CN103113461A - Novel defensin BD2, and gene and application thereof - Google Patents

Abstract

The invention relates to the field of gene engineering, particularly a novel defensin BD2, and a gene and application thereof. The gene sequence of the defensin BD2 is disclosed as SEQ ID NO.2 or SEQ ID NO.3, and the amino acid sequence is disclosed as SEQ ID NO.1 or SEQ ID NO.4. The defensin provided by the invention has an antibacterial action on both Aeromonas hydrophila and Bacillus subtilis, and has antibacterial activity for both Gram-negative bacteria and Gram-positive bacteria. As a novel defensin, the defensin provided by the invention has an application value in the industries of aquaculture feed additives, fishery medicines and the like.

Description

A kind of novel alexin BD2 and gene and application

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of novel alexin BD2 and gene and application.

Background technology

Antibacterial peptide (antimicrobial peptides, AMPs) is the important member of natural immune system, in defence, has vital role during extraneous pathogen infection, has the advantages such as fast, resistance of reaction times, broad spectrum antibiotic activity.Beta-alexin (defensin) belongs to the antibacterial peptide that a class contains 30-45 amino-acid residue, beta-alexin mainly is distributed in a kind of cation micro molecule antibacterial peptide that is rich in halfcystine in respiratory tract, gi tract, reproductive tract surface and body of gland, forms the important defence line of first of opposing pathogenic agent.Beta-alexin the earliest is the self-conceit pipe antibacterial peptide (TAP) (Diamond G et al., Proc Natl Acad Sci USA, 88 (9): 3952-3956,1991) of finding in the tracheal mucosa epithelial cell of ox.

Alexin is the low molecule small peptide of non-glycosyl amphiphilic (hydrophilic, lipophilic) that the molecule amount is 3-4KDa, generally is rich in arginine, positively charged.6 cysteine residues are arranged in molecule, form 3 pairs of disulfide linkage, the β-pleated sheet structure chip architecture that contains 3 corresponding symmetries, lack αhelix territory (Schneider JJ, et al.Journal of MolecularMedicine 83:587-95,2005; Dhople V, et al.Biochimica et Biophysica Acta (BBA)-Biomembranes 1758:1499-512,2006).By means of this medium of cell, no matter in cell or extracellular, alexin is the pathogenic micro-organisms such as killing bacteria, fungi, virus directly.In intracellular environment, they facilitate the death of anerobe by engulfing microorganism.In being released to extracellular environment, they are by attack micro organisms adventitia performance anti-microbial activity.In addition, alexin is also carried out the reparation of immunomodulatory and wound and nerve injury as immune inside and outside body " arbitrator ".Alexin plays an important role in host's neutrophilic granulocyte, mucomembranous surface, skin and other epithelial cell immunity; its location and regulation and control are to realize (De Yang by the invasion and attack of opposing cause of disease and two kinds of approach of growth of endogenetic bacteria; et al.Trends in Immunology 23:291-296,2002; Zasloff.Nature 415:389-395,2002; Lai Y, et al.Trends in Immunology 30:131-41,2009).

Now increasing pathogenic bacteria produces resistance to microbiotic, and antibacterial peptide becomes irresistible trend as antibiotic substitute.Beta-alexin, as the important member of antibacterial peptide, has wide spectrum antibacterium and antiviral activity, for the infringement of defending extraneous pathogenic micro-organism, has vital role.At present more to the research of mankind's beta-alexin, the report of the beta-alexin research in fish, especially function aspects is also fewer.And all concentrate on the research (Guo Hongyan etc., life science, 24 (4): 321-327,2012) of seawater fish.In the last few years, along with the deterioration of breeding environment, the increase of cultivation density, fish diseases constantly occurred, and had caused huge financial loss, and the application of beta-alexin on culture fishery is also the follow-up problem that will solve.Loach is a kind of novel and representative research objects as the fresh-water fishes of a kind of Cypriniformes, and the application that has the alexin of broad spectrum antibiotic activity and devote the industries such as aquatic feeds, fishing medicine for exploitation provides the foundation.Although beta-alexin has broad spectrum antibiotic activity, its expression amount is still lower, expect large-scale application, and the beta-alexin that obtains having biologic activity by engineered method is also to need at present the large problem solved.

