Application of echinoderm MLKL

Abstract

The invention relates to the field of biology, in particular to construction and application of controllable expression vectors of MLKL (ApMLKL and ArMLKL) of two echinoderm animals. The ApMLKL and the ArMLKL are respectively shown as an amino acid sequence in a sequence table SEQ ID No. 1 and an amino acid sequence in a sequence table SEQ ID No. 2. The construction and induced expression method of the expression vectors of the ApMLKL and the ArMLKL comprises the following steps: respectively connecting genes of the ApMLKL and the ArMLKL to a pET30a expression vector to obtain recombinant plasmids pETApMLKL and pETArMLKL, transforming escherichia coli, and carrying out induced expression at 37 DEG C by using 0.2 mM of isopropyl-beta-D-thiogalactoside for 15-20 hours. After the ApMLKL and the ArMLKL are subjected to induced expression, survival and growth of host bacteria can be inhibited, and the ApMLKL and the ArMLKL have a remarkable bacteriostatic effect.

Description

Technical Field

[0001] This invention relates to the field of biology, specifically the application of two echinoderms, MLKL. Background Technology

[0002] MLKLs are the executors of necroptosis, capable of disrupting cell membranes and ultimately leading to cell lysis. In mammals, MLKL-mediated necroptosis plays a crucial role in regulating hematopoiesis, anti-infection, pulmonary edema, and a range of inflammatory responses. Unlike mammals, functional studies of MLKLs in echinoderms are limited, and related reports are scarce. Whether echinoderm MLKLs possess antibacterial functions is also unknown. Summary of the Invention

[0003] The purpose of this invention is to provide applications for two echinoderms, MLKL (ApMLKL and ArMLKL).

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] Applications of echinoderms MLKL, and its antibacterial properties.

[0006] The application of the echinoderm MLKL in inhibiting host bacteria.

[0007] The application of the echinoderm MLKL in inhibiting host bacteria under the inducer IPTG.

[0008] The echinoderm MLKL is either ApMLKL or ArMLKL; wherein the sequences of ApMLKL and ArMLKL are respectively the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 of the sequence listing.

[0009] The genes of ApMLKL and ArMLKL were respectively ligated into the pET30a expression vector to obtain recombinant plasmids pETApMLKL and pETArMLKL, which were transformed into Escherichia coli and induced to express with isopropyl-β-D-thiogalactoside, and then used for antibacterial applications in host bacteria.

[0010] The present invention has the following advantages:

[0011] The two echinoderms of the present invention, MLKL, after being induced and expressed in Escherichia coli, can inhibit the growth and survival of host bacteria; the antibacterial effect of MLKL of the present invention is controllable and only occurs when the inducer IPTG (isopropyl-β-D-thiogalactoside) is added. Attached Figure Description

[0012] Figure 1To illustrate the antibacterial effects of two echinoderms, MLKL, provided in this embodiment of the invention, DE3 / pET30a (control), DE3 / pETApMLKL, and DE3 / pETArMLKL were cultured on plates containing IPTG and without IPTG for 18 hours. Bacterial growth was observed (A), and bacterial counts were calculated (B). The experimental data in Figure B represent the results of three parallel experiments. * P<0.05. Detailed Implementation

[0013] The present invention will be further described below with reference to embodiments. These embodiments are intended to illustrate the invention and are not intended to limit the invention in any way.

[0014] Example 1

[0015] Construction of controllable expression vectors for ApMLKL and ArMLKL

[0016] (1) Construction of ApMLKL expression plasmid pETApMLKL

[0017] The ApMLKL protein sequence of this invention is the amino acid sequence listed in SEQ ID No. 1. SEQ ID No. 1 is:

[0018] MEVVKLAVDILQIAKKIFDVVEEVQHFKNRSRRLKHRIENLTPAIEKLASSEQAVPENARTPDGLKLFSKSLGQVLQLLIKIRDFIKSLDKWTFINKLLYKDRIGNQFDEYNDQLDCLAQAIH FSLAVENREIMYQFKNDKTDQDKDESDLIGEFQKGKEQILEQVARIAQGQEEVMKELHKIIAQRPALPTVFPTRQLMTTSDEDQRIKAELREIDPEQLESMSVMCTSRFGKIYKATYKQETVA VKSFSVSIDDSVKKVLLREAKNLLYLNSSYIVRLWGVCTRTDRCLLVFEYMEKGNLRDVLDEHGRDWTELPWARRIQMALDGALGLYRMHNSEPRPLMHQDLASDVFLVNASWGVKLSDVGFA ETRTTIEKHSKPLRTNRTTLNYISPERLANINLRPTAHTDIYSYGIVMWEIATGSRPYEGYTDQQIEDFVLEKKDKLALPDDCLDGFKALIDQCRSVDPLTRPTSGEIVAKLQELRASLEQTWC

[0019] (a) Sequence characteristics:

[0020] ·Length: 493

[0021] Type: Amino acid sequence

[0022] • Chain type: Single chain

[0023] • Topology: Linear

[0024] (b) Molecular type: protein

[0025] (c) Assumption: No

[0026] (d) Antonym: No

[0027] (e) Original source: Acanthasterplanci

[0028] Based on the ApMLKL gene sequence published by NCBI, the ApMLKL gene was synthesized by Beijing Tsingke Biotech Co., Ltd., and then ligated into the pET30a expression vector to obtain the recombinant plasmid pETApMLKL. In this plasmid, ApMLKL expression is only initiated in the presence of the inducer IPTG.

