization / World Health Organization (FAO / WHO) recognized it as a safe, efficient and reliable food preservative, and was subsequently recognized by more than ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/315C12N15/62C12N15/75C12N1/21C12P21/02C12P21/06C12R1/125
Inventor 张日俊余占桥马青山
Owner CHINA AGRI UNIV
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