阅读说明：本技术 一种中华鳖的趋化因子蛋白的表达方法及应用 (Expression method and application of chemotactic factor protein of Chinese softshell turtle ) 是由 徐洁皓 钱国英 徐程 于 2019-11-16 设计创作，主要内容包括：本发明提供一种中华鳖的抗细菌病相关蛋白及其应用,即制备重组蛋白并鉴定重组表达产物的抑菌活性,并制备绿色饲料添加剂以及抗菌剂。本发明所提供对的中华鳖的趋化因子蛋白,其氨基酸序列为SEQ ID NO:1。本发明还提供一种用于在大肠杆菌中重组表达的上述趋化因子蛋白的核苷酸片段,其核苷酸序列为SEQ ID NO:3。本发明提供的基因与免疫抗病相关,其体外重组表达的蛋白能够明显抑制革兰氏阴性菌、革兰氏阳性菌的增殖,因此,在中华鳖病害防治、饲料添加剂及杀菌剂等方面有应用价值。(The invention provides an antibacterial disease related protein of Chinese softshell turtles and application thereof, namely preparing a recombinant protein, identifying the bacteriostatic activity of a recombinant expression product, and preparing a green feed additive and an antibacterial agent. The amino acid sequence of the chemotactic factor protein of the Chinese softshell turtle provided by the invention is SEQ ID NO. 1. The invention also provides a nucleotide fragment of the chemotactic factor protein for recombinant expression in escherichia coli, and the nucleotide sequence is SEQ ID NO. 3. The gene provided by the invention is related to immune disease resistance, and the in vitro recombinant expressed protein can obviously inhibit the proliferation of gram-negative bacteria and gram-positive bacteria, so that the gene has application values in the aspects of disease control of the Chinese softshell turtle, feed additives, bactericides and the like.)

1. A chemokine protein of a trionyx sinensis, said protein comprising:

1) protein with amino acid sequence of SEQ ID NO. 1;

2) the protein sequence in 1) is substituted, deleted or added with one or more amino groups, has 1) protein functions and is derived from 1).

2. A gene encoding the chemokine protein of claim 1.

3. The gene of claim 2, wherein one nucleotide sequence of the gene is SEQ ID NO 2.

4. A nucleotide fragment for recombinant expression of the chemokine protein of claim 1 in E.coli, wherein the nucleotide sequence of said fragment is SEQ ID NO 3.

5. A recombinant expression vector carrying the nucleotide fragment of claim 4.

6. A recombinant Escherichia coli engineering bacterium, which is transformed with the recombinant expression vector of claim 5.

7. The use of the recombinant engineered Escherichia coli of claim 6 in the preparation of products, feed additives or antibacterial agents for preventing and treating bacterial diseases of Chinese softshell turtles.

Technical Field

The invention belongs to the technical field of aquatic product immune control, and particularly relates to an antibacterial disease-related protein of Chinese softshell turtles and application thereof.

Background

Chinese soft-shelled turtle (Pelodiscus sinensis) is a traditional freshwater aquaculture variety in China, has high yield in Zhejiang, Hunan, Hubei, Jiangxi, Anhui, Jiangsu and other provinces, is popular with consumers due to delicious meat and rich nutrition, and has good market demand. In recent years, due to the fact that the culture density is increased and the culture environment is increasingly poor, the Chinese soft-shelled turtles are damaged by various disease microorganisms in the culture process, and huge economic losses are caused to farmers.

At present, farmers mostly adopt chemical drugs such as antibiotics for treatment, and have larger hidden dangers of ecological environment pollution and food safety. Therefore, the research on the immune mechanism of the Chinese soft-shelled turtle is focused, and more environment-friendly and effective antibiotic substitutes or immune control products are developed, so that a novel culture management mode beneficial to disease control and continuous industry development of the Chinese soft-shelled turtle is formed, and the healthy development of the whole Chinese soft-shelled turtle culture industry is maintained.

Chemokines are small molecule proteins with chemoattractant and secretory properties that can induce migration and activation of leukocyte populations and modulate immune responses by activating chemokine receptors. The chemotactic factor has bactericidal and bacteriostatic activity and is a potential antibiotic substitute; and can recruit leucocytes, activate immune cells and promote polarization of CD4+ T cells. In addition, the chemotactic factor is taken as an immune regulatory factor produced by a host, and in the immune response triggered by the vaccine, the chemotactic factor can play a role of antigen presentation at the injection site in the vaccine induction stage and can play a role of immune effector molecules in the later effect stage, so that the activity and the function of the chemotactic factor are enhanced or the expression and the secretion of bioactive molecules in cells are promoted, and the chemotactic factor has good immune adjuvant performance.

At the early stage, the chemotactic factor A of the Chinese softshell turtle capable of responding to external LPS immune stimulation is obtained by analyzing and screening based on transcriptomics technology and bioinformatics. Chemokine a, secreted by macrophages and leukocytes, was first isolated from LPS-stimulated macrophages, plays an important role in cell migration, Th1 cell differentiation, and regulation of CD8+ T cell signaling. In aquatic animals, chemokine a has been shown to be involved in innate and adaptive immunity in certain teleost fish, where LPS is capable of inducing upregulation of chemokine a gene transcript levels in the intestinal, ovarian and spleen of the body; in Trachinotus ovatus (Trachinotus ovatus), has antibacterial effect; in megalobrama amblycephala, a scholar preliminarily proves that the chemotactic factor A has a potential pro-inflammatory effect; in grouper (Epinephalus coioides), researchers find that LPS and poly (I: C) can promote the increase of the mRNA expression amount of the chemokine A, and meanwhile prove that the chemokine A can activate leukocytes, induce inflammatory response and promote lymphocyte differentiation to a Th1 pathway.

Because chemokine proteins contain transmembrane domains and are of small molecular weight, there are certain difficulties in prokaryotic expression and protein purification. The invention optimizes the nucleotide of the chemotactic factor A, solves the problems of difficult expression and difficult purification and further improves the expression quantity. Meanwhile, the research on the chemotactic factor is not available in the Chinese softshell turtle, and the research on the application of the chemotactic factor to antibacterial immunity is not available. Therefore, the project not only provides a feasible nucleotide modification method to obtain in-vitro protein, but also further researches the immune response of the chemotactic factor A in vivo, finds out whether the chemotactic factor A has the application and popularization values as an antibacterial drug and an immunologic adjuvant in vitro, and lays a foundation for the immune control of the diseases of the Chinese softshell turtles.

Disclosure of Invention

The invention provides an antibacterial disease related protein of Chinese softshell turtles and application thereof, namely preparing a recombinant protein, identifying the bacteriostatic activity of a recombinant expression product, and preparing a green feed additive and an antibacterial agent.

The invention firstly provides a chemotactic factor protein of Chinese softshell turtle, comprising:

1) protein with amino acid sequence of SEQ ID NO. 1;

2) the protein sequence in 1) is substituted, deleted or added with one or more amino groups, has 1) protein function and is derived from 1);

MKVSVAALAVLLIAAFCSQASSAPIGSDPPTACCFTYASRKIPRTLVE DYYDTNSMCSQPAIVFITKKGREICANPQADWVQEYVAYLDQN(SEQ ID NO:1)；

the nucleotide sequence of the gene for coding the chemotactic factor protein is SEQ ID NO. 2;

ATGAAGGTCTCCGTGGCTGCCCTCGCCGTCCTGCTCATCGCGGCC TTCTGCTCCCAGGCCTCCTCCGCCCCAATTGGCTCCGACCCCCCGACT GCCTGCTGCTTTACCTACGCCTCTCGGAAGATCCCGCGCACCTTGGTGGAGGATTATTATGATACCAACAGCATGTGCTCCCAGCCTGCCATTGTCT TCATCACAAAGAAGGGCCGGGAGATCTGTGCCAACCCCCAGGCGGA CTGGGTCCAGGAGTATGTGGCTTACTTGGATCAGAACTGA；

the invention also provides a nucleotide fragment of the chemotactic factor protein for recombinant expression in escherichia coli, and the nucleotide sequence of the nucleotide fragment is SEQ ID NO: 3:

ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTG CCGCGCGGCAGCCATATGACCTATGCGAGCCGCAAAATTCCGCGCAC CCTGGTGGAAGATTATTATGATACCAACAGCATGTGCAGCCAGCCGGC GATTGTGTTCATTACCAAAAAAGGCCGCGAAATTTGCGCGAACCCGC AGGCGGATTGGGTGCAGGAATATGTGGCGTATCTGGATCAGAACACCT ATGCGAGCCGCAAAATTCCGCGCACCCTGGTGGAAGATTATTATGATA CCAACAGCATGTGCAGCCAGCCGGCGATTGTGTTCATTACCAAAAAA GGCCGCGAAATTTGCGCGAACCCGCAGGCGGATTGGGTGCAGGAATA TGTGGCGTATCTGGATCAGAACCTCGAGCACCACCACCACCACCACT GA；

in still another aspect, the invention provides a recombinant expression vector, wherein the recombinant expression vector carries the fragment with the nucleotide sequence of SEQ ID NO. 3;

the invention also provides a recombinant escherichia coli engineering bacterium, wherein the recombinant escherichia coli engineering bacterium is transformed with the recombinant expression vector;

the recombinant Escherichia coli engineering bacteria recombinant expression protein is used for preparing products for preventing and treating bacterial diseases of Chinese softshell turtles, feed additives and antibacterial agents.