Summary of the invention

The object of the present invention is to provide a kind of alexin BD2.

A further object of the present invention is to provide the above-mentioned alexinic gene bd2 of coding.

A further object of the present invention is to provide the recombinant vectors that comprises above-mentioned phylaxin gene.

A further object of the present invention is to provide the recombinant plasmid that comprises above-mentioned phylaxin gene.

A further object of the present invention is to provide a kind of alexinic method for preparing.

The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, and the new alexin that a kind of character is good is provided.The present inventor obtains a new alexin with anti-Gram-negative bacteria and gram-positive bacteria activity from loach (Paramisgurnus dabryanus).Be suitable for using in the industries such as aquatic feeds, fishing medicine.

Obtained a kind of alexin BD2 from loach, this albumen contains 62 amino acid and a terminator codon, the signal peptide sequence that 19 amino acid of front end are.Its aminoacid sequence is as shown in SEQ ID NO.1:

1 MRVLRLLVIT LLLLTAGEAY DTEIQGWTCG SRGLCRKHCY AQEHTVGYHG

51CPRRYRCCAL RF*

The present invention also provides the above-mentioned alexinic genomic dna sequence of coding and cDNA sequence.Genomic dna sequence is as shown in SEQ ID NO.2, and total length 1002bp, comprise three exons and two introns.Wherein 52-164bp is First Intron, and 282-981bp is second intron.CDNA sequence total length 189bp, as shown in SEQ ID NO.3.

SEQ ID NO.2：

1 ATGAGAGTAC TAAGACTGCT TGTTATCACT TTACTGCTGC TGACAGCTGG AAAAGGTACA

61 AGATACACGG TCATTTGAAT GCCTACAGAT TATAGCTATA GTTTACTATG TGTGCTACCA

121CACCAGATAT GGTTATGTTT TAATATAGTT GTGATCTATT CCTTACAGCT TATGACACAG

181GAATACAGGG ATGGACTTGT GGATCTCGAG GTCTGTGCAG GAAGCACTGC TATGCACAAG

241AGCATACGGT CGGTTATCAT GGCTGCCCTC GAAGATACAG GTGCTCTTCA GATCTTATGA

301ATTACATTTA TGTTTTTGAA TTATTGTGTT GTTGAATAAT CCTGTAAATG ATATGAACAC

361ATATAGATTG GAAGATATAT ATAACAATGA GCTATAAAAG AGCTGTTACT GGGGTGGTAC

421CCTTACAAA AAGTACCTAAA GGGTACATAT TAGTACCTCA AAGGTACATA TTGGTATTTC

481AAAGGTACAT CTGTATCTAT GGTTTAAATA TATTAGGACT TTTTGAAAGG GTATCATCAC

541AGTGACATTT ATTGCCCCAA AGGATCTAAA AACTTTTTAC TGTAGATTTA AATGAACATT

601AAACATTAAC AAGTCTTTAT CTTTACAAAA TAAAACTATG AAATAACAGC CTTATGCAAA

661GCATTCTGGG AACCAAAAAT CCTCATCAAC CTTTTTCTGT TATTTCCTTC AGATTTTGTT

721TCAATGCTTT GCATGAGGCT GTTATTTAAA AAAATTTCTG TAAAGATAAA GACTTATTCA

781TGTTTAATGT TCGTGTTAAC TTTGATCAAA CATCTGCCAG TAATATTGTT TTTCTACAGT

841GATGTTGACA GAGCTTAATT TTAACTCAAA AATATTCCCG TAAAGCCGAT TTTTTATAAA

901TGTTAAAGTA AGTTTGTGAT TCATTGAGTG AGTTATATTA GCATTGACTC AGCAACCTTA

961CAATCTTCTT TTTTTATGAA GTGCTGTGCT TTGCGATTTT AA

SEQ ID NO.3

1 ATGAGAGTAC TAAGACTGCT TGTTATCACT TTACTGCTGC TGACAGCTGG AGAAGCTTAT

61 GACACAGAAA TACAGGGATG GACTTGTGGA TCTCGAGGTC TGTGCAGGAA GCACTGCTAT

121GCACAGGAGC ATACGGTCGG TTATCATGGA TGCCCTCGAA GATACAGGTG CTGTGCTTTG

181CGATTTTAA

Sequence after 19 amino acid of alexin BD2 removal front end is as shown in SEQ ID NO.4:

