CN100344763C - G-type lysozyme gene of Argopecten irradians and encoded protein and cloning method thereof - Google Patents
G-type lysozyme gene of Argopecten irradians and encoded protein and cloning method thereof Download PDFInfo
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- CN100344763C CN100344763C CNB2004100506522A CN200410050652A CN100344763C CN 100344763 C CN100344763 C CN 100344763C CN B2004100506522 A CNB2004100506522 A CN B2004100506522A CN 200410050652 A CN200410050652 A CN 200410050652A CN 100344763 C CN100344763 C CN 100344763C
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Abstract
The present invention relates to G type lysozyme, particularly to a G type lysozyme gene of an argopecten irradian and encoded protein and a cloning method thereof. The G type lysozyme gene of the argopecten irradian 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 argopecten irradian, which is cloned by the present invention, is provided with a polyadenylic acid tailing signal and a polyadenylic acid tail. The gene has a critical function on an immune defence aspect of the irradian, 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 the argopecten irradian and the further development of medical products.
Description
Technical field
The present invention relates to G type N,O-Diacetylmuramidase, particularly bay scallop G type lysozyme gene (cDNA complete sequence and aminoacid sequence) reaches the cloning process that clones the bay scallop N,O-Diacetylmuramidase from bay scallop 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, the white shrimp c-lysozyme of Rogerio report, 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, bay scallop G type lysozyme gene is studied, from bay scallop, 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.
The present invention is cloned into G type lysozyme gene and is had the base sequence shown in the SEQ ID NO.1 from bay scallop; 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 bay scallop comprises:
1) purifying of the extraction of the total RNA of bay scallop and mRNA;
2) bay scallop cDNA library construction;
3) the extensive mensuration of bay scallop cDNA library est sequence;
4) homology analysis of bay scallop est sequence and the segmental screening of bay scallop G type lysozyme gene;
5) carry out 3 ' and 5 ' RACE with gene-specific primer AILyzF 5 '-GAGCCACTTACTGTATCCAACC-3 ' and AILyzR 5 '-TGACTCACGGCTGGCTAAGG-3 ' and be cloned into bay scallop G type lysozyme gene cDNA full length sequence.
The extraction of the total RNA of described bay scallop is to extract total RNA from the bay scallop hemolymph that has infected Vibrio anguillarum.
The cDNA sequence of the bay scallop G type lysozyme gene that the present invention clone obtains and the aminoacid sequence of this genes encoding and partial sequence wherein, this gene cDNA total length 659bp, the open reading frame that a 600bp is arranged, 200 amino acid of encoding, the long 18bp of 5 ' non-coding region, the long 41bp 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 bay scallop, can be used for the recombinant expressed and transgenosis of N,O-Diacetylmuramidase, and for the disease control of bay scallop, gene assist-breeding and further be developed as pharmaceutical prod and lay the foundation.Prepared bay scallop G type lysozyme gene can be in the application in the bay scallop 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 bay scallop 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: 659 base pairs
Type: nucleic acid
Chain: two strands
Topological framework: linear
Source: bay scallop (Argopecten irradians)
Sequence description:
GACGAGGCTGATTTGACAATGAATGCCCTAGTGGTTATAACGCTTCTT
GCTTTCAGCACTGGCGCCTGGGCAGCGTCCTACACGTGCCATGGTGA
CGTCAGGCGTCTTCATCCAACCGGAGAGCACAACGGAGGTAACGCTG
CCTCTCATAACGATGTTAAATATGACTACAACGACCTCCTTAACAAGA
AGTCTTGTTACGACCAGGCTGGAGCCACTTACTGTATCCAACCATCGG
TGATCGCTGCCTTAGCCAGCCGTGAGTCACGTGGTGGTCGCCTGTTGC
ATTCAACCAATGGATGGGGAGACCATCACCATGCTTACGGAATTTTAC
AGTGTGACATCCGTTACCACAGCTGTACCCAGCACGCATGGGACAGC
TGTGCACACATTTCCCAGATGGTTCAAGAAGTCCTAGTGCCCTACATC
AATCAGGTGGCCCACAAACATCCCACATGGTCAAAGGAGCAACAACT
CCTAGGTGGAATCGCTGCTTATAACTCTGGAGTCGGAAACGTCCAGAC
CTGGAGTGGCCTTGACATTGGAACAACCGGAAATGACTACAGCAATG
ACGTAGTTGCACGTGCTCAATATCTCATCAGCCATTATGGATGGCATTA
ATAAAATATATGGCTACAGTTAAAAAAAAAAAAAAAAAA
(2) information of sequence table SEQ ID NO.2
Sequence signature:
Length: 200 amino acid
Type: amino acid
Chain: strand
Topological framework: linear
Source: bay scallop (Argopecten irradians)
Sequence description:
MNALVVITLLAFSTGAWAASYTCHGDVRRLHPTGEHNGGNAASHNDVK
YDYNDLLNKKSCYDQAGATYCIQPSVIAALASRESRGGRLLHSTNGWG
DHHHAYGILQCDIRYHSCTQHAWDSCAHISQMVQEVLVPYINQVAHKHP
TWSKEQQLLGGIAAYNSGVGNVQTWSGLDIGTTGNDYSNDVVARAQYL
ISHYGWH
The cloning process of embodiment 2. bay scallop G type lysozyme genes
1) purifying of the extraction of the total RNA of bay scallop and mRNA: from the closed shell flesh of the bay scallop that infected Vibrio anguillarum, gather hemolymph with syringe, centrifugal 10 minutes of 4 ℃ of 700g, utilize the Trizol reagent of Invitrogen company and from the bay scallop hemolymph that has infected Vibrio anguillarum, extract total RNA, utilize the Oligotex mRNA purification kit purified mRNA of QIAGENE company with reference to its explanation;
2) bay scallop 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 bay scallop 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 bay scallop est sequence and the segmental screening of bay scallop 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 bay scallop G type lysozyme gene cDNA full length sequence (utilizing 3 ' and 5 ' RACE technology): according to designing gene-specific primer AILyzF 5 '-GAGCCACTTACTGTATCCAACC-3 ' and AILyzR 5 '-TGACTCACGGCTGGCTAAGG-3 ' with G type lysozyme gene homologous est sequence, utilize carrier universal primer T3 respectively, T7 and AILyzF, AILyzR 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 AILyzF and AILyzR 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 AILyzF (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 60 ℃, 72 ℃ were extended 50 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 AILyzR (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 55 ℃, 72 ℃ were extended 40 seconds, carry out 34 circulations altogether, last 72 ℃ were extended 10 minutes.
The application of sequence in the bay scallop hereditary and selection of embodiment 3. bay scallop G type lysozyme genes
According to sequences Design PCR and the RT-PCR primer of SEQ ID NO.1 in the sequence table among the embodiment 1, the partial sequence of bay scallop Different Individual G type lysozyme gene is increased and sequencing, relatively 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 bay scallop with this.
The application of the recombinant expressed and recombination expression product of embodiment 4. bay scallop G type lysozyme genes
According to the corresponding cDNA sequences Design of bay scallop 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 bay scallop G type N,O-Diacetylmuramidase of vivoexpression.This product can be used as medicine, fodder additives, sanitas and preservation agent and uses.
Bay scallop
SEQUENCE?LISTING
＜110〉Institute of Oceanology of the Chinese Academy of Sciences
＜120〉bay scallop G type lysozyme gene and proteins encoded and cloning process thereof
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>659
<212>DNA
＜213〉bay scallop (Argopecten irradians)
<220>
<221>CDS
<222>(19)..(618)
<223>
<400>1
gacgaggctg?atttgaca?atg?aat?gcc?cta?gtg?gtt?ata?acg?ctt?ctt?gct 51
Met?Asn?Ala?Leu?Val?Val?Ile?Thr?Leu?Leu?Ala
1 5 10
ttc?agc?act?ggc?gcc?tgg?gca?gcg?tcc?tac?acg?tgc?cat?ggt?gac?gtc 99
Phe?Ser?Thr?Gly?Ala?Trp?Ala?Ala?Ser?Tyr?Thr?Cys?His?Gly?Asp?Val
15 20 25
agg?cgt?ctt?cat?cca?acc?gga?gag?cac?aac?gga?ggt?aac?gct?gcc?tct 147
Arg?Arg?Leu?His?Pro?Thr?Gly?Glu?His?Asn?Gly?Gly?Asn?Ala?Ala?Ser
30 35 40
cat?aac?gat?gtt?aaa?tat?gac?tac?aac?gac?ctc?ctt?aac?aag?aag?tct 195
His?Asn?Asp?Val?Lys?Tyr?Asp?Tyr?Asn?Asp?Leu?Leu?Asn?Lys?Lys?Ser
45 50 55
tgt?tac?gac?cag?gct?gga?gcc?act?tac?tgt?atc?caa?cca?tcg?gtg?atc 243
Cys?Tyr?Asp?Gln?Ala?Gly?Ala?Thr?Tyr?Cys?Ile?Gln?Pro?Ser?Val?Ile
60 65 70 75
Bay scallop
gct?gcc?tta?gcc?agc?cgt?gag?tca?cgt?ggt?ggt?cgc?ctg?ttg?cat?tca 291
Ala?Ala?Leu?Ala?Ser?Arg?Glu?Ser?Arg?Gly?Gly?Arg?Leu?Leu?His?Ser
80 85 90
acc?aat?gga?tgg?gga?gac?cat?cac?cat?gct?tac?gga?att?tta?cag?tgt 339
Thr?Asn?Gly?Trp?Gly?Asp?His?His?His?Ala?Tyr?Gly?Ile?Leu?Gln?Cys
95 100 105
gac?atc?cgt?tac?cac?agc?tgt?acc?cag?cac?gca?tgg?gac?agc?tgt?gca 387
Asp?Ile?Arg?Tyr?His?Ser?Cys?Thr?Gln?His?Ala?Trp?Asp?Ser?Cys?Ala
110 115 120
cac?att?tcc?cag?atg?gtt?caa?gaa?gtc?cta?gtg?ccc?tac?atc?aat?cag 435
His?Ile?Ser?Gln?Met?Val?Gln?Glu?Val?Leu?Val?Pro?Tyr?Ile?Asn?Gln
125 130 135
gtg?gcc?cac?aaa?cat?ccc?aca?tgg?tca?aag?gag?caa?caa?ctc?cta?ggt 483
Val?Ala?His?Lys?His?Pro?Thr?Trp?Ser?Lys?Glu?Gln?Gln?Leu?Leu?Gly
140 145 150 155
gga?atc?gct?gct?tat?aac?tct?gga?gtc?gga?aac?gtc?cag?acc?tgg?agt 531
Gly?Ile?Ala?Ala?Tyr?Asn?Ser?Gly?Val?Gly?Asn?Val?Gln?Thr?Trp?Ser
160 165 170
ggc?ctt?gac?att?gga?aca?acc?gga?aat?gac?tac?agc?gat?gac?gta?gtt 579
Gly?Leu?Asp?Ile?Gly?Thr?Thr?Gly?Asn?Asp?Tyr?Ser?Asn?Asp?Val?Val
175 180 185
gca?cgt?gct?caa?tat?ctc?atc?agc?cat?tat?gga?tgg?cat?taataaaata 628
Ala?Arg?Ala?Gln?Tyr?Leu?Ile?Ser?His?Tyr?Gly?Trp?His
190 195 200
tatggctaca?gttaaaaaaa?aaaaaaaaaa?a 659
<210>2
<211>200
<212>PRT
＜213〉bay scallop (Argopecten irradians)
<400>2
Met?Asn?Ala?Leu?Val?Val?Ile?Thr?Leu?Leu?Ala?Phe?Ser?Thr?Gly?Ala
1 5 10 15
Trp?Ala?Ala?Ser?Tyr?Thr?Cys?His?Gly?Asp?Val?Arg?Arg?Leu?His?Pro
20 25 30
Thr?Gly?Glu?His?Asn?Gly?Gly?Asn?Ala?Ala?Ser?His?Asn?Asp?Val?Lys
35 40 45
Tyr?Asp?Tyr?Asn?Asp?Leu?Leu?Asn?Lys?Lys?Ser?Cys?Tyr?Asp?Gln?Ala
50 55 60
Bay scallop
Gly?Ala?Thr?Tyr?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?His?Ser?Thr?Asn?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?Thr?Gln?His?Ala?Trp?Asp?Ser?Cys?Ala?His?Ile?Ser?Gln?Met
115 120 125
Val?Gln?Glu?Val?Leu?Val?Pro?Tyr?Ile?Asn?Gln?Val?Ala?His?Lys?His
130 135 140
Pro?Thr?Trp?Ser?Lys?Glu?Gln?Gln?Leu?Leu?Gly?Gly?Ile?Ala?Ala?Tyr
145 150 155 160
Asn?Ser?Gly?Val?Gly?Asn?Val?Gln?Thr?Trp?Ser?Gly?Leu?Asp?Ile?Gly
165 170 175
Thr?Thr?Gly?Asn?Asp?Tyr?Ser?Asn?Asp?Val?Val?Ala?Arg?Ala?Gln?Tyr
180 185 190
Leu?Ile?Ser?His?Tyr?Gly?Trp?His
195 200
Claims (4)
1. bay scallop G type lysozyme gene is characterized in that: be the base sequence shown in the SEQ ID NO.1.
2. the described bay scallop 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 bay scallop G of claim 1 a type lysozyme gene is characterized in that, comprising:
1) purifying of the extraction of the total RNA of bay scallop and mRNA;
2) bay scallop cDNA library construction;
3) the extensive mensuration of bay scallop cDNA library est sequence;
4) homology analysis of bay scallop est sequence and the segmental screening of bay scallop G type lysozyme gene;
5) utilize 3 ' with 5 ' RACE technology clone bay scallop G type lysozyme gene cDNA full length sequence.
4. according to the cloning process of the described bay scallop G of claim 3 type lysozyme gene, it is characterized in that the extraction of the total RNA of described bay scallop is to extract total RNA from the bay scallop hemolymph that has infected Vibrio anguillarum.
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| CNB2004100506522A CN100344763C (en) | 2004-10-22 | 2004-10-22 | G-type lysozyme gene of Argopecten irradians and encoded protein and cloning method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100506522A CN100344763C (en) | 2004-10-22 | 2004-10-22 | G-type lysozyme gene of Argopecten irradians and encoded protein and cloning method thereof |
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| CN100344763C true CN100344763C (en) | 2007-10-24 |
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| CN102061291B (en) * | 2010-11-05 | 2012-10-03 | 中国科学院海洋研究所 | Lysozyme as well as preparation and application thereof |
| 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 CNB2004100506522A patent/CN100344763C/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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