The gene provided by the invention is related to immune disease resistance, and the in vitro recombinant expressed protein can obviously inhibit the proliferation of gram-negative bacteria and gram-positive bacteria, so that the gene has application values in the aspects of disease control of the Chinese softshell turtle, feed additives, bactericides and the like.

Drawings

FIG. 1: the chemokine gene amplification map of the invention, wherein lanes 1, 3 are beta-actin electrophoresis in small intestine and large intestine samples; lanes 2, 4 are chemokine a in small and large intestine samples;

FIG. 2: the qRT-PCR quantitative detection gene expression level In each tissue of adult healthy Chinese soft-shelled turtle is shown In the drawing, wherein the abscissa represents different tissues of the Chinese soft-shelled turtle (Ki: kidney Lu: lung IC: large intestine Li: liver In: small intestine Sp: spleen He: heart Mu: muscle), and the ordinate represents the expression level of the gene In different tissues;

FIG. 3: expression analysis chart of chemotactic factor A gene of Chinese softshell turtle in immune related tissue after infection stimulated by LPS; the abscissa represents the time points after infection, and the ordinate represents the expression amounts of the genes associated with bacterial diseases in different tissues. A total of 6 tissues were examined, including: intestine (Intestine), Lung (Lung), Liver (Liver), Spleen (Spleen).

FIG. 4: inducible expression profiling of recombinant proteins was performed using a nucleotide fragment of sequence SEQ ID NO 2, wherein lanes 1-6 are: 1. non-induced bacterial liquid; 2.0.5 mmol of IPTG inducing at 28 ℃ for 12h 3.0.5 mmol of IPTG inducing at 37 ℃ for 12h 4.1 mmol of IPTG inducing at 28 ℃ for 12h 5.1 mmol of IPTG inducing at 37 ℃ for 12h 6.1 mmol of IPTG inducing at 37 ℃ for 24 h;

FIG. 5: inducing expression of recombinant protein by using modified nucleotide fragment with sequence of SEQ ID NO. 3, wherein a is an electrophoresis picture of expressing BL21 expression protein by a small amount of a chemotactic factor A-like prokaryotic expression plasmid, M: high molecular weight protein marker; 1: not inducing; 2: inducing for 1 hour; 3: inducing for 2 hours; 4: inducing for 4 hours; 5: inducing overnight;

panel b is a soluble analysis of the recombinant protein, M: high molecular weight protein marker; 1: non-induced bacteria liquid; 2: performing whole bacteria; 3: precipitating after ultrasonic crushing; 4: supernatant after ultrasonic crushing;

FIG. 6: separating and purifying the reconstructed chemotactic factor A recombinant protein of the trionyx sinensis, wherein M in the picture a is a high molecular weight protein marker; 1-6: sequentially eluting the obtained solution every 0.5mL of Elution Buffer D; 7: non-induced bacteria liquid; 8: inducing bacterial liquid; m in FIG. b: high molecular weight protein marker; the resulting solutions were eluted sequentially every 0.5mL of Elution Buffer E from 1 to 6.

FIG. 7: an in vitro antibacterial activity analysis diagram of the recombinant protein, wherein (1) the effect schematic diagram of the recombinant protein of the chemokine A for inhibiting gram-negative bacteria escherichia coli (E.coli) and aeromonas hydrophila (A.hydrophylla) is shown, and 0 mu g/mL of PBS is used as a negative control group; the rest are 3 recombinant proteins with different concentrations as experimental groups: the effect of the recombinant proteins of 1 mug/mL, 2 mug/mL and 4 mug/mL (2) of the chemokine A on inhibiting gram-positive bacteria Bacillus thuringiensis (Bacillus thuringiensis) is shown schematically, and PBS is used as a negative control group at 0 mug/mL; the rest are 3 recombinant proteins with different concentrations as experimental groups, which are 1. mu.g/mL, 2. mu.g/mL and 4. mu.g/mL respectively.

Detailed Description

In order to make the objects, technical lines and advantages of the present invention more apparent, examples of the present invention are described in detail below with reference to the accompanying drawings.