CN1766109A - Chinese prawn C-type agglutinin gene and its coded C-type agglutinin peptide and uses - Google Patents
Chinese prawn C-type agglutinin gene and its coded C-type agglutinin peptide and uses Download PDFInfo
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- CN1766109A CN1766109A CN 200510044586 CN200510044586A CN1766109A CN 1766109 A CN1766109 A CN 1766109A CN 200510044586 CN200510044586 CN 200510044586 CN 200510044586 A CN200510044586 A CN 200510044586A CN 1766109 A CN1766109 A CN 1766109A
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Abstract
The invention discloses a C-type agglutinin gene of China prawn with its coded polypeptide as well as application of the gene in preparing recombination protein with antibacterial activity. This product can be used in feedstuff additive, food preserving and drug exploitation.
Description
Technical field
The present invention relates to a kind of agglutinin gene and encoded protein thereof and application, relate in particular to the C-type agglutinin peptide and the application of a kind of Chinese prawn C-type agglutinin gene and coding thereof; Belong to gene engineering technology field.
Background technology
Invertebrates suffers can activate various kinds of cell and humoral immune reaction behind the pathogen infection, generally will pass through following several steps from microbiological attack to final removing cause of disease: at first these invertebratess must can discern external foreign matter, are called the non-own identification (Recognition of infectious non-self) of infection; Subsequently, caused the extracellular cascade reaction that activates serine protease and remove serpin, thereby the signal that will be infected is enlarged into stronger " danger " signal or removes false alarm, and this process is called the adjustment and the amplification (Signal modulationand amplification) of signal; Priming signal transduction pathway (Signal transduction pathway) then, and cause the transcriptional activity of goal gene; At last, activated the effector reactive system.
Though there is the function of a lot of effector genes also not known, it is very clearly that 3 big class effector systems are arranged: i.e. antibacterial peptide, phenol oxidase dependency melanism system and apoptosis-related genes system.
Below main initial step---the progress of immune identification receptor research aspect of setting forth congenital immunity.
In the past invertebrates is discerned the mechanism that infects microorganism and understand seldom, the progress of this respect was very fast in recent years.Now basic confirm this " non-oneself " identification be because exist some specific, soluble or with cytolemma bonded pattern recognition acceptor (pattern recognition receptors PRRs), is also referred to as pattern recognition albumen or pattern recognition molecule.Though they do not have higher animal B cell and the significant specificity of T cell system, but can discern or in conjunction with those microorganisms surface conservative and in the host non-existent pathogen-associated molecular pattern, as lipopolysaccharides (LPS), peptidoglycan (peptidoglycans) and mannosans (mannans) etc.After the identification, these acceptors are present in the proteolytic enzyme of hemolymph and utilize the intracellular signal transduction approach of immune response tissue to cause immune response by activation.The immunity identification receptor can also also can be the initiation factor of immunologic processes such as aggegation, melanism or other protein modification cascade reactions as the Opsonin that promotes to engulf.Classification about these immune identification receptors, different authors have different opinions, someone comprises that with higher animal the PRR that finds among the mankind is divided into 7 families, i.e. C type lectin, rich leucine albumen, removing acceptor, pentamer albumen, lipid transferring enzyme, integration element and Complement Regulatory Protein.The pattern recognition acceptor that the scholar who has recently will find in fruit bat and anopheles is divided into 6 types: peptidoglycan recognition protein, contain thioester bond albumen, and gram-negative bacteria is conjugated protein, removes acceptor, C-type lectin and sulphur dependent form lectin.
The research of pattern recognition acceptor, be actually research innate immune system identification " oneself " with " non-own " essence, this is to the function of the congenital immunity of understanding invertebrates in depth and regulation mechanism, have important significance for theories to the integrity and the discipline development of immunology research, and the formulation that can be economic animal immune protection strategy simultaneously provides guidance.Therefore the research of carrying out this aspect has important theory and practical significance.
Summary of the invention
The purpose of this invention is to provide a kind of pattern recognition acceptor of from prawn, cloning---Chinese prawn C-type agglutinin gene sequence, and its cloning process and carrier are provided.
Chinese prawn C-type agglutinin gene of the present invention has the nucleotide sequence shown in the SEQ ID NO.1; Information shown in it is:
(a) sequence signature:
* length: 557 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: prawn (Fenneropenaeius chinensis)
(f) sequence description: SEQ ID NO.1
atgaagttcc tggcgccggt tattcttact acgttgatct ccgtcgctgc gtcagccagt 60
gtccgtgcga ccgagtgccc ctccccctac gagcccctgg acgaaacgag gtgcattttc 120
ttggacgcct tcgtcagcta cacttggcaa gagacggttg acttgtgcaa gagccacggc 180
ggagaaatcc tgacaatcga ggactgcgag acgttcgccc ttgtctatga ctacatcagg 240
agccaggatg tcacgaaagg aaaacactac tggttaggag caaccgatga agtagaagaa 300
ggcacatgga aatttgtaaa caacagactc actcccatgg gcattcctta ctggggtgta 360
aacgagccta acaatgggaa tacctacaac tgcgctatga tgcatgctag ttataaccat 420
tattggtacg atgccgcgtg cggaagcaag tataacccca tttgtctcaa gaattattaa 480
aaagtgcatg ggcaagggag cactgtgata aaaggtacga cttggaaatg aagagacagc 540
taaaaaaaaa aaaaaaa 557
Another object of the present invention provides a kind of C-type agglutinin peptide by Chinese prawn C-type agglutinin gene coding, and it has the aminoacid sequence shown in the SEQ ID NO.2; Information shown in it is:
(a) sequence signature
* length: 159 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
(c) sequence description: SEQ ID NO.2
Met Lys Phe Leu Ala Pro Val Ile Leu Thr Thr Leu Ile Ser Val
5 10 15
Ala Ala Ser Ala Ser Val Arg Ala Thr Glu Cys Pro Ser Pro Tyr
20 25 30
Glu Pro Leu Asp Glu Thr Arg Cys Ile Phe Leu Asp Ala Phe Val
35 40 45
Ser Tyr Thr Trp Gln Glu Thr Val Asp Leu Cys Lys Ser His Gly
50 55 60
Gly Glu Ile Leu Thr Ile Glu Asp Cys Glu Thr Phe Ala Leu Val
65 70 75
Tyr Asp Tyr Ile Arg Ser Gln Asp Val Thr Lys Gly Lys His Tyr
80 85 90
Trp Leu Gly Ala Thr Asp Glu Val Glu Glu Gly Thr Trp Lys Phe
95 100 105
Val Asn Asn Arg Leu Thr Pro MET Gly Ile Pro Tyr Trp Gly Val
110 115 120
Asn Glu Pro Asn Asn Gly Asn Thr Tyr Asn Cys Ala Met Met His
125 130 135
Ala Ser Tyr Asn His Tyr Trp Tyr Asp Ala Ala Cys Gly Ser Lys
140 145 150
Tyr Asn Pro Ile Cys Leu Lys Asn Tyr
155
Wherein, also comprise the varient of the aminoacid sequence shown in the SEQ ID NO.2, its coding has the homologous variation albumen that is less than 8 amino acid changes, and amino acid change is that conservative amino acid changes.
The cloning process of Chinese prawn C-type Lectin cDNA of the present invention is:
Adopt single stage method or RNA kit from prawn, to extract total RNA, perhaps use mRNA kit separation and Extraction mRNA.Utilize total RNA or mRNA reverse transcription to synthesize cDNA then;
Wherein: the primer according to the design of the conserved sequence of prawn C-type lectin is:
Forward primer: 5 ' GGT GCA TTT TCT TGG ACG CCT-3 '
Reverse primer: 5 ' ATG GGA GTG AGT CTG TTG TTT-3 '
The PCR reaction conditions is: at first 94 ℃ of sex change are 2 minutes, enter following circulation then: 94 ℃ 30 seconds, 53 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 30 circulations altogether, last 72 ℃ were extended 10 minutes.
Purifying obtains dna fragmentation by chain polymerization enzyme reaction (PCR) amplification;
Get above-mentioned purified product and be cloned into pGEM-T Easy carrier (Promega company product) or pMD 18-T carrier (TaKaRa company product), transform DH5 α cell (common carrier host cell), the dull and stereotyped cultivation;
Extract and plasmid purification amplification plasmid and order-checking;
Through 3 ' and 5 ' terminal rapid amplifying,, promptly get the agglutinin gene of Chinese prawn C-type shown in SEQ ID NO.1 full length nucleotide sequence again with the splicing of 3 ' and 5 ' terminal sequence.
Chinese prawn C-type agglutinin gene of the present invention has application in the recombinant protein of anti-microbial activity in preparation.
Wherein, the method for above-mentioned application is by gene recombination technology described gene to be expressed in intestinal bacteria, yeast and insect nuclear polyhedrosis virus, obtains to have the recombinant protein of anti-microbial activity.
For example: the Chinese prawn C-type agglutinin gene that obtains is cloned into pET-30a (Novagen company) expression vector, and transformed into escherichia coli BL21 DE3 cell carries out abduction delivering, purifying; And utilize the acquisition recombinant protein to carry out bacteriostatic experiment.
Its antimicrobial spectrum and minimal inhibitory concentration (concentration of not growing with thalline is minimal inhibitory concentration) see Table 1.
Table 1: Chinese prawn C-type lectin minimal inhibitory concentration
| Bacterial classification | Concentration (μ M) |
| Bacillus subtilis Bacillus cercus bacillus megaterium Dipel micrococcus luteus staphylococcus aureus e coli | 5 5 2.5 2.5 5 10 20 |
| Streptococcus pneumoniae | 20 |
Utilize Chinese prawn C-type agglutinin gene of the present invention to change it over to crop, can obtain to have the crop of antibacterial ability by gene engineering method.
Utilize method of the present invention to modify Chinese prawn C-type agglutinin gene, also can be used for other researchs and production by existing gene engineering method.
Utilizing the C-type lectin of the reorganization that the present invention obtains to can be used for antibacterial feed additive, Food preservation, animal-plant gene transforms and drug development.
Description of drawings
Fig. 1 Chinese prawn C-type lectin recombinant expressed
Wherein: 1 induces preceding bacterial strain, and 2 induce back bacterial strain, the supernatant liquor after 3 cytoclasises, the precipitation after 4 cytoclasises, the C-type lectin of 5 purifying, M standard Marker.
Embodiment
Embodiment 1: the clone of Chinese prawn C-type Lectin cDNA
1) extraction of total RNA: adopt the prior art single stage method to extract total RNA.
2) cDNA first chain is synthetic: the total RNA of 6 micrograms, add reaction solution 20 microlitres, reaction solution is 50 mmole Repone K and 3 mmole magnesium chloride mixed solutions, 10 mmole tris-HCI buffer (Tris-HCl) pH8.3,1 mmole dithiothreitol (DTT) (DTT), 5 micromole's oligomerization dT acid (Oligod (T)
17), 500 micromole's deoxyribonucleotide mixtures (dNTP), 25 RNA of unit enzyme inhibitorss, 8 AMV of unit reversed transcriptive enzymes, 42 ℃ were reacted 70 ℃ of 10 minutes termination reactions 90 minutes.
3) PCR reaction: chain polymerization enzyme reaction (PCR) reagent and condition:
At first following reagent is mixed:
10xTaq dna polymerase buffer liquid 5 microlitres (μ l)
Template cDNA 1 μ l
Forward primer (1.25 μ g/ μ l) 1 μ l
Reverse primer (1.25 μ g/ μ l) 1 μ l
Deoxynucleoside acid mixture (dNTP) 4 μ l
Taq archaeal dna polymerase 0.25 μ l
Aqua sterilisa 37.75 μ l
Cumulative volume 50 μ l
Primer according to the design of the conserved sequence of prawn C-type lectin is:
Forward primer: 5 ' GGT GCA TTT TCT TGG ACG CCT-3 '
Reverse primer: 5 ' ATG GGA GTG AGT CTG TTG TTT-3 '
The PCR reaction conditions is: at first 94 ℃ of sex change are 2 minutes, enter following circulation then: 94 ℃ 30 seconds, 53 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 30 circulations altogether, last 72 ℃ were extended 10 minutes.
4) reaction product purifying: utilize the product QIAquick Gel Extraction Kit of German Quinn (QIAGEN) company, operation steps is undertaken by product description.
5) prawn C-type Lectin cDNA clone: get purified product 3 microlitres, be connected in pGEM-T Easy carrier (Promega company product).Be transformed into e.colistraindh5, in the dull and stereotyped grow overnight that contains penbritin (100 mcg/ml), 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal 0.2 mcg/ml) and isopropylthiogalactoside (IPTG0.1 mole/milliliter), 3 hickies of picking, overnight incubation in LB liquid nutrient medium (5 milliliters contain 100 mcg/ml peace penicillin G).
6) plasmid purification: collect 2 milliliters of incubated overnight bacterium liquid, centrifugal (10000 rev/mins, 1 minute) collecting cell.With minim DNA purification kit (Wizard plus SV Minipreps DNA Purification System, U.S. Pu Luomaige Promega company) plasmid purification, the purification step by specification carries out.
7) sequencing and homology retrieval: get plasmid purification 4 microlitres, with carrier primer T7 automatically check order (originally being operated in Shanghai life worker company finishes).Institute's calling sequence and gene pool sequence are compared.
8) prawn C-type Lectin cDNA 3 ' end rapid amplifying
After obtaining the antibacterial peptide gene fragment, design a specificity forward primer F3:5 '-GTT TAC AAA TTTCCA AGT CCC TTC-3 ' again, carry out cDNA 3 ' end rapid amplifying, operation steps is undertaken by precious biological 3 ' RACE test kit specification sheets.
Get the about 20 μ g of the total RNA of hemocyte, the modulation of other reagent and reaction conditions by specification carry out.Carry out 3 ' end pcr amplification with Auele Specific Primer F3 and 3 ' joint, adopt 55 ℃ of annealing, all the other are identical with above-mentioned pcr amplification program, and products therefrom is cloned, verified and check order by preceding method.
9) prawn C-type Lectin cDNA 5 ' end clone
Press the SMART of CLONTECH company (U.S.)
TMPCR cDNA library construction test kit specification sheets carries out, and comprises the following steps:
The first chain cDNA is synthetic:
1.0 μ l RNA sample (the total RNA of 0.05-1.0 μ g)
1.0 μ l SMART oligonucleotide (5 ' TACGGCTGCGAGAAGACGACAGAAGGG-3 ')
1 μ l CDS/3 ' PCR primer
2.0 μ l water
With mentioned reagent mixing in the 0.5ml centrifuge tube, hatched 2 minutes for 72 ℃.Ice bath is 2 minutes again, adds following reagent then:
2.0 the μ l 5x first chain damping fluid
1.0μl DTT(20mM)
1.0μl dNTP(10mM)
1.0 μ l MMLV reversed transcriptive enzyme
10 μ l cumulative volumes
Mixing was hatched the ice bath termination reaction 1 hour for 42 ℃.
With above-mentioned cDNA is template, and 5 ' the PCR primer and reverse primer 5 ' the TGT CCT TAC CCCTAC GAG CCC CTG 3 ' that provide with test kit are that primer carries out pcr amplification, obtains 5 ' sequence of prawn C-type Lectin cDNA.
With the splicing of 3 ' and 5 ' terminal sequence, promptly obtain the agglutinin gene of Chinese prawn C-type shown in SEQ ID NO.1 full length nucleotide sequence.
Embodiment 2: recombinant expression vector structure, expression and bacteria resistance function are measured
(1) according to the sequence of Chinese prawn C-type lectin and the cloning site of expression vector pPET30a (Novagen company), the design primer:
LEC-pet F：5’AAT ATT
GAA TTC ATG AAG TTC CTG GCG CCG GTT 3’(EcoR1)
LEC-pet R：5’GCC CGA
CTC GAG TTA ATA ATT CTT GAG ACA AAT G 3’(Xhol)
The present invention has selected the EcoR I and the Xho I restriction enzyme site of pET30a cloning site, therefore, has introduced EcoR I restriction enzyme site at upstream primer during the design primer, has introduced Xho I restriction enzyme site on the downstream primer.
(2) gene amplification, clone and recombinant plasmid screening
With pMD-18T-Lec is template, carries out the PCR reaction with above-mentioned primer, and amplification condition is: 94 ℃, and the pre-sex change of 2min; 94 ℃, 30s, 56 ℃, 45s, 72 ℃, 45s, 35 circulations; 72 ℃ are extended 10min.
2% agarose gel electrophoresis PCR product detects.
The PCR product is made the preparation electrophoresis, with UNIQ-5 Column DNA Gel Extraction Kit (chemical product is given birth in Shanghai) recovery, purified pcr product, through EcoR I and Xho I endonuclease digestion, the cutting-out two ends have the housefly phylaxin mature peptide cDNA fragment of Xho I and EcoR I restriction enzyme site, same expression vector pET30a exposes the Xho I and the EcoR I restriction enzyme site at multiple clone site two ends through EcoR I and Xho I endonuclease digestion.Then, the amplified production after enzyme cut is connected with the T4 dna ligase with expression vector, transforms DH5 α competent cell, the dull and stereotyped PCR screening positive clone of LB+Amp.The bacterium colony that picking PCR is sieved to, 37 ℃ of vibrate amplification cultivation and extracting plasmids after EcoR I and Xho I double digestion and the sequence verification, are recombinant expression plasmid pET30a-Lec.Switching through E.coli expression strain BL21 DE3 competent cell, coating LB+Amp flat board is inverted incubated overnight for 37 ℃.
(3) screening expression strain
8 mono-clonal bacterium colonies of picking from the above-mentioned LB+Amp flat board, 37 ℃ of shaken overnight of 2ml LB+Amp liquid nutrient medium are cultivated, and get 20 μ l incubated overnight liquid and join the transfer of 2ml LB+Amp liquid nutrient medium and cultivate next day, and 37 ℃ of shaking culture 3h are to OD
600Between 0.5～0.7, add then IPTG to final concentration be 1mmol, continue 37 ℃ of shaking culture abduction delivering 4h.Before inducing, from a sample, take out 0.5ml bacterium liquid at random, do not induce contrast during electrophoresis detection.
After having expressed, respectively get 0.5ml bacterium liquid, the centrifugal 5min collecting cell of 5000r/min comprises not inductive sample, is resuspended in the 100 μ l deionized waters, makes 12.5% SDS-PGAE as the electrophoresis sample.According to electrophoresis result, identify expression strain.
(5) recombinant C-type lectin expression and purification
Picking expression strain mono-clonal 37 ℃ of overnight shakings in the LB+Amp liquid nutrient medium are cultivated, next day, join the transfer of 100ml LB+Amp substratum at 1: 100 according to volume ratio and cultivate, behind 37 ℃ of shaking culture 3h, adding IPTG again is 1mmol to final concentration, 37 ℃ of vibration inducing culture 4h again.Take out the bacterium liquid of 0.5ml before inducing, as inducing preceding control sample.After inducing culture is intact, the centrifugal 10min collecting cell of bacterium liquid 7000r/min in suitable centrifuge tube, cell is resuspended in 1 * PBS of 5ml precooling, adds the Triton X-100 of 50 μ l 20%, fully ice bath 30min behind the mixing.The ultrasonic disruption cell, ultrasonic circulating is: ultrasonic 1s; Interval 1s; Omnidistance 40s.Repeat 4 times, with bacterium liquid mixing in ice bath, avoid local temperature too high during each gap, make protein denaturation.At last, with the centrifugal 15min of bacterium liquid 10000r/min after the fragmentation, collect supernatant liquor and precipitation, supernatant liquor and precipitation keep sample respectively, standby.
Recombinant protein exists with the form of inclusion body, through inclusion body purifying, renaturation and affinity chromatography, has promptly acquired recombinant protein with ordinary method---recombinant C-type lectin (see figure 1).
(6) recombinant protein determination of activity
Activity identification adopts liquid growth-inhibiting method.
Get single bacterium colony (seeing Table 1) LB incubated overnight liquid such as test bacterium intestinal bacteria, streptococcus aureus, micrococcus luteus respectively, be diluted to OD with the LB liquid nutrient medium
600=0.01 (about 10
6About) for testing bacterium liquid.Get 100 μ l test bacterium liquid respectively with the recombinant C-type lectin sample mixing of 10 times of dilutions, in 30 ℃ of shaking culture of 96 well culture plates, survey A with microplate reader every 1h
630Absorption value with Microsoft Excel processing data, is drawn growth curve chart, is contrast with penbritin and Elution Buffer.
The bacteriostatic experiment result:
Its antimicrobial spectrum and minimal inhibitory concentration (concentration of not growing with thalline is minimal inhibitory concentration) see Table 1.
Table 1: Chinese prawn C-type lectin minimal inhibitory concentration
| Bacterial classification | Concentration (μ M) |
| Bacillus subtilis Bacillus cercus bacillus megaterium Dipel micrococcus luteus staphylococcus aureus e coli | 5 5 2.5 2.5 5 10 20 |
| Streptococcus pneumoniae | 20 |
Sequence table
SEQ ID NO.1
＜110〉Shandong University
＜120〉the C-type agglutinin peptide and the application of Chinese prawn C-type agglutinin gene and coding thereof
<141>2005-9-1
<160>2
<210>1
<211>557
<212>cDNA
＜213〉prawn (Fenneropenaeius chinensis)
＜221〉Chinese prawn C-type agglutinin gene
<222>(1)…(557)
<400>1
atgaagttcc tggcgccggt tattcttact acgttgatct ccgtcgctgc gtcagccagt 60
gtccgtgcga ccgagtgccc ctccccctac gagcccctgg acgaaacgag gtgcattttc 120
ttggacgcct tcgtcagcta cacttggcaa gagacggttg acttgtgcaa gagccacggc 180
ggagaaatcc tgacaatcga ggactgcgag acgttcgccc ttgtctatga ctacatcagg 240
agccaggatg tcacgaaagg aaaacactac tggttaggag caaccgatga agtagaagaa 300
ggcacatgga aatttgtaaa caacagactc actcccatgg gcattcctta ctggggtgta 360
aacgagccta acaatgggaa tacctacaac tgcgctatga tgcatgctag ttataaccat 420
tattggtacg atgccgcgtg cggaagcaag tataacccca tttgtctcaa gaattattaa 480
aaagtgcatg ggcaagggag cactgtgata aaaggtacga cttggaaatg aagagacagc 540
taaaaaaaaa aaaaaaa 557
SEQ ID NO.2
<210>2
<211>159
<212>PRT
＜221〉the C-type agglutinin peptide of Chinese prawn C-type agglutinin gene coding
<222>(1)…(159)
<400>2
Met Lys Phe Leu Ala Pro Val Ile Leu Thr Thr Leu Ile Ser Val
5 10 15
Ala Ala Ser Ala Ser Val Arg Ala Thr Glu Cys Pro Ser Pro Tyr
20 25 30
Glu Pro Leu Asp Glu Thr Arg Cys Ile Phe Leu Asp Ala Phe Val
35 40 45
Ser Tyr Thr Trp Gln Glu Thr Val Asp Leu Cys Lys Ser His Gly
50 55 60
Gly Glu Ile Leu Thr Ile Glu Asp Cys Glu Thr Phe Ala Leu Val
65 70 75
Tyr Asp Tyr Ile Arg Ser Gln Asp Val Thr Lys Gly Lys His Tyr
80 85 90
Trp Leu Gly Ala Thr Asp Glu Val Glu Glu Gly Thr Trp Lys Phe
95 100 105
Val Asn Asn Arg Leu Thr Pro MET Gly Ile Pro Tyr Trp Gly Val
110 115 120
Asn Glu Pro Asn Asn Gly Asn Thr Tyr Asn Cys Ala Met Met His
125 130 135
Ala Ser Tyr Asn His Tyr Trp Tyr Asp Ala Ala Cys Gly Ser Lys
140 145 150
Tyr Asn Pro Ile Cys Leu Lys Asn Tyr
155
Claims (5)
1. Chinese prawn C-type agglutinin gene, it has the nucleotide sequence shown in the SEQ ID NO.1; Information shown in it is:
(a) sequence signature:
* length: 557 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: prawn (Fenneropenaeius chinensis)
(f) sequence description: SEQ ID NO.1
atgaagttcc tggcgccggt tattcttact acgttgatct ccgtcgctgc gtcagccagt 60
gtccgtgcga ccgagtgccc ctccccctac gagcccctgg acgaaacgag gtgcattttc 120
ttggacgcct tcgtcagcta cacttggcaa gagacggttg acttgtgcaa gagccacggc 180
ggagaaatcc tgacaatcga ggactgcgag acgttcgccc ttgtctatga ctacatcagg 240
agccaggatg tcacgaaagg aaaacactac tggttaggag caaccgatga agtagaagaa 300
ggcacatgga aatttgtaaa caacagactc actcccatgg gcattcctta ctggggtgta 360
aacgagccta acaatgggaa tacctacaac tgcgctatga tgcatgctag ttataaccat 420
tattggtacg atgccgcgtg cggaagcaag tataacccca tttgtctcaa gaattattaa 480
aaagtgcatg ggcaagggag cactgtgata aaaggtacga cttggaaatg aagagacagc 540
taaaaaaaaa aaaaaaa 557
2. the C-type agglutinin peptide of the described Chinese prawn C-of claim 1 type agglutinin gene coding, it has the aminoacid sequence shown in the SEQ IDNO.2; Information shown in it is:
(a) sequence signature
* length: 159 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
(c) sequence description: SEQ ID NO.2
Met Lys Phe Leu Ala Pro Val Ile Leu Thr Thr Leu Ile Ser Val
5 10 15
Ala Ala Ser Ala Ser Val Arg Ala Thr Glu Cys Pro Ser Pro Tyr
20 25 30
Glu Pro Leu Asp Glu Thr Arg Cys Ile Phe Leu Asp Ala Phe Val
35 40 45
Ser Tyr Thr Trp Gln Glu Thr Val Asp Leu Cys Lys Ser His G1y
50 55 60
Gly Glu Ile Leu Thr Ile Glu Asp Cys Glu Thr Phe Ala Leu Val
65 70 75
Tyr Asp Tyr Ile Arg Ser G1n Asp Val Thr Lys Gly Lys His Tyr
80 85 90
Trp Leu Gly Ala Thr Asp Glu Val Glu Glu Gly Thr Trp Lys Phe
95 100 105
Val Asn Asn Arg Leu Thr Pro MET G1y Ile Pro Tyr Trp Gly Val
110 115 120
Asn Glu Pro Asn Asn Gly Asn Thr Tyr Asn Cys Ala Met Met His
125 130 135
Ala Ser Tyr Asn His Tyr Trp Tyr Asp Ala Ala Cys Gly Ser Lys
140 145 150
Tyr Asn Pro Ile Cys Leu Lys Asn Tyr
155
3. the varient of the aminoacid sequence shown in the described SEQ ID of claim 2 NO.2, its coding has the homologous variation albumen that is less than 8 amino acid changes, and amino acid change is that conservative amino acid changes.
4. the described Chinese prawn C-of claim l type agglutinin gene has application in the recombinant protein of anti-microbial activity in preparation.
5. has application in the recombinant protein of anti-microbial activity as Chinese prawn C-type agglutinin gene as described in the claim 4 in preparation, its method is by gene recombination technology described gene to be expressed in intestinal bacteria, yeast and insect nuclear polyhedrosis virus, obtains to have the recombinant protein of anti-microbial activity.
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| CNB2005100445862A CN100420750C (en) | 2005-09-13 | 2005-09-13 | Chinese prawn C-type agglutinin gene and its coded C-type agglutinin peptide and uses |
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| CNB2005100445862A CN100420750C (en) | 2005-09-13 | 2005-09-13 | Chinese prawn C-type agglutinin gene and its coded C-type agglutinin peptide and uses |
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| CN102321628A (en) * | 2011-08-01 | 2012-01-18 | 武汉凯肽来生物科技有限公司 | C-type agglutinin gene of procambarus clarkia as well as preparation method and applications thereof |
| CN103833839A (en) * | 2012-11-27 | 2014-06-04 | 沈阳药科大学 | C-type lectin as well as preparation method and application thereof |
| CN104623631A (en) * | 2015-01-09 | 2015-05-20 | 中国科学院海洋研究所 | Application of C-type lectin |
| CN106589098A (en) * | 2017-01-24 | 2017-04-26 | 中国科学院海洋研究所 | Deep sea shrimp C-type lectin and application thereof |
| CN107365372A (en) * | 2017-08-28 | 2017-11-21 | 中国科学院南海海洋研究所 | A kind of Environment of Litopenaeus vannamei Low L-type agglutinin and its encoding gene and application |
| CN107857805A (en) * | 2017-10-27 | 2018-03-30 | 中国科学院南海海洋研究所 | A kind of Environment of Litopenaeus vannamei Low fibrinogen LvFREP and its encoding gene and application |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6133507A (en) * | 1991-11-12 | 2000-10-17 | Board Of Trustees Operating Michigan State University | Nettle lectin cDNA |
| US5969104A (en) * | 1996-07-30 | 1999-10-19 | Incyte Pharmaceuticals, Inc. | Human C-type lectin |
| CN1073623C (en) * | 1998-10-06 | 2001-10-24 | 中国农业科学院生物技术研究所 | Composite coded homoptera pest-resisting gene of galanthus agglutinin and its application |
| CN1621413A (en) * | 2003-11-24 | 2005-06-01 | 中国人民解放军军事医学科学院放射医学研究所 | C-type agglutinin and genes encoding same |
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| CN101864425A (en) * | 2010-05-17 | 2010-10-20 | 山东大学 | Silkworm C-type agglutinin recombination expression method and application thereof |
| CN102321628A (en) * | 2011-08-01 | 2012-01-18 | 武汉凯肽来生物科技有限公司 | C-type agglutinin gene of procambarus clarkia as well as preparation method and applications thereof |
| CN103833839A (en) * | 2012-11-27 | 2014-06-04 | 沈阳药科大学 | C-type lectin as well as preparation method and application thereof |
| CN103833839B (en) * | 2012-11-27 | 2016-08-03 | 沈阳药科大学 | C-type agglutinin and its preparation method and application |
| CN104623631A (en) * | 2015-01-09 | 2015-05-20 | 中国科学院海洋研究所 | Application of C-type lectin |
| CN106589098A (en) * | 2017-01-24 | 2017-04-26 | 中国科学院海洋研究所 | Deep sea shrimp C-type lectin and application thereof |
| CN107365372A (en) * | 2017-08-28 | 2017-11-21 | 中国科学院南海海洋研究所 | A kind of Environment of Litopenaeus vannamei Low L-type agglutinin and its encoding gene and application |
| CN107365372B (en) * | 2017-08-28 | 2020-06-30 | 中国科学院南海海洋研究所 | L-type agglutinin of Litopenaeus vannamei, and coding gene and application thereof |
| CN107857805A (en) * | 2017-10-27 | 2018-03-30 | 中国科学院南海海洋研究所 | A kind of Environment of Litopenaeus vannamei Low fibrinogen LvFREP and its encoding gene and application |
| CN107857805B (en) * | 2017-10-27 | 2021-01-05 | 中国科学院南海海洋研究所 | Litopenaeus vannamei fibrinogen LvFREP and coding gene and application thereof |
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| CN100420750C (en) | 2008-09-24 |
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