Y DTEIQGWTCG SRGLCRKHCY AQEHTVGYHG CPRRYRCCAL RF 43

Its nucleotide sequence is as shown in SEQ ID NO.5:

tatgacacag aaatacaggg atggacttgt ggatctcgag gtctgtgcag gaagcactgc 60

tatgcacagg agcatacggt cggttatcat ggatgccctc gaagatacag gtgctgtgct 120

ttgcgatttt aa 132

The DNA sequence dna of encoding mature alexin protein gene bd2 and the aminoacid sequence derived are carried out to the BLAST comparison in GenBank, determine that BD2 is a kind of new alexin, for the transformation of its encoding gene and in various heterologous gene expression systems high efficient expression good genetic material is provided.

The present invention also provides the recombinant vectors that comprises above-mentioned phylaxin gene bd2, is preferably pcDNA3.1 (+)-bd2-s.Gene of the present invention is inserted between the restriction enzyme site that expression vector is suitable, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, be preferably phylaxin gene is inserted between the EcoRI and BamHI restriction enzyme site on plasmid pcDNA3.1 (+), obtain recombined eukaryotic cell expression plasmid pcDNA3.1 (+)-bd2.

The present invention also provides the express cell that comprises above-mentioned phylaxin gene bd2 system, is preferably HEK293T clone.

The present invention also provides the application of above-mentioned alexin BD2 for the preparation of Gram-negative bacteria and Gram-positive bacteria inhibitor.

The present invention also provides a kind of method for preparing above-mentioned alexin BD2, comprises the following steps:

1) with above-mentioned recombinant vectors transformed host cell, obtain recombinant plasmid;

2) transfection plasmid, obtain alexin BD2 by the HEK293T cell expressing.

Alexin of the present invention all has anti-microbial effect to Aeromonas hydrophila and subtilis.As a kind of novel alexin, in industries such as feeding additive aquatic animal and fishing medicines, using value is arranged.

The accompanying drawing explanation

The gene structure figure of Fig. 1 phylaxin gene bd2.

Fig. 2 phylaxin gene bd2 analyzes at the RT-PCR of HEK293T cells.

Fig. 3 alexin BD2 of the present invention detects the activity of Aeromonas hydrophila.

Fig. 4 alexin BD2 of the present invention detects the activity of subtilis.

Embodiment

Test materials and reagent

1, fish, carrier and cell: loach is purchased from Beijing fish market, and carrier pcDNA3.1 (+) is purchased from Invitrogen company, and clone HEK293T is purchased from American type culture collection(ATCC).

2, enzyme and other biochemical reagents: DNA restriction enzyme, ligase enzyme are purchased from TakaRa company, and reverse transcription test kit and SYBR mix are purchased from TOYOBO company, and large upgrading grain test kit is purchased from sky, Beijing root biochemical technology company limited, liposome Lipofetmiane ²⁰⁰⁰purchased from Invitrogen company, the cell cultures related reagent is purchased from Invitrogen and Nuck company.Other is all domestic reagent (all can buy and obtain from common biochemical reagents company).

3, substratum: Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).Illustrate: do not make the experimental methods of molecular biology illustrated in following examples, all with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book, carry out, or carry out according to test kit and product description.

The clone of embodiment 1 loach alexin encoding gene bd2

Utilizing the TRIZON method to extract loach gill section organizes RNA and is reversed to the first chain cDNA.Find two conserved amino acid sequence WTCGYRGLC and GCPRRYRCC according to the alexinic sequence alignment result of the fish of delivering, and design has been synthesized degenerated primer bd2-F and bd2-R(primer sequence in Table 1), the cDNA of loach of take carries out pcr amplification as template.The PCR reaction parameter is: 95 ℃ of 5min; 94 ℃ of 30sec, 55-50 ℃ of 30sec (wherein after each circulation, the renaturation temperature descends 0.5 ℃), 72 ℃ of 30s, then 10 circulations enter second cycling program: 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 30s, after 28 circulations; 72 ℃ of 10min, agarose electrophoresis detects, and obtains the fragment of about 100bp, and after reclaiming, pGEM-T Easy carrier is connected and transforms e. coli jm109, send order-checking after positive after testing.

According to measuring sequence results, in the GenBank of NCBI, utilize BLASTX( http:// www.ncbi.nlm.nih.gov/BLAST) carry out sequence alignment, tentatively judge that this gene fragment is the alexin fragment, and carry out the Study on Similarity of this fragment.

The nucleotide sequence obtained according to order-checking, upstream and downstream designs respectively three TAIL-PCR Auele Specific Primers and two RACE-PCR Auele Specific Primers and they is distinguished to called after D1, D2, D3 (upstream Auele Specific Primer) and U1, U2 (downstream Auele Specific Primer) is in Table 1.Obtain respectively the 5 ' distolateral wing sequence and 3 ' the end UTR sequence of known fragment sequence by TAIL-PCR and RACE-PCR, amplification obtains after product reclaims checking order.

The core fragment that degenerated primer is obtained and the flanking sequence obtained through TAIL-PCR and 3 ' the end UTR sequence obtained through RACE-PCR are spliced and are obtained the bd2 full-length gene.Result shows, this based encode district total length 189bp(SEQID NO.1), 62 amino acid of encoding (SEQ ID NO.3) and a terminator codon.The signal peptide sequence of prediction is 19 amino-acid residues of front end.This encoding gene is a new gene.

Table 1. phylaxin gene bd2 clones primer

^ay=C/T, K=T/G, R=A/G, N=A/T/G/C; The line part is restriction enzyme site.

The tissue distribution of embodiment 2 loach alexin BD2 and attack poison after the expression amount variance analysis

Extract healthy loach and respectively organize RNA, comprise eye, the gill, liver, spleen, brain, spermary, ovary, muscle, intestines, skin, head-kidney, heart, be reversed to the first chain cDNA, analyze alexin by Q-PCR the highest at the eyes expression amount, minimum at ovary, spleen expression amount.

After attacking malicious loach with Aeromonas hydrophila, extracted respectively the RNA of the gill, liver and spleen at 6,12,24 hours, by Q-PCR analyze attack poison after the alexinic expression amount of alexin and healthy loach change the trend over time that reaches, result, after attacking malicious loach with Aeromonas hydrophila, the gill, liver and spleen alexin all are up-regulated expression trend, and the gill is up-regulated expression gradually in time, and liver and spleen descend subsequently 12 hours expression amounts are the highest.Illustrate that the loach alexin may play the effect of defence in the process that infects of antagonism Aeromonas hydrophila.

The expression of embodiment 3 loach phylaxin gene bd2

Phylaxin gene bd2 is connected with pcDNA3.1 (+) and expresses in the HEK293T cell.

According to order-checking and the consequence devised primer pcDNA-F of sequential analysis and pcDNA-R(primer sequence in Table 1), the cDNA of take carries out pcr amplification as template.The gene bd2 of plasmid pcDNA3.1 (+) and removal signal peptide encoding mature albumen carries out being connected to form carrier pcDNA3.1 (+)-bd2 after double digestion (EcoRI and BamHI).Preparation TransI competent cell, transform Host Strains by recombinant vectors pcDNA3.1 (+)-bd2 thermal shock.Identify positive recombinant, extract recombinant plasmid transfectional cell.By recombinant plasmid and empty carrier pcDNA3.1 (+) transfection simultaneously HEK293T cell, adopt Lipofetmiane ²⁰⁰⁰the liposome transfection method, grow to plating efficiency approximately 90% the time until cell, the ratio transfection plasmid and the liposome that according to mass volume ratio, are 1 to 2, and transfection, after 6 hours, sucks liposome and plasmid, adds nutrient solution to cultivate 72 hours again.After transient transfection 72 hours, collecting cell, detect its expression for being RT-PCR.Extract cell total rna, be reversed to the first chain cDNA, can amplify the purpose band as masterplate with Auele Specific Primer, and under the same terms, control group pcDNA empty carrier can not amplify respective strap, and two groups of masterplates all can amplify internal reference β-actin (Fig. 2).

Equally, the fragment (SEQ ID No.5) of removing signal peptide sequence is carried out to above-mentioned identical test, obtain the restructuring alexin.

Embodiment 4 alexin anti-microbial activities detect

Embodiment 3 obtains alexinic anti-microbial activity and detects: the target bacterial classification that anti-microbial activity detects is gram-positive microorganism subtilis and Gram-negative bacteria Aeromonas hydrophila, concrete detection method is: get two kinds of bacterium, the 37 ℃ of incubated overnight of being rule, after the mono-clonal that grows about 2mm, picking is in the LB nutrient solution of 20ml, continue to be cultured to its logarithmic phase, the OD value is about 0.6, 1% inoculum size of take again is connected to (NaCl concentration is 0.22mM) in less salt LB substratum, get 50ul after mixing in 96 orifice plates, the albumen (pcDNA3.1 (+)-bd2 and contrast pcDNA3.1 (+)) that simultaneously adds embodiment 3 separation of equivalent, the treatment process of albumen is: after collecting cell, with not containing the PB damping fluid re-suspended cell of NaCl, after ultrasonication, the centrifuging and taking supernatant carries out protein quantification, guarantee that recombinant protein is the same with the Tot Prot of control group empty carrier, get subsequently supernatant and carry out the activity detection, after hatching about 14h together with bacterium, it detects OD ₆₀₀reading, for Aeromonas hydrophila, buffered soln PB is hatched rear average OD value with it be 0.495, empty carrier is hatched rear average OD value with it be 0.4685, average OD value after BD2 is hatched with it is 0.245, same is respectively 0.623 for three numerical value of subtilis, 0.598, 0.177, data are after the spass software statistics, bacterium OD value after BD2 is hatched is significantly lower than PB and empty carrier contrast (P<0.01), can find out that recombinant protein pcDNA3.1 (+)-BD2 all has extremely remarkable restraining effect (referring to Fig. 3 to Aeromonas hydrophila and subtilis with respect to control group pcDNA3.1 (+), Fig. 4).

Removing by expression recombinant protein that the fragment (SEQ ID No.5) of signal peptide sequence obtains in embodiment 3 also is proved to be Aeromonas hydrophila and subtilis is all had to extremely significantly restraining effect.

Claims (10)

1. an alexin BD2, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.4.

2. a phylaxin gene bd2, is characterized in that, the alexin claimed in claim 1 of encoding.

3. phylaxin gene bd2 according to claim 2, is characterized in that, its base sequence is as shown in SEQ ID NO.2 or SEQ ID NO.3.

4. the recombinant vectors that comprises the described phylaxin gene bd2 of claim 2 or 3.

5. recombinant vectors according to claim 4, is characterized in that, described recombinant vectors is pcDNA3.1 (+)-bd2.

6. the recombinant plasmid that comprises the described phylaxin gene bd2 of claim 2 or 3.

7. recombinant plasmid according to claim 6, is characterized in that, the expressive host of described recombinant plasmid is HEK293T clone.

8. a method for preparing phylaxin gene bd2, is characterized in that, comprises the following steps:

1) with the recombinant vectors transformed host cell of claim 4, obtain recombinant plasmid;

2) transfection plasmid, obtain alexin BD2 by the HEK293T cell expressing.

9. the described alexin BD2 of claim 1 is for the preparation of the application of Gram-negative bacteria and Gram-positive bacteria inhibitor.

10. application according to claim 9, is characterized in that, Gram-negative bacteria is Aeromonas hydrophila, and described gram-positive microorganism is subtilis.

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