CN100344762C - G-type lysozyme gene of Chlamys farreri and encoded protein and cloning method thereof - Google Patents
G-type lysozyme gene of Chlamys farreri and encoded protein and cloning method thereof Download PDFInfo
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- CN100344762C CN100344762C CNB2004100506518A CN200410050651A CN100344762C CN 100344762 C CN100344762 C CN 100344762C CN B2004100506518 A CNB2004100506518 A CN B2004100506518A CN 200410050651 A CN200410050651 A CN 200410050651A CN 100344762 C CN100344762 C CN 100344762C
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Abstract
The present invention relates to G type lysozyme, particularly to a G type lysozyme gene of chlamys farreri and encoded protein and a cloning method thereof. The G type lysozyme gene of the Chlamys farreri has a base sequence which is shown by SEQ ID NO. 1 and the encoded protein of the G type lysozyme gene has an amino acid sequence which is shown by SEQ ID NO. 2. The G type lysozyme gene of the Chlamys farreri, which is cloned by the present invention, has a polyadenylic acid tailing signal and a polyadenylic acid tail. The gene has a critical function on a chlamys farreri immune defence aspect, can be used for the recombinant expression and the gene transfer of lysozyme and establishes a basis for the disease prevention and control and the gene assistance breeding of chlamys farreri and the further development of medical products.
Description
Technical field
The present invention relates to G type N,O-Diacetylmuramidase, particularly chlamys farreri G type lysozyme gene (cDNA complete sequence and aminoacid sequence) reaches the cloning process that clones the chlamys farreri N,O-Diacetylmuramidase from chlamys farreri cDNA.
Background technology
N,O-Diacetylmuramidase is found (Proc.R.Soc.Lond.B.Biol.Sci.1922) by Fu Laiming in nineteen twenty-two.This enzyme full name is 1; 4-β-N-N,O-Diacetylmuramidase; claim mucopeptide N-ethanoyl muramyl lytic enzyme again; β-1 in the energy hydrolytic bacteria cell walls between N-acetylglucosamine and the-acetylmuramic acid, the 4-glycosidic link destroys the peptidoglycan support; the cell spalling is opened under the effect that internal penetration is pressed; cause the bacterium cracking, so claim muramidase again, i.e. N-acetyl murein glucosides glycan lytic enzyme.Some Gram-negative bacteria as dust Xi Shi intestinal bacteria, salmonella typhi, also can be subjected to the destruction of N,O-Diacetylmuramidase.The acellular wall construction of humans and animals cell does not also have peptidoglycan, so N,O-Diacetylmuramidase is to the human body cell free of toxic effects.N,O-Diacetylmuramidase is a kind of small molecular basic protein that extensively is present in nature animal, plant and the microorganism, plays non-special defense mechanism in body.The anti-microbial effect that is used for N,O-Diacetylmuramidase just still medically all has important use in industry and is worth.At first N,O-Diacetylmuramidase is pharmaceutically acceptable, and tool is antibiotic, remove effects such as local necrosis tissue, hemostasis, detumescence, anti-inflammatory and recovery organization function, medically can be used to make antiphlogistic drug etc.In foodstuffs industry, can be used as sanitas, also can be added in the toothpaste as preventing and treating the medicinal toothpaste of carious tooth.Be a kind of important bacteriolysant on fermentation industry, be used to deposit and make cell walls, prepare no thalline extracting solution.
N,O-Diacetylmuramidase can be divided into four types: c type (hen egg-white lysozyme, chicken egg-white lysozyme, cly), the g type (the goose hen egg white lysozyme, goose egg-white lysozyme, gly), i type (invertebratelysozyme), phage lysozyme.Most of N,O-Diacetylmuramidase all belongs to the c type, and cly is made up of 130 amino-acid residues, and relative molecular mass is 14700.Gly finds that by Jolles and Canfield contain 185 amino-acid residues approximately, relative molecular mass is 21000 the earliest.Olsen, the shellfish i type N,O-Diacetylmuramidase and the c type N,O-Diacetylmuramidase of reports such as Nilson have certain homology.Phage lysozyme is positioned at various phages, and dissolving host bacterium is played an important role, and it contains 164 amino-acid residues. and relative molecular mass is 18700.The report that in invertebrates, does not also have at present the g-N,O-Diacetylmuramidase, the carp g-lysozyme that the Savan report is arranged of relevant marine organisms report, Rogerio report from shrimp c-lysozyme, the zebra fish c-lysozyme of FengLiu report, the lefteye flounder g-lysozyme of Hikima report, and Olsen, the shellfish i-lysozyme that has homology with cly of reports such as Nilson.
N,O-Diacetylmuramidase is resisted at seashells and is played the part of important role in the pathogenic bacterial infection, and because its this exclusive low temperature, high salt action environment can make it to be different from existing Mammals N,O-Diacetylmuramidase, have important application prospects, it can be applicable to different field such as marine organisms medicine, marine organisms immunity, aquatic product low temperature storage and transport.
Summary of the invention
The present invention utilizes construction cDNA library, EST analysis, 3 ' and 5 ' RACE technology, chlamys farreri G type lysozyme gene is studied, from chlamys farreri, be cloned into G type lysozyme gene first, this gene can be used for the in-vitro recombination expression and the transgenosis of lysozyme gene, for the exploitation of pharmaceutical prod exploitation, the research of scallop immunology, hereditary and selection and improvement, disease control and fresh-keeping product and sanitas is laid a good foundation.
G type lysozyme gene is cloned in the present invention from chlamys farreri, it has the base sequence shown in the SEQ ID NO.1; Its proteins encoded has the aminoacid sequence shown in the SEQ ID NO.2.
The method of being cloned into G type lysozyme gene from chlamys farreri comprises:
(1) purifying of the extraction of the total RNA of chlamys farreri and mRNA;
(2) chlamys farreri cDNA library construction;
(3) the extensive mensuration of chlamys farreri cDNA library est sequence;
(4) homology analysis of chlamys farreri est sequence and the segmental screening of chlamys farreri G type lysozyme gene;
(5) carry out 3 ' and 5 ' RACE with gene-specific primer CFLyzF 5 '-CATCCGCTACCACTCCTGTC-3 ' and CFLyzR 5 '-CAACTACGTCATTGCTGTAGTC-3 ' and be cloned into chlamys farreri G type lysozyme gene cDNA full length sequence.
The extraction of the total RNA of described chlamys farreri is to extract total RNA from the chlamys farreri hemolymph that has infected Vibrio anguillarum.
The chlamys farreri G type lysozyme gene that the present invention clone obtains, this gene cDNA total length 829bp has the open reading frame of a 600bp, 200 amino acid of encoding, the long 21bp of 5 ' non-coding region, the long 208bp of 3 ' non-coding region has polyadenylic acid tailing signal and polyadenylic acid tail; This gene has critical function aspect the scallop immune defense.
The present invention utilizes expressed sequence tag (EST) technology, cDNA 3 ' and 5 ' rapid amplifying technology (RACE) to be cloned into G type lysozyme gene from chlamys farreri, can be used for the recombinant expressed and transgenosis of N,O-Diacetylmuramidase, and for the disease control of chlamys farreri, gene assist-breeding and further be developed as pharmaceutical prod and lay the foundation.The sequence of prepared chlamys farreri G type lysozyme gene can be in the application in the chlamys farreri hereditary and selection; Also can be in the expression in bacterium, yeast and the insect cell line; Its recombination expression product also can be in the application in drug manufacture, fodder additives and sanitas and the preservation agent production simultaneously.
Embodiment
The chlamys farreri G type lysozyme gene that 1. 1 kinds of clones of embodiment obtain has following sequence:
(1) information of SEQ ID NO.1 in the sequence table
Sequence signature
Length: 829 base pairs
Type: nucleic acid
Chain: two strands
Topological framework: linear
Source: chlamys farreri (Chlamys farreri)
Sequence description:
GCACGAGGCTAGATTCCAACGATGAACCCACTGGCAGTACTCACACTTCTTGCTATCAGCACTG
GTGCCTGGGCAGCGTCCTACACCTGCCATGGTGACGTCACACGACTTCATCCCCATGGACAACA
CAATAGAGGTGTCGCTGCATCCAACCGTGGCGTAGATTACGATTACCATGACCTGTTAGCCAAG
AAAAGTTGTTACGAAGCATCAGGCGCACGACACTGTATTCAGCCGTCTGTGATTGCCGCCTTGG
CCAGTCGAGAATCACGTGGAGGGCGTCTTCTGACGTCAACAGGAGGATGGGGAGATCATCACCA
TGCCTACGGTATATTACAGTGTGACATCCGCTACCACTCCTGTCAGCAGTACGCTTGGAACAGT
TGTGAACACATAGAACAAATGGTGAAGGAGGTCCTTGTGGCATACATCGGTCAGGTGGCGCGTA
AACATCCCACGTGGTCACGAGATCAGCAACTCCAAGGTGGTATCGCCGCCTACAACTCCGGAGT
TGGCAACGTCCAGACCTGGGCCCACCTCGACGTCGGCACAACCGGAAATGACTACAGCAATGAC
GTAGTTGCGCGTGCTAAACACCTTATTTCAAGCCACGGCTGGCATTAAATGCGATTAAGCACGA
AAGAGAGCCACCCATGCTAATTTCAAGATATAATATTCAAAAGAGCGATATTTTTACTTTGAAG
AGATAATCGTTTTAAGGACAGGACATAGAAATCATGCATTGTTGGTAATGGCTTGCATATTACA
GTAATAAACGGTTGTACTAATGTTCTTCAAAACCAAAAAAAAAAAAAAAAAAAAAAAAAAA
(2) information of sequence table SEQ ID NO.2
Sequence signature:
Length: 200 amino acid
Type: amino acid
Chain: strand
Topological framework: linear
Source: chlamys farreri (Chlamys farreri)
Sequence description:
MNPLAVLTLLAISTGAWAASYTCHGDVTRLHPHGQHNRGVAASNRGVDYDYHDLLAKKSCYEAS
GARHCIQPSVIAALASRESRGGRLLTSTGGWGDHHHAYGILQCDIRYHSCQQYAWNSCEHIEQM
VKEVLVAYIGQVARKHPTWSRDQQLQGGIAAYNSGVGNVQTWAHLDVGTTGNDYSNDVVARAKH
LISSHGWH
The clone of embodiment 2. chlamys farreri G type lysozyme genes
1) purifying of the extraction of the total RNA of chlamys farreri and mRNA: from the closed shell flesh of the chlamys farreri that infected Vibrio anguillarum, gather hemolymph with syringe, centrifugal 10 minutes of 4 ℃ of 700g, utilize the Trizol reagent of Invitrogen company and extract total RNA, utilize the Oligotex mRNA purification kit purified mRNA of QIAGENE company with reference to its explanation.
2) chlamys farreri cDNA library construction: utilize cDNA Synthesis Kit of Stratagene company and ZAP-cDNA Synthesis Kit (Stratagene) and consult and use explanation and carry out the synthetic of cDNA, double-stranded cDNA is through end-filling, EcoR I joint connects, EcoR I terminal phosphateization, utilize the QIAEX II Agarose Gel Extraction Kit of QIAGEN company that the endonuclease bamhi greater than 100bp is reclaimed behind the Xho I endonuclease digestion, be connected with Invetrogen company Uni-ZAP XR vector carrier, utilize the ZAP-cDNA Gigapack III Gold Cloning Kit test kit of Stratagene company to carry out the library packing, utilize Exassist Helper Phage and SOLR bacterial strain from Uni-ZAP
Cut pBluescript outside the XR Vector upper body and become plasmid library.
3) the extensive mensuration of chlamys farreri cDNA library est sequence: screening positive clone from the library, use carrier universal primer T3 on the MegaBACE1000 sequenator, to carry out sequencing, with parent mass peak map file (the * .abi that obtains, * .abd file) data are converted into sequential file (* .seq) and quality document (* .seq.qual) through the Phred routine processes, the numerical value that provides according to quality document determines to obtain the word error probability of sequence, remove low-quality base, with the carrier sequence in the cross-mach program mask data, from the data that obtain, choose continuous base quality greater than Q13 (accuracy rate is greater than 95%) and length greater than the sequence of 100bp as the EST data.
4) homology analysis of chlamys farreri est sequence and the segmental screening of chlamys farreri G type lysozyme gene: whole effectively EST data that will obtain are carried out the cluster splicing, generate Contigs and Singletons, respectively Contigs and the Singletons that is obtained carried out BLASTn and BLASTx analysis in database, seek out and G type lysozyme gene homologous est sequence according to the similarity analysis result.
5) clone of chlamys farreri G type lysozyme gene cDNA full length sequence: utilize 3 ' and 5 ' RACE technology clone chlamys farreri G type lysozyme gene cDNA full length sequence, according to designing gene-specific primer CFLyzF 5 '-CATCCGCTACCACTCCTGTC-3 ' and CFLyzR 5 '-CAACTACGTCATTGCTGTAGTC-3 ' with G type lysozyme gene homologous est sequence, utilize carrier universal primer T3 respectively, T7 and CFLyzF, CFLyzR carries out 3 ' and 5 ' terminal amplification, the PCR product detects with 1.5% agarose electrophoresis, reclaim recovery and the purifying that test kit (go up marine Ke Kairui Biochip company) carries out the PCR product with glue, be connected with pMD-18T carrier (the precious biotechnology in Dalian company limited) again, transform the XL1-blue bacterial strain, select positive colony and extract plasmid, carry out PCR with CFLyzF and CFLyzR primer and carrier primer and detect, carry out sequencing after confirming to insert clip size.The sequence that records obtains full length sequence after CLUSTER analyzes splicing;
3 ' end used system of pcr amplification and reaction conditions: 25 μ l reaction systems: 2.5 μ l, 10 * PCRbuffer, 1.0 μ l MgCl
2(2.5mM), 2.0 μ l dNTP (2.5mM), 1 μ l primer CFLyzF (10pmol/ μ l), 1 μ l primer T7 (10pmol/ μ l), 16.3 μ l of PCR-grade water, 0.2 μ l (1U) Taq polymerase (Promega), 1 μ l cDNA template.
Be reflected among the PTC-100 Programmable Thermal Controller Cycler (MJ Research) and carry out, reaction conditions is: at first 94 ℃ of pre-sex change are 5 minutes, enter following circulation then: 94 ℃ of sex change 30 seconds, annealed 30 seconds for 59 ℃, 72 ℃ were extended 55 seconds, carry out 31 circulations altogether, last 72 ℃ were extended 10 minutes.
5 ' end used system of pcr amplification and reaction conditions: 25 μ l reaction systems: 2.5 μ l, 10 * PCRbuffer, 1.0 μ l MgCl
2(2.5mM), 2.0 μ l dNTP (2.5mM), 1 μ l primer CFLyzR (10pmol/ μ l), 1 μ l primer T3 (10pmol/ μ l), 16.3 μ l of PCR-grade water, 0.2 μ l (1U) Taq polymerase (Promega), 1 μ l cDNA template.
Be reflected among the PTC-100 Programmable Thermal Controller Cycler (MJ Research) and carry out, reaction conditions is: at first 94 ℃ of pre-sex change are 5 minutes, enter following circulation then: 94 ℃ of sex change 30 seconds, annealed 30 seconds for 57 ℃, 72 ℃ were extended 55 seconds, carry out 34 circulations altogether, last 72 ℃ were extended 10 minutes.
The application of sequence in the chlamys farreri hereditary and selection of embodiment 3. chlamys farreri G type lysozyme genes
Sequences Design PCR and RT-PCR primer according to SEQ ID NO.1 in the sequence table among the embodiment 1, partial sequence to chlamys farreri Different Individual G type lysozyme gene increases and sequencing, compare the difference of Different Individual on G type lysozyme gene sequence, compare the difference of Different Individual G type lysozyme gene mRNA expression level simultaneously, instruct the hereditary and selection of chlamys farreri with this.
The application of the recombinant expressed and recombination expression product of embodiment 4. chlamys farreri G type lysozyme genes
According to the corresponding cDNA sequences Design of chlamys farreri G type N,O-Diacetylmuramidase among sequence table SEQ ID NO.2 primer, introduce two different restriction enzyme mutational sites, be cloned into pQE series then, pET series, pGEX-4T series, pPIC series, serial expression vector such as pGAPZ, again with the recombinant expression vector transformed into escherichia coli and the yeast that are built into, screening positive clone and with IPTG or methanol induction Recombinant Protein Expression.Utilize affinity chromatography that expression product is carried out multistage purifying, obtain the reorganization chlamys farreri G type N,O-Diacetylmuramidase of vivoexpression.This product can be used as medicine, fodder additives, sanitas and preservation agent and uses.
Chlamys farreri
SEQUENCE?LISTING
＜110〉Institute of Oceanology of the Chinese Academy of Sciences
＜120〉chlamys farreri G type lysozyme gene and proteins encoded and cloning process thereof
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>829
<212>DNA
＜213〉chlamys farreri (Chlamys farreri)
<220>
<221>CDS
<222>(22)..(621)
<223>
<400>1
gcacgaggct?agattccaac?g?atg?aac?cca?ctg?gca?gta?ctc?aca?ctt?ctt 51
Met?Asn?Pro?Leu?Ala?Val?Leu?Thr?Leu?Leu
1 5 10
gct?atc?agc?act?ggt?gcc?tgg?gca?gcg?tcc?tac?acc?tgc?cat?ggt?gac 99
Ala?Ile?Ser?Thr?Gly?Ala?Trp?Ala?Ala?Ser?Tyr?Thr?Cys?His?Gly?Asp
15 20 25
gtc?aca?cga?ctt?cat?ccc?cat?gga?caa?cac?aat?aga?ggt?gtc?gct?gca 147
Val?Thr?Arg?Leu?His?Pro?His?Gly?Gln?His?Asn?Arg?Gly?Val?Ala?Ala
30 35 40
tcc?aac?cgt?ggc?gta?gat?tac?gat?tac?cat?gac?ctg?tta?gcc?aag?aaa 195
Ser?Asn?Arg?Gly?Val?Asp?Tyr?Asp?Tyr?His?Asp?Leu?Leu?Ala?Lys?Lys
45 50 55
agt?tgt?tac?gaa?gca?tca?ggc?gca?cga?cac?tgt?att?cag?ccg?tct?gtg 243
Ser?Cys?Tyr?Glu?Ala?Ser?Gly?Ala?Arg?His?Cys?Ile?Gln?Pro?Ser?Val
60 65 70
Chlamys farreri
att?gcc?gcc?ttg?gcc?agt?cga?gaa?tca?cgt?gga?ggg?cgt?ctt?ctg?acg 291
Ile?Ala?Ala?Leu?Ala?Ser?Arg?Glu?Ser?Arg?Gly?Gly?Arg?Leu?Leu?Thr
75 80 85 90
tca?aca?gga?gga?tgg?gga?gat?cat?cac?cat?gcc?tac?ggt?ata?tta?cag 339
Ser?Thr?Gly?Gly?Trp?Gly?Asp?His?His?His?Ala?Tyr?Gly?Ile?Leu?Gln
95 100 105
tgt?gac?atc?cgc?tac?cac?tcc?tgt?cag?cag?tac?gct?tgg?aac?agt?tgt 387
Cys?Asp?Ile?Arg?Tyr?His?Ser?Cys?Gln?Gln?Tyr?Ala?Trp?Asn?Set?Cys
110 115 120
gaa?cac?ata?gaa?caa?atg?gtg?aag?gag?gtc?ctt?gtg?gca?tac?atc?ggt 435
Glu?His?Ile?Glu?Gln?Met?Val?Lys?Glu?Val?Leu?Val?Ala?Tyr?Ile?Gly
125 130 135
cag?gtg?gcg?cgt?aaa?cat?ccc?acg?tgg?tca?cga?gat?cag?caa?ctc?caa 483
Gln?Val?Ala?Arg?Lys?His?Pro?Thr?Trp?Ser?Arg?Asp?Gln?Gln?Leu?Gln
140 145 150
ggt?ggt?atc?gcc?gcc?tac?aac?tcc?gga?gtt?ggc?aac?gtc?cag?acc?tgg 531
Gly?Gly?Ile?Ala?Ala?Tyr?Asn?Ser?Gly?Val?Gly?Asn?Val?Gln?Thr?Trp
155 160 165 170
gcc?cac?ctc?gac?gtc?ggc?aca?acc?gga?aat?gac?tac?agc?aat?gac?gta 579
Ala?His?Leu?Asp?Val?Gly?Thr?Thr?Gly?Asn?Asp?Tyr?Ser?Asn?Asp?Val
175 180 185
gtt?gcg?cgt?gct?aaa?cac?ctt?att?tca?agc?cac?ggc?tgg?cat 621
Val?Ala?Arg?Ala?Lys?His?Leu?Ile?Ser?Ser?His?Gly?Trp?His
190 195 200
taaatgcgat?taagcacgaa?agagagccac?ccatgctaat?ttcaagatat?aatattcaaa 681
agagcgatat?ttttactttg?aagagataat?cgttttaagg?acaggacata?gaaatcatgc 741
attgttggta?atggcttgca?tattacagta?ataaacggtt?gtactaatgt?tcttcaaaac 801
caaaaaaaaa?aaaaaaaaaa?aaaaaaaa 829
<210>2
<211>200
<212>PRT
＜213〉chlamys farreri (Chlamys farreri)
<400>2
Met?Asn?Pro?Leu?Ala?Val?Leu?Thr?Leu?Leu?Ala?Ile?Ser?Thr?Gly?Ala
1 5 10 15
Trp?Ala?Ala?Ser?Tyr?Thr?Cys?His?Gly?Asp?Val?Thr?Arg?Leu?His?Prc
20 25 30
His?Gly?Gln?His?Asn?Arg?Gly?Val?Ala?Ala?Ser?Asn?Arg?Gly?Val?Asp
35 40 45
Chlamys farreri
Tyr?Asp?Tyr?His?Asp?Leu?Leu?Ala?Lys?Lys?Ser?Cys?Tyr?Glu?Ala?Ser
50 55 60
Gly?Ala?Arg?His?Cys?Ile?Gln?Pro?Ser?Val?Ile?Ala?Ala?Leu?Ala?Ser
65 70 75 80
Arg?Glu?Ser?Arg?Gly?Gly?Arg?Leu?Leu?Thr?Ser?Thr?Gly?Gly?Trp?Gly
85 90 95
Asp?His?His?His?Ala?Tyr?Gly?Ile?Leu?Gln?Cys?Asp?Ile?Arg?Tyr?His
100 105 110
Ser?Cys?Gln?Gln?Tyr?Ala?Trp?Asn?Ser?Cys?Glu?His?Ile?Glu?Gln?Met
115 120 125
Val?Lys?Glu?Val?Leu?Val?Ala?Tyr?Ile?Gly?Gln?Val?Ala?Arg?Lys?His
130 135 140
Pro?Thr?Trp?Ser?Arg?Asp?Gln?Gln?Leu?Gln?Gly?Gly?Ile?Ala?Ala?Tyr
145 150 155 160
Asn?Ser?Gly?Val?Gly?Asn?Val?Gln?Thr?Trp?Ala?His?Leu?Asp?Val?Gly
165 170 175
Thr?Thr?Gly?Asn?Asp?Tyr?Ser?Asn?Asp?Val?Val?Ala?Arg?Ala?Lys?His
180 185 190
Leu?Ile?Ser?Ser?His?Gly?Trp?His
195 200
Claims (4)
1. chlamys farreri G type lysozyme gene is characterized in that: be the base sequence shown in the SEQ ID NO.1.
2. the described chlamys farreri G of claim 1 a type lysozyme gene proteins encoded is characterized in that: be the aminoacid sequence shown in the SEQ ID NO.2.
3. the cloning process of the described chlamys farreri G of claim 1 a type lysozyme gene is characterized in that, comprising:
(1) purifying of the extraction of the total RNA of chlamys farreri and mRNA;
(2) chlamys farreri cDNA library construction;
(3) the extensive mensuration of chlamys farreri cDNA library est sequence;
(4) homology analysis of chlamys farreri est sequence and the segmental screening of chlamys farreri G type lysozyme gene;
(5) utilize 3 ' with 5 ' RACE technology clone chlamys farreri G type lysozyme gene cDNA full length sequence.
4. according to the cloning process of the described chlamys farreri G of claim 3 type lysozyme gene, it is characterized in that the extraction of the total RNA of described chlamys farreri is to extract total RNA from the chlamys farreri hemolymph that has infected Vibrio anguillarum.
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| CNB2004100506518A CN100344762C (en) | 2004-10-22 | 2004-10-22 | G-type lysozyme gene of Chlamys farreri and encoded protein and cloning method thereof |
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|---|---|---|---|
| CNB2004100506518A CN100344762C (en) | 2004-10-22 | 2004-10-22 | G-type lysozyme gene of Chlamys farreri and encoded protein and cloning method thereof |
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| CN1763201A CN1763201A (en) | 2006-04-26 |
| CN100344762C true CN100344762C (en) | 2007-10-24 |
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| CN103160526B (en) * | 2011-12-15 | 2014-05-14 | 中国科学院烟台海岸带研究所 | Mytilus edulis G-type lysozyme gene and recombinant protein and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6268164B1 (en) * | 1998-06-26 | 2001-07-31 | Incyte Genomics, Inc. | Human goose-type lysozyme |
| CN1373211A (en) * | 2001-03-02 | 2002-10-09 | 复旦大学 | Human G-type lysozyme and its coding sequence, preparing process and application |
-
2004
- 2004-10-22 CN CNB2004100506518A patent/CN100344762C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6268164B1 (en) * | 1998-06-26 | 2001-07-31 | Incyte Genomics, Inc. | Human goose-type lysozyme |
| CN1373211A (en) * | 2001-03-02 | 2002-10-09 | 复旦大学 | Human G-type lysozyme and its coding sequence, preparing process and application |
Non-Patent Citations (2)
| Title |
|---|
| genbank accession number Araki t et al,Genbank accession number 2003 * |
| 溶菌酶的研究进展 贾向志等，生物技术通讯，第13卷第5期 2002 * |
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