CN110684788B - Goldfish c-type lysozyme subtype and coding gene and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种金鱼c型溶菌酶两个亚型及其所编码基因和应用。本发明首次从金鱼肾脏组织中克隆到了一种包含两个亚型的c型溶菌酶(gfLyz‑C1、gfLyz‑C2)，两个亚型的核苷酸序列分别如SEQ ID NO.1和SEQ NO.2所示，其编码的c型溶菌酶氨基酸序列分别如SEQ ID NO.5和SEQ NO.6所示。本发明发现通过大肠杆菌重组表达获得的gfLyz‑C1、gfLyz‑C2两个重组蛋白，均对水产动物常见的几种病原细菌表现出抑菌作用和溶菌活性，尤其是当两个溶菌酶亚型蛋白联合使用时，对迟钝爱德华氏菌的溶解作用会呈现显著的叠加效应，在淡水水产养殖中具有应用前景。

The invention discloses two subtypes of a goldfish c-type lysozyme, the encoded genes and the application. The present invention clones a c-type lysozyme (gfLyz-C1, gfLyz-C2) containing two subtypes from goldfish kidney tissue for the first time, and the nucleotide sequences of the two subtypes are as shown in SEQ ID NO.1 and SEQ ID NO.1 respectively. As shown in NO.2, the encoded c-type lysozyme amino acid sequences are shown in SEQ ID NO.5 and SEQ NO.6, respectively. The present invention finds that two recombinant proteins, gfLyz-C1 and gfLyz-C2 obtained by recombinant expression of Escherichia coli, both exhibit bacteriostatic effect and bacteriolytic activity on several common pathogenic bacteria of aquatic animals, especially when two lysozyme subtypes When the protein is used in combination, the dissolution effect on Edwardsiella tarda has a significant additive effect, which has application prospects in freshwater aquaculture.

Description

Goldfish c-type lysozyme subtype and coding gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering and aquaculture, in particular to goldfish c-type lysozyme containing two subtypes and a coding gene and application thereof.

Background

Goldfish (Carassius auratus), also known as golden crucian, belongs to the order Cyprinales and family Cyprinaceae, and is an ornamental fish domesticated from wild crucian. Goldfish originates from China and is one of three famous ornamental fishes in the world. At present, the goldfish breeding industry is rapidly developed, and all provinces and cities except Tibet have goldfish production and breeding, wherein Beijing, Suzhou, Fuzhou and Guangzhou are the main breeding areas. The germplasm resources of Chinese goldfish are rich, including 46 strains and 315 varieties, wherein 70 percent of the varieties belong to undeveloped basic varieties and have great development potential. However, the current goldfish breeding industry has the problems of low breeding technology level, laggard disease control technology, lack of special feed and functional feed and the like, and brings great obstacles to the breeding development of goldfishes. Compared with other common edible fishes, the problems in the cultivation of ornamental fishes, particularly the problem of frequently outbreak bacterial diseases, are relatively lacked. Among them, Edwardsiella tarda (Edwardsiella tarda) is also called Edwardsiella tarda or Edwardsiella tarda, is a pathogenic bacterium which seriously harms aquatic animals, can infect more than twenty common economic fishes, such as eel, paralichthys olivaceus, tilapia, carp, goldfish and the like, and causes huge loss to aquaculture. Edwardsiella tarda of goldfishes can occur all the year round, mainly occurs at the water temperature of more than 15 ℃, the peak of disease occurrence is more than 25-30 ℃, and the goldfishes are infected mainly by contacting with diseased fish with Edwardsiella tarda.

Lysozyme, also known as muramidase, is an alkaline enzyme that hydrolyzes mucopolysaccharides in pathogenic bacteria. The bacterial lysis is achieved by breaking the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the bacterial cell wall, breaking down the insoluble mucopolysaccharide into soluble glycopeptides, causing the contents of the broken cell wall to escape. Lysozyme in animals mainly includes three types, i.e., c-type (chicken-type), g-type (goose-type) and i-type (invertebrate-type) lysozyme, in which vertebrates have both c-type and g-type lysozyme genes. The research on the bacteriolytic and antibacterial functions of fish lysozyme can understand the innate immune defense mechanism of fish, can also be used as the basis for preparing novel antibacterial peptide products, and has wide application prospect in the aspect of fish non-antibiotic immune defense products. However, the current research on fish lysozyme mainly focuses on fishes such as turbot, tilapia, grouper and the like, for example: chinese patent CN201210452265.6 discloses a turbot C-type lysozyme and construction and application thereof; chinese patent CN201410185795.8 discloses a preparation method of lysozyme-transgenic tilapia; the Yushao et al disclose the cloning and sequence analysis research of 3C-type lysozyme genes of Oria tilapia (Yushao, leaf star, Zulili, etc.. cloning and sequence analysis of 3C-type lysozyme genes of Oria tilapia [ J ]. agricultural biotechnology bulletin, 2010,18(1): 66-74.); qinhiwei et al disclose the cloning and research of two lysozyme genes of grouper (molecular cloning and function research of two lysozyme genes of grouper [ C ]// "ecological safety of ocean and lake and marsh under global change" academic exchange paper abstract collection 2014.). At present, no related research report about goldfish lysozyme is found, and the research on the goldfish lysozyme has great significance for preventing and treating aquatic animal diseases.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a coding gene of a goldfish c-type lysozyme subtype.