[0029] (2) Construction of the expression plasmid pETArMLKL for ArMLKL

[0030] The ArMLKL protein sequence of this invention is the amino acid sequence listed in SEQ ID No. 2. SEQ ID No. 2 is:

[0031] MVDIIEPAIMVLNVIKEIYTIAEKVKSFKSSSETLQGRIAKLTPAIKKLATSENKEMNQKSLKKAAVEGLLVLVEKIKNFLASLTHDSFFKKFVNANKIGKRFDEYSEQLDNLLND FQFLSLVQIEEASFNRIKQDEEDRKEDLKYFLQNTQRLEGLESGQQQIIDEIRKTSEKSEHNFYPGQDGRPEIDVHKLENKRTLSLDERFIYKASYMKETVVLKVYEKGSNLNEGR LFREIKNLIRLDSKNVVRIWGVCSTSIEPMIIMEYMERGNLRHVLDTTDPSMLPFHRRIQMALDGALGLYRIHNMDRAMVHRDLASHRFLVNAKWEVKISGLKMAKAYVSVWGSEI EKQRRRRMCYISPERTGHGLRDIDAPMEVYSYGIIMWEIATGLRPYEGKDCKEIEKFVRKKKKLPFPSTVTNEGLKELIDNCRSFRPLDRPTSSEIVDTLQGLRSAIEKSSGVPWQ

[0032] (a) Sequence characteristics:

[0033] · Length: 465

[0034] Type: Amino acid sequence

[0035] • Chain type: Single chain

[0036] • Topology: Linear

[0037] (b) Molecular type: protein

[0038] (c) Assumption: No

[0039] (d) Antonym: No

[0040] (e) Original source: Asterias rubens

[0041] Based on the ArMLKL gene sequence published by NCBI, the ArMLKL gene was synthesized by Beijing Tsingke Biotech Co., Ltd., and then ligated into the pET30a expression vector to obtain the recombinant plasmid pETArMLKL. In this plasmid, ArMLKL expression is only initiated in the presence of the inducer IPTG.

[0042] Example 2

[0043] Antibacterial effects of ApMLKL and ArMLKL

[0044] Step 1) Preparation of bacterial dilution solution

[0045] Plasmids pETApMLKL, pETArMLKL, and pET30a (control plasmid) were transformed into *E. coli* Transetta(DE3) using standard methods to obtain transformants DE3 / pETApMLKL, DE3 / pETArMLKL, and DE3 / pET30a. DE3 / pETApMLKL, DE3 / pETArMLKL, and DE3 / pET30a were cultured in LB liquid medium containing kanamycin (50 μg / ml) at 37°C until OD600. 600 The concentration should be 0.4-0.6. Dilute the bacterial culture with LB medium at a concentration of 10 μL. 4 The dilutions are DE3 / pETApMLKL, DE3 / pETArMLKL, and DE3 / pET30a.

[0046] Step 2) Detection of the antibacterial effects of ApMLKL and ArMLKL

[0047] Six LB agar plates containing kanamycin (50 μg / ml) were prepared and labeled af, with plates d, e, and f containing 0.2 mM IPTG. DE3 / pET30a dilution was spread on plates a and d, 600-800 CFU (Colony Forming Unit) per plate; DE3 / pETApMLKL dilution was spread on plates b and e, 600-800 CFU per plate; and DE3 / pETApMLKL dilution was spread on plates c and f, 600-800 CFU per plate. All plates were incubated at 37°C for 15-20 hours, and the bacterial count (represented by CFU) was observed. The results showed no significant difference in bacterial count between plates a and d, but the bacterial count on plate e was significantly lower than that on plate b, and the bacterial count on plate f was significantly lower than that on plate c. Figure 1 These results indicate that when IPTG induces the expression of ApMLKL and ArMLKL, it can inhibit the growth and survival of host bacteria, thus ApMLKL and ArMLKL have significant antibacterial effects.

[0048] The LB liquid culture medium consists of the following components by weight percentage: 1.0% peptone, 0.5% yeast extract, 1.0% sodium chloride, and 97.5% distilled water.

[0049] The LB solid culture medium consists of the following components by weight percentage: 1.0% peptone, 0.5% yeast extract, 1.0% sodium chloride, 1.5% agarose, and 96.0% distilled water.

Claims

1. The use of echinoderm MLKL, characterized in that: Application of echinoderm MLKL in antibacterial activities.

2. The use of echinoderm MLKL according to claim 1, characterized in that: The use of the echinoderm MLKL in inhibiting host bacteria.

3. The use of echinoderm MLKL according to claim 2, characterized in that: The use of the echinoderm MLKL in inhibiting host bacteria under the inducer IPTG.

4. The use of echinoderm MLKL according to any one of claims 1 to 3, characterized in that: The echinoderm MLKL is ApMLKL or ArMLKL; wherein the sequences of ApMLKL and ArMLKL are respectively shown in the amino acid sequences of SEQ ID No. 1 and SEQ ID No. 2 in the sequence table.

5. The use of echinoderm MLKL according to claim 3, characterized in that: The genes of ApMLKL and ArMLKL are connected to the pET30a expression vector respectively to obtain recombinant plasmids pETApMLKL and pETArMLKL, which are transformed into Escherichia coli and induced to express with isopropyl-β-D-thiogalactoside, and then used for the antibacterial application of host bacteria.

Images