CN100404680C - Duck B lymphocyte stimulating factor cDNA and its clone method and recombinant use - Google Patents
Duck B lymphocyte stimulating factor cDNA and its clone method and recombinant use Download PDFInfo
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- CN100404680C CN100404680C CNB2006100396663A CN200610039666A CN100404680C CN 100404680 C CN100404680 C CN 100404680C CN B2006100396663 A CNB2006100396663 A CN B2006100396663A CN 200610039666 A CN200610039666 A CN 200610039666A CN 100404680 C CN100404680 C CN 100404680C
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Abstract
The present invention relates to a duck B lymphocyte stimulating factor cDNA and a cloning method and recombined application thereof, which belongs to the field of biological genetic engineering. A gene of the duck B lymphocyte stimulating factor comprises sequences shown by SEQ ID No. 1 and SEQ ID No. 2. The duck B lymphocyte stimulating factor has the following cloning method that total RNA is extracted from a duck spleen according to a conserved sequence design primer of the B lymphocyte stimulating factor, a section of cDNA is enlarged by an RT-PCR method and is named P1 cloned in a pMD18-T carrier, and the sequence of P1 is detected; a 3'-5'-full length tail end region of the cDNA is enlarged by respectively using a sequence design positive sense primer and a negative sense primer with a cDNA tail end fast enlargement method, enlarged segments are respectively named P2 and P3 which are respectively cloned in the pMD18-T carrier, and the sequences of P2 and P3 are detected; P1, P2 and P3 are spliced into a cDNA full length sequence, a cDNA full length sequence is enlarged by a single step by designing a head primer and terminal primer and is cloned in the pMD18-T carrier; and the sequence of the cDNA full length sequence is detected by picking positive cloning. The cDNA of the duck B lymphocyte stimulating factor can produce recombination duck BAFF by the existing genetic engineering methods and is used as a duck type immunity reinforcing agent.
Description
Technical field
The present invention relates to the biological gene engineering field, be specifically related to duck B lymphocyte stimulating factor cDNA and clone thereof, expression technology.
Background technology
Human B lymphocyte stimulating factor (hBAFF) is that newfound a kind of and human immunity in 1999 are regulated and control closely-related tnf family cytokines member, mainly expresses in peripheral blood lymphocytes, spleen, lymphoglandula and marrow.BAFF has very strong B cell chemotaxis, external it as the costimulating factor of B cell proliferation and differentiation, can induce activatory B emiocytosis a large amount of IgG, IgA, IgM etc., for dormancy B cell, though can not independently activating, BAFF makes it to enter the cell cycle circulation, by raising inhibitor of apoptosis protein Bcl-2, the Bcl-X of cell surface
LExpression, prolong life-span of dormancy B cell.It can promote that the T1-B cell is to the growth of T2-B cell and mature B cell in the spleen in the body.The normal expression of BAFF is GC formation, affinity matured antibody, memory B cell forms and the necessary condition of B cell normal development.BAFF-/-the secondary lymphatic organ capsule of mouse reaches zone of transition B cell outward and lacks fully.BAFF stimulates B cell proliferation, differentiation justacrine antibody, and the antibody of sophisticated B cell and generation thereof has constituted body to be resisted infection and suppress the key ingredient that tumour is grown.Because the immunostimulant specific activity of BAFF has just shown huge potential applicability in clinical practice since being found.In November, 2000, U.S. FDA official approval recombinant human B AFF enters the human clinical trial, is used for the treatment of common variation immunodeficiency disease (CVID) patient.
Learn according to GenBank, EMBL and DDBJ international three big main nucleic acid sequence data library searching results, have only the BAFF cDNA of people, mouse, chicken to be cloned at present, and in the GenBank database, applied for sequence number respectively, sequence number is respectively AF116456, AF119383 and AF506010.Duck B lymphocyte stimulating factor (duBAFF) cDNA is not also come out by the clone, also is in complete space state about the research of duck BAFF gene both at home and abroad whole.Duck is crucial bird, because bone-marrow-derived lymphocyte stimulating factor (BAFF) has stronger immunostimulant ability, genetically engineered duck BAFF albumen might be developed to a kind of biotechnological formulation that can strengthen duck para-immunity ability, be used for that physique is weak, the sick duck of immunologic hypofunction, increase duck class anti-virus ability, especially in today that bird flu has constituted a threat to the mankind.In recent years, along with deepening continuously and the development of Protocols in Molecular Biology of pair cell factor research, a large amount of avian cytokines is cloned and is recombinant expressed, many cytokine gene engineering medicines appear on the market, new means are provided for the treatment poultry disease, created huge economic and social benefit simultaneously, therefore, reorganization duck BAFF albumen has potential great market using value.Simultaneously, bone-marrow-derived lymphocyte stimulating factor (BAFF) is the newfound immunity system important regulating and controlling factor, and the research of duck BAFF is helped to explore the immunoregulation mechanism of duck class, promotes the development of its immunity system research.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of bone-marrow-derived lymphocyte irritation factor CDNA that extracts from duck is provided, and the cloning process and the recombinant technology of bone-marrow-derived lymphocyte irritation factor CDNA are provided.
The bone-marrow-derived lymphocyte irritation factor CDNA is cloned in the present invention from duck, it has the sequence shown in the SEQ ID NO.1.
Duck B lymphocyte stimulating factor, it has the sequence shown in the SEQ ID NO.2.
The cloning process of duck B lymphocyte stimulating factor cDNA of the present invention is as follows:
(1) conserved sequence according to the bone-marrow-derived lymphocyte stimulating factor designs primer:
Positive MODN degenerate primer du1:5 '-CTGNNTGCANCTGATTGCNGACAG-3 ' (N=A, T, G, C)
Antisense oligonucleotide degenerate primer du2:5 '-GCACCAAANAANGTNNCNTCTCC-3 ' (N=A, T, G, C)
(2) extract total RNA from the duck spleen,
(3) by the RT-PCR method, be Auele Specific Primer with above-mentioned primer du1 and du2, amplify one section cDNA sequence, called after P1 is cloned into the pMD18-T carrier, and its base sequence is measured,
(4) use terminal rapid amplifying (RACE) method of cDNA, according to acquired P1 base sequence design sense primer GSP2 (5 '-GAAGGCCCACGTGTTTGGTGATGATCTG-3 ') and antisense primer GSP1 (5 '-CAGATCATCACCAAACACGTGGGCCTTC-3 ') respectively in order to amplify above-mentioned cDNA 3 '-and 5 '-total length stub area, amplified fragments is called after P2 and P3 respectively, be cloned into the pMD18-T carrier respectively, its base sequence is measured;
(5) splice the full length sequence of cDNA according to the overlapping region of P1, P2, P3, and design head and the tail primers F L1 (5 '-GGAGGAAAGAGCAGCGATGAAATCC-3 ') and FL2 (5 '-GGATGGAACAAAATTAAAATGCACT-3 ') step amplifies full length cDNA sequence, be cloned into the pMD18-T carrier, the picking positive colony carries out base sequence and measures.
The cloning process concrete operations of above-mentioned duck B lymphocyte stimulating factor cDNA are as follows:
(1) according to the positive MODN degenerate primer du1 of two sections high conserved region territories of bone-marrow-derived lymphocyte stimulating factor full-length cDNA design homologous clone (5 '-CTGNNTGCANCTGATTGCNGACAG-3 ' (N=A, T, G, C)) and antisense oligonucleotide degenerate primer du2 (5 '-GCACCAAANAANGTNNCNTCTCC-3 ' (N=A, T, G, C))
(2) use RNA extraction agent TRIzol (Invitrogen company),, extract total RNA of duck spleen cell according to operational manual,
(3) using the RT-PCR method, is Auele Specific Primer with above-mentioned du1 and du2, amplifies one section cDNA sequence, and total length is 408bp, and called after P1 is cloned into pMD 18-T carrier, and its base sequence is measured.The reaction conditions of RT-PCR is: with total RNA is template, in 50 μ l reaction systems, adds 5 * RT-PCR reaction buffer, 10 μ l, 25mM MgSO
4Each 2.5 μ l of the upstream and downstream primer of 2 μ l, 10mM dNTP mixture 1 μ l, 20 μ M, AMV reversed transcriptive enzyme (Takara company) 1 μ l, Tf1 archaeal dna polymerase 1 μ l and RNA template 2 μ l, moisturizing to 50 μ l.The RT-PCR loop parameter is: 48 ℃ of reverse transcription 45min, 94 ℃ of sex change 2min carry out 40 PCR circulations (68 ℃ are extended 2min for 94 ℃ of sex change 30s, 57 ℃ of annealing 1min) again, extend 7min in 68 ℃ at last.The RT-PCR product reclaims the DNA band that test kit reclaims about 408bp with glue after separating with 1.5% agarose gel electrophoresis, puts 4 ℃ of preservations.
(4) use terminal rapid amplifying (RACE) method of cDNA, adopt SMART RACE Kit (Clontech company) according to acquired P1 base sequence design sense primer GSP2 (5 '-GAAGGCCCACGTGTTTGGTGATGATCTG-3 ') and antisense primer GSP1 (5 '-CAGATCATCACCAAACACGTGGGCCTTC-3 ') respectively in order to amplify above-mentioned cDNA3 '-and 5 '-total length stub area, amplified fragments is called after P2 (642bp) and P3 (697bp) respectively, be cloned into the pMD18-T carrier respectively, its base sequence is measured.
(5) splice full length sequence according to the overlapping region of P1, P2, P3, and design head and the tail primers F L1 (5 '-GGAGGAAAGAGCAGCGATGAAATCC-3 ') and FL2 (5 '-GGATGGAACAAAATTAAAATGCACT-3 ') step amplifies full length cDNA sequence, be cloned into the pMD18-T carrier, 8 positive colonies of picking carry out base sequence and measure.
Institute's calling sequence and GenBank database sequence are carried out similarity searching, find with known chicken, people, mouse BAFF sequence similarity rate the highest, be respectively 91%, 53%, 50%, can determine that this sequence is the full-length cDNA of duck B lymphocyte stimulating factor (duBAFF), its open reading frame is shown in SEQ ID NO.1.
To further studies show that of this gene: this gene mainly is expressed in the duck para-immunity organ, comprises bursa of Fabricius, spleen and thymus gland.Wherein the bursa of Fabricius expression level is the highest, and spleen takes second place, and thymus gland is minimum; The recombination fusion protein of this gene has the high reactivity function of dsBAFF; The albumen of this genes encoding has significantly short survival/multiplication effect to duck B lymphocyte; The biological function that the bone-marrow-derived lymphocyte of people and mouse is all had short survival/propagation; Show that fully the present invention clones the cDNA that the gene that obtains is exactly duck B lