CN102952185B

CN102952185B - Novel defensin pdBD, and gene and application thereof

Info

Publication number: CN102952185B
Application number: CN201210418553.XA
Authority: CN
Inventors: 郭庆文; 王兴吉; 王忠连; 李芳芳; 刘文龙; 孙硕; 钱娟娟
Original assignee: Shandong Longkete Enzyme Preparation Co Ltd
Current assignee: Shandong Lonct Enzymes Co ltd
Priority date: 2012-10-29
Filing date: 2012-10-29
Publication date: 2014-04-30
Anticipated expiration: 2032-10-29
Also published as: CN102952185A

Abstract

The invention relates to the field of gene engineering, and in particular relates to a novel defensin pdBD, and gene and application thereof. The defensin pdBD provided by the invention has an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO.3. The defensin provided by the invention has antibacterial activity against Gram-negative bacteria (Escherichia coli, Aeromonas hydrophila) and Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus). As a novel defensin, it has application value in industries such as aquatic feed additives and fishery medicine.

Description

A kind of novel alexin pdBD and gene thereof and application

Technical field

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

Background technology

Occurring in nature different kind organism all has its inherent system of defense, and when body is subject to external microbe and infects, this system just can produce the defensive peptide class of class active substance, is called as antibacterial peptide (antimicrobial peptides).And alexin (defensin) belongs to the antibacterial peptide that a class contains 30-45 amino-acid residue, mainly be distributed in skin and mucosal epithelium, stop the field planting of pathogenic micro-organism and by being incorporated on the after birth of pathogenic micro-organism, killed (Taylor K, et al.Biopolymers 90:1-7,2008).Alexin is a kind of little cationic peptide, is the basis of natural host identification microorganism.Research up to now finds that alexinic kind has: Research of Mammalian Defensins, insect defensin and plant alexin, and in mammalian body, find 3 kinds of alexin subfamilies, be α-alexin, beta-alexin and θ-alexin, and θ-alexin exists only in (Guan í-Guerra E in primate, et al.Clinical Immunology 135:1-11,2010).

Alexin is that molecule amount is the low molecule small peptide of non-glycosyl amphiphilic (hydrophilic, lipophilic) of 3-4KDa, 30-45 amino acid, consists of, and is generally rich in arginine, positively charged.In molecule, have 6 cysteine residues, form 3 pairs of disulfide linkage, the β-pleated sheet structure chip architecture that contains 3 corresponding symmetries, lacks αhelix territory (Schneider JJ, et al.Journal of Molecular Medicine 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 can directly be killed the pathogenic micro-organisms such as bacterium, fungi, virus.In intracellular environment, they facilitate the death of anerobe by engulfing microorganism.When being released in 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 opposing invasion and attack of cause of disease and two kinds of approach of the growth of endogenetic bacteria; et al.Trends in Immunology 23:291-296,2002; Zasloff.Nature415:389-395,2002; Lai Y, et al.Trends in Immunology 30:131-41,2009).

Fish alexin is relatively late with respect to Research of Mammalian Defensins research starting, and all concentrate on the research of seawater fish, loach is a kind of novel and representative research objects as the fresh-water fishes of a kind of Cypriniformes, for developing a kind of application that has the alexin of broad spectrum antibiotic activity and devote the industries such as aquatic feeds, fishing medicine, provides some theoretical basiss.

Summary of the invention

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

A further object of the present invention is to provide the above-mentioned alexinic gene pdBD 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 cell lines that comprises above-mentioned phylaxin gene.

A further object of the present invention is to provide one and prepares alexinic method.

The present invention's technical problem 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 people 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.

From loach, obtained a kind of alexin pdBD, this albumen contains 67 amino acid and a terminator codon, and the signal peptide sequence of prediction is 24 amino-acid residues of front end.Theoretical molecular is 7.28kDa.Its aminoacid sequence is as shown in SEQ ID NO.1:

MKPQCILLLALVVILVLHSKVNEAVSFPWGCASISGVCRQGTCLPSELYFGPLGCG

KGFQ 60

CCVSHFL 67

The present invention also provides coding above-mentioned alexinic genome sequence, and total length 627bp, comprises three exons and two introns.Wherein 1-60bp is first exon, and 61-334bp is First Intron, and 335-454bp is second exon, and 455-603bp is second intron, and 604-627bp is the 3rd exon.Whole genome sequence is as shown in SEQ ID NO.2:

atgaaacctc aatgtatact tctactggct ctcgtggtca tcttggtatt gcacagtaag 60

ttttatattc ttgcttttgg taaaagatta gtagctttgt gtaaacctga aattatgatt 120

cttatcacat ttttaagcca aaaaacagca ataataaaac ttgctttatt gtgatgtatt 180

ggaagtgaca ttactcatgg atggcaaagg acaaagcagc aaatgccagc tgtattcctc 240

tagatcacat ttggctgatt ctgtatgtag attgttttca tctgtctgtg ttgatctgtg 300

tggagccaaa acatttattt ttgtctaagg caaggtgaat gaggctgtgt cgtttccctg 360

gggctgtgcg agcatcagtg gagtttgcag acaaggaacg tgtctacctt ctgaactcta 420

ctttggaccg ttaggctgtg gcaagggatt ccagtaagtt ccaatattta tacattccag 480

ataaacacac acacgcatat atcatatgca catataaatg acatattgtg tgatattctt 540

tacacatcaa gtaaatgatt acaaatctat ttgtttttga tttcttcctt gtttttttcc 600

agatgctgtg tatcacattt tctttga 627

The present invention also provides coding above-mentioned alexinic gene.The method separating clone of the present invention by PCR the cDNA sequence of this phylaxin gene as shown in SEQ ID NO.3:

atgaaacctc aatgtatact tctactggct ctcgtggtca tcttggtatt gcacagcaag 60

gtgaatgagg ctgtgtcgtt tccctggggc tgtgcaagca tcagtggagt ttgcagacaa 120

ggaacgtgtc taccttctga actctacttt ggaccgttag gctgtggcaa gggattccag 180

tgctgtgtat cacattttct ttga 204

PdBD sequence after 24 amino acid of removal front end is as shown in SEQ ID NO.4:

VSFPWGCASISGVCRQGTCLPSELYFGPLGCGKGFQCCVSHFL 43

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

gtgtcgtttc cctggggctg tgcaagcatc agtggagttt gcagacaagg aacgtgtcta 60

ccttctgaac tctactttgg accgttaggc tgtggcaagg gattccagtg ctgtgtatca 120

cattttcttt ga 132

By the DNA sequence dna of phylaxin gene pdBD and the aminoacid sequence derived are carried out to BLAST comparison in GenBank, determine that pdBD is a kind of new alexin, for the transformation of phylaxin gene pdBD 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 pdBD, is preferably pcDNA3.1 (+)-pdBD.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 (+)-pdB and pcDNA3.1 (+)-pdBD-s.

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

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

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

2) transfection plasmid, obtains alexin pdBD by HEK293T cell expressing;

3) anti-microbial activity of detection gained alexin pdBD.

Alexin of the present invention all has anti-microbial activity to Gram-negative bacteria (intestinal bacteria, Aeromonas hydrophila) and gram-positive microorganism (subtilis, streptococcus aureus).As a kind of novel alexin, in industries such as feeding additive aquatic animal and fishing medicines, there is using value.

Accompanying drawing explanation

Fig. 1 phylaxin gene pdBD analyzes at the alexinic western blot of HEK293T cells, the pdBD that 1 cell expressing obtains; The expression of the maturation protein encoding gene pdBD-s of 2 removal signal peptides in cell; 3 expression of contrast empty carrier pcDNA3.1 (+) in cell.

Embodiment

Test materials and reagent

1, carrier and cell: carrier pcDNA3.1 (+) is purchased from Invitrogen company, and clone HEK293T is purchased from ATCC.

2, enzyme and other biochemical reagents: restriction endonuclease, ligase enzyme be purchased from Takara company, reverse transcription test kit is purchased from TOYOBO company, liposome Lipofetmiane ²⁰⁰⁰purchased from Invitrogen company, 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: the experimental methods of molecular biology that in following examples, work illustrates, 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 pdBD

Utilizing TRIZON method to extract loach gill portion organizes RNA and is reversed to the first chain cDNA.According to the alexinic sequence alignment result of the fish of delivering, find two conserved amino acid sequence ENEAASFP and GCGKGFLCC, and design has been synthesized degenerated primer DP-BD1-F and DP-BD1-R(primer sequence in Table 1), take the cDNA of loach as template, carry out pcr amplification.PCR reaction parameter is: 95 ℃ of 5min; 94 ℃ of 30sec, 55-50 ℃ of 30sec (wherein after each circulation, renaturation temperature declines 0.5 ℃), 72 ℃ of 30s, 10 circulations, then 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, obtain long 125bp fragment, 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 alexin fragment.The nucleotide sequence obtaining 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 1.5 ' the end flanking sequence and 3 ' the end UTR sequence that by TAIL-PCR and RACE-PCR, obtain respectively known fragment sequence, amplification obtains product and reclaims rear order-checking.

The core fragment that degenerated primer is obtained and the flanking sequence that obtains through TAIL-PCR and 3 ' the end UTR sequence obtaining through RACE-PCR are spliced and are obtained pdBD full-length gene.Result shows, this based encode district total length 204bp(SEQ ID NO.2), encode 67 amino acid (SEQ ID NO.1) and a terminator codon.Signal peptide sequence is 24 amino-acid residues of front end, and without the identical gene of sequence, this encoding gene is a new gene.

Table 1. alexin pdBD Auele Specific Primer

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

The expression of embodiment 3 loach phylaxin gene pdBD

Phylaxin gene pdBD is connected with pcDNA3.1 (+) and expresses in HEK293T cell.According to order-checking and the consequence devised primer pcDNA-F of sequential analysis and pcDNA-R(primer sequence in Table 1), take cDNA as template, carry out pcr amplification.Plasmid pcDNA3.1 (+) and gene (comprising the gene of band signal peptide-coding sequence and the gene of removal signal peptide encoding mature albumen) carry out being connected to form carrier pcDNA3.1 (+)-pdBD and pcDNA3.1 (+)-pdBD-s after double digestion (EcoRI and BamHI).Preparation TransI competent cell, transforms Host Strains by recombinant vectors pcDNA3.1 (+)-pdBD or pcDNA3.1 (+)-pdBD-s thermal shock.Identify positive recombinant, extract recombinant plasmid transfectional cell.By recombinant plasmid and empty carrier pcDNA3.1 (+) transfection simultaneously HEK293T cell, adopt Lipofetmiane ²⁰⁰⁰liposome transfection method, when cell grows to plating efficiency approximately 90%, the ratio transfection plasmid and the liposome that according to mass volume ratio, are 1 to 2, transfection, after 6 hours, sucks liposome and plasmid, adds nutrient solution to cultivate 72 hours again.After transient transfection 72 hours, collecting cell, for being RT-PCR and western blot detects its expression.Extract cell total rna, be reversed to the first chain cDNA, as masterplate, with Auele Specific Primer, can amplify object band, and under the same terms, control group pcDNA empty carrier can not amplify respective strap.It is by after collecting cell that Western blot detects, resuspended with HANKS buffer, the centrifugal 5min of 12000rpm after ultrasonication, get supernatant and carry out Tricine-SDS-PAGE, 100V transferring film 1h subsequently, through primary antibodie two is anti-hatch after, colour developing can find out that recombinant protein pcDNA3.1 (+)-pdBD has an obvious band near 7KDa, and control group pcDNA empty carrier (Fig. 1).

Embodiment 4 alexin anti-microbial activities detect

The recombinant protein anti-microbial activity that embodiment 3 obtains detects: subtilis, the streptococcus aureus of intestinal bacteria, Aeromonas hydrophila and gram-positive microorganism that the target bacterial classification that anti-microbial activity detects is Gram-negative bacteria.Concrete detection method is: get two kinds of bacterium, the 37 ℃ of incubated overnight of ruling, after growing the mono-clonal of about 2mm, picking is in the LB nutrient solution of 20ml, continue to be cultured to its logarithmic phase, OD value is about 0.6, take 1% inoculum size, be connected to (NaCl concentration is as 0.22mM) in less salt LB substratum again, after mixing, get 50ul in 96 orifice plates, the recombinant protein (gene fragment of pcDNA3.1 (+)-pdBD, removal signal peptide that simultaneously adds the embodiment 3 of equivalent to obtain.And contrast pcDNA3.1 (+)), the treatment process of albumen is: after collecting cell, with the PB damping fluid re-suspended cell that does not contain NaCl, after ultrasonication, 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 activity detection, after it hatches about 14h together with bacterium, detect OD ₆₀₀reading, for the intestinal bacteria of Gram-negative bacteria, buffered soln PB is hatched rear average OD value with it be 0.762, empty carrier is hatched rear average OD value with it be that average OD value after 0.735, pdBD is hatched with it is 0.351, for Aeromonas hydrophila, buffered soln PB is hatched rear average OD value with it be 0.495, and empty carrier is hatched rear average OD value with it be that average OD value after 0.463, pdBD is hatched with it is 0.140, same is respectively 0.623 for three numerical value of subtilis, 0.595, 0.102, for three numerical value of streptococcus aureus, be respectively 0.670, 0.625, 0.319, data are after spass software statistics, bacterium OD value after pdBD is hatched is significantly lower than PB and empty carrier contrast (P<0.01), data are after spass software statistics, can find out recombinant protein intestinal bacteria to Gram-negative bacteria with respect to control group pcDNA3.1 (+) that embodiment 3 obtains, Aeromonas hydrophila and gram-positive microorganism streptococcus aureus Aeromonas hydrophila have remarkable restraining effect, subtilis is had to extremely significantly restraining effect.

Claims

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

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

3. phylaxin gene pdBD according to claim 2, is characterized in that, its base sequence is as shown in SEQ IDNO.2, SEQ ID NO.3 or SEQ ID NO.5.

4. comprise the recombinant vectors of phylaxin gene pdBD described in claim 2 or 3.

5. recombinant vectors according to claim 4, it is characterized in that, described recombinant vectors is pcDNA3.1 (+)-pdBD, wherein, described phylaxin gene pdBD is inserted between the EcoRI and BamHI restriction enzyme site on plasmid pcDNA3.1 (+), obtains recombinant expression vector pcDNA3.1 (+)-pdBD.

6. comprise the recombinant plasmid of phylaxin gene pdBD described in claim 2 or 3.

7. a method of preparing alexin pdBD, 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, obtains alexin pdBD by HEK293T cell expressing.

Described in claim 1 alexin pdBD for the preparation of the application of Gram-negative bacteria and Gram-positive bacteria inhibitor, wherein said Gram-negative bacteria is intestinal bacteria and/or Aeromonas hydrophila, and described gram-positive microorganism is subtilis and/or streptococcus aureus.