The invention also aims to provide the goldfish c-type lysozyme.

The invention further aims to provide application of the goldfish c-type lysozyme.

The above object of the present invention is achieved by the following technical solutions:

the nucleotide sequences of encoding genes gfLyz-C1 and/or gfLyz-C2 and gfLyz-C1 of the goldfish C-type lysozyme subtype are shown in SEQ ID NO.1 and SEQ ID NO. 3; the nucleotide sequence of gfLyz-C2 is shown in SEQ ID NO.2 and SEQ ID NO. 4.

The invention clones two subtypes (gfLyz-C1 and gfLyz-C2) of goldfish C-type lysozyme from goldfish kidney tissues for the first time, the nucleotide sequences of the two subtypes are respectively shown as SEQ ID NO.1 and SEQ NO.2, and the CDS coding regions are respectively shown as SEQ ID NO.3 and SEQ NO. 4; the corresponding coded c-type lysozyme amino acid sequences are respectively shown as SEQ ID NO.5 and SEQ NO. 6. It is also within the scope of the present invention to modify the nucleotide sequence of the above-described encoding gene without changing the amino acid sequence, taking into account the degeneracy of the codon.

The invention also claims a primer pair for amplifying the gfLyz-C1 or gfLyz-C2 gene, which is characterized in that the amplification primer pair of the gfLyz-C1 gene is shown as SEQ ID NO.7 and SEQ ID NO.8, and the amplification primer pair of the gfLyz-C2 gene is shown as SEQ ID NO.9 and SEQ ID NO. 10.

The invention also claims a recombinant expression vector containing the gfLyz-C1 and/or gfLyz-C2 gene.

Preferably, the recombinant expression vector is a prokaryotic expression vector pET-28 a.

The invention also claims a host bacterium containing the recombinant expression vector.

Preferably, the host bacterium is escherichia coli BL 21.

The invention also claims a cell line containing the host bacterium.

The research of the invention finds that the two recombinant proteins of gfLyz-C1 and gfLyz-C2 obtained by recombinant expression of escherichia coli are respectively applied to several common pathogenic bacteria of aquatic animals: the micrococcus muralis, the vibrio parahaemolyticus, the escherichia coli or the edwardsiella tarda and the like show bacteriostatic action and bacteriolytic activity, so that the invention firstly protects the application of the gfLyz-C1 and/or the gfLyz-C2 in preventing and treating pathogenic bacteria of aquatic animals, or the application in preparing medicaments for preventing and treating bacterial diseases of aquatic animals, or the application in preparing bacteriolytic medicaments for preventing and treating pathogenic bacteria of aquatic animals.

Specifically, the aquatic animal bacterial diseases are diseases caused by one or more pathogenic bacteria of micrococcus muralis, vibrio parahaemolyticus, escherichia coli or edwardsiella tarda.

The invention also finds that when two lysozyme protein subtypes of gfLyz-C1 and gfLyz-C2 are used in combination, the dissolving effect on Edwardsiella tarda can show a remarkable additive effect. Therefore, the goldfish C-type lysozyme subtypes gfLyz-C1 and gfLyz-C2 are jointly used for preparing the prevention and treatment medicines for aquatic animal bacterial diseases, particularly the diseases caused by Edwardsiella tarda; the combined use ratio is 1: 1.

Preferably, the aquatic animal is a goldfish.

Compared with the prior art, the invention has the following beneficial effects:

the invention clones a C-type lysozyme (gfLyz-C1, gfLyz-C2) containing two subtypes from goldfish kidney tissues for the first time, the nucleotide sequences of the two subtypes are respectively shown as SEQ ID NO.1 and SEQ NO.2, and the amino acid sequences of the coded C-type lysozyme are respectively shown as SEQ ID NO.5 and SEQ NO. 6. The invention discovers that the two recombinant proteins of gfLyz-C1 and gfLyz-C2 obtained by recombinant expression both show bacteriostatic action and bacteriolytic activity on several common pathogenic bacteria of aquatic animals, and particularly, when two lysozyme subtype proteins are used in combination, the two recombinant proteins can show obvious additive effect on the lysis of Edwardsiella tarda, so that the two recombinant proteins have application prospects in freshwater aquaculture.

Drawings

FIG. 1 shows the nucleotide sequences of goldfish C-type lysozyme gfLyz-C1 and gfLyz-C2 and the proteins encoded by the nucleotide sequences.

FIG. 2 shows the domains and three-dimensional protein structures of goldfish C-type lysozyme gfLyz-C1 and gfLyz-C2

FIG. 3 shows the evolution analysis of goldfish C-type lysozyme gfLyz-C1, gfLyz-C2 and other lysozyme proteins

FIG. 4 shows the recombinant expression and purification of Goldfish C-type lysozyme gfLyz-C1 and gfLyz-C2 in E.coli

FIG. 5 shows the killing and lysis effects of recombinant proteins of Goldfish C-type lysozyme gfLyz-C1 and gfLyz-C2 on Micrococcus muralyticus (M. lysodeikticus), Vibrio parahaemolyticus (V. parahaemolyticus), Escherichia coli (E.coli) and Edwardsiella tarda in plate bacteriolysis assay and bacteriolysis turbidity assay.

FIG. 6 shows the combined effect of recombinant proteins of Goldfish C-type lysozyme gfLyz-C1 and gfLyz-C2 in lysis of Edwardsiella tarda.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 cDNA cloning and sequencing of Goldfish C-type Lyz-C1, gfLyz-C2 Lyz-lysozyme

Screening the sequence of the gfLyz-C1 and gfLyz-C2 transcripts from the constructed goldfish kidney transcriptome library, and designing an upstream primer (5'-ATGAG GGTGG CTGTT GTT-3') and a downstream primer (5'-GTGCT CCTCA CATCC TTTCA CCCAG CGTGT-3') according to the sequence for amplifying the gfLyz-C1; the forward primer (5'-ATGAA GGTGG CGATT GCG-3') and the reverse primer (5'-GTGCT CCTCA CAACC TTTCA CCCAG CGTGA-3') were used to amplify gfLyz-C2.

Total RNA was extracted from the kidney tissue of Goldfish by Trizol method (Invitrogen). Using extracted RNA of goldfish kidney tissue as template, PrimeScript^TMRT Kit (TaKaRa) was reverse transcribed into first strand cDNA. PCR-amplifying a gfLyz-C1 and a gfLyz-C2 cDNA fragment using cDNA as a template and the above-mentioned forward primer and reverse primer; the PCR reaction system is as follows: rTaq mixture (TaKaRa) 12.5. mu.L,cDNA template 1. mu.L, upstreamprimer 1. mu.L,downstream primer 1. mu.L, and sterilized water 9.5. mu.L; the PCR reaction program is: pre-denaturation at 94 ℃ for 3 min; 30 cycles: denaturation at 94 ℃ for 15 seconds, renaturation at 56 ℃ for 15 seconds, and extension at 72 ℃ for 1 minute; final extension at 72 ℃ for 3 min. The band of interest was recovered by agarose gel electrophoresis using an agarose gel recovery Kit (Tiangen organism), and the amplified fragments of gfLyz-C1 and gfLyz-C2 were TA cloned using pMD18-T Ligation Kit (TaKaRa). And transforming the ligation product into escherichia coli DH5 alpha competent cells by using a heat shock method, obtaining positive clones by blue-white screening, carrying out enlarged culture, extracting plasmids and sequencing.

The result is shown in FIG. 1, the obtained gfLyz-C1 nucleotide sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO. 3; the obtained gfLyz-C2 nucleotide sequences are respectively shown as SEQ ID NO.2, and the corresponding amino acid sequences are shown as SEQ ID NO. 4. The domain prediction and three-dimensional protein structure prediction of goldfish C-type lysozyme gfLyz-C1 and gfLyz-C2 are shown in FIG. 2. Evolutionary analysis of goldfish C-type lysozyme gfLyz-C1, gfLyz-C2 and other lysozyme proteins is shown in FIG. 3, which indicates that gfLyz-C1 and gfLyz-C2 are all newly obtained C-type lysozyme genes.

Example 2 prokaryotic recombinant expression of the gfLyz-C1, gfLyz-C2 proteins

Respectively subcloning the mature peptide parts of gfLyz-C1 and gfLyz-C2 to a prokaryotic expression vector pET-28a by adopting a PCR amplification and restriction enzyme digestion method, obtaining positive clones by screening kanamycin, transforming the positive clones to BL21(DE3) escherichia coli, and carrying out amplification culture. The gfLyz-C1, gfLyz-C2 recombinant proteins were obtained by induction with 0.1mM IPTG at 28 ℃ for 24 hours. The pure proteins of gfLyz-C1 and gLyz-C2 were obtained by His-Bind Kits (Novagen) affinity chromatography. Desalting was by PD-10Desalting Columns (GE Healthcare). Induced expression and purification of the gfLyz-C1 and gfLyz-C2 recombinant proteins are shown in FIG. 4, the size of the gfLyz-C1 recombinant protein is about 16.3kDa, and the size of the gfLyz-C2 recombinant protein is about 16.4kDa, which is consistent with the expected size, indicating that the gfLyz-C1 and gfLyz-C2 recombinant proteins are successfully obtained.

Example 3 plate lysis assay and lysis turbidity assay of gfLyz-C1, gfLyz-C2 recombinant proteins

Lysis analysis was performed on plates containing Micrococcus muralis, Vibrio parahaemolyticus, Escherichia coli, and Edwardsiella tarda at OD600 ═ 0.4, respectively, using a 1% agarose gel prepared in PBS (50mM, pH 6.2). The recombinant proteins gfLyz-C1 and gfLyz-C2 at a concentration of 20. mu.g/. mu.L in a volume of 50. mu.L were added to round wells of 6 mm in diameter, incubated at 30 ℃ for 24 hours and the size of the lysosome was measured. The plate bacteriolytic capacity of the gfLyz-C1 and gfLyz-C2 recombinant proteins is shown in FIG. 5. The results show that: the gfLyz-C1 and gfLyz-C2 recombinant proteins have an antibacterial effect on Micrococcus muralis, Vibrio parahaemolyticus, Escherichia coli and Edwardsiella tarda, and especially have an obvious antibacterial effect on Micrococcus muralis, Vibrio parahaemolyticus and Edwardsiella tarda.

Turbidity analysis was used to detect the lytic activity of the gfLyz-C1, gfLyz-C2 recombinant proteins. 2mL of each of Micrococcus muralis, Vibrio parahaemolyticus, Escherichia coli, and Edwardsiella tarda suspension having an OD600 of 0.4 was prepared using PBS (50mM, pH 6.2). Mu.g of gfLyz-C1 or gfLyz-C2 recombinant protein was added and incubated at 28 ℃. The optical density was measured at OD450 wavelengths at 1 minute intervals for 30 minutes by means of a microplate spectrophotometer (Thermo Scientific) in a 24-well plate. The lytic activity of the gfLyz-C1 and gfLyz-C2 recombinant proteins as shown by turbidity analysis is shown in FIG. 5. The results show that: the gfLyz-C1 and gfLyz-C2 recombinant proteins show bacteriolytic action on micrococcus muralis, Vibrio parahaemolyticus, Escherichia coli and Edwardsiella tarda, and particularly show obvious bacteriolytic activity on the micrococcus muralis, the Vibrio parahaemolyticus and the Edwardsiella tarda.

Example 4 combination of gfLyz-C1, gfLyz-C2 recombinant proteins Edwardsiella tarda

2mL of edwardsiella tarda suspension having an OD600 of 0.4 was prepared using PBS (50mM, pH 6.2). The three groups,group 1 added 20. mu.g of gfLyz-C1 recombinant protein (final concentration 10. mu.g/mL),group 2 added 20. mu.g of gfLyz-C1 recombinant protein (final concentration 10. mu.g/mL), and group 3 added 10. mu.g of gfLyz-C1 recombinant protein and 10. mu.g of gfLyz-C2 recombinant protein (final concentrations of both recombinant proteins were 5. mu.g/mL, respectively, and totalfinal concentration 10. mu.g/mL), and incubated at 28 ℃. The optical density was measured at OD450 wavelengths at 1 minute intervals for 30 minutes by means of a microplate spectrophotometer (Thermo Scientific) in a 24-well plate. As a result, as shown in FIG. 6, it was found that the bacteriolytic activity of group 3(gfLyz-C1, gfLyz-C2 combined group) against Edwardsiella tarda was significantly higher than that of group 1(gfLyz-C1 alone) and group 2(gfLyz-C2 alone) at the same concentration of total protein.

Sequence listing

<110> Ling south college of learning

<120> goldfish c-type lysozyme subtype and coding gene and application thereof

<141> 2019-10-24

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Claims (2)

1. The application of the encoding gene of goldfish C-type lysozyme subtype gfLyz-C1 and/or gfLyz-C2 or the application of goldfish C-type lysozyme subtype gfLyz-C1 and/or gfLyz-C2 in preparing the medicines for preventing and treating the bacterial diseases of aquatic animals is characterized in that the nucleotide sequence of the encoding gene of gfLyz-C1 is shown as SEQ ID No.1 or SEQ ID No. 3; the nucleotide sequence of the gfLyz-C2 coding gene is shown in SEQ ID NO.2 or SEQ ID NO. 4; the amino acid sequences of gfLyz-C1 and gfLyz-C2 are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively; the aquatic animal bacterial diseases are diseases caused by one or more pathogenic bacteria of micrococcus muralis, vibrio parahaemolyticus, escherichia coli or edwardsiella tarda.

2. The use according to claim 1, wherein the bacterial disease in aquatic animals is caused by Edwardsiella tarda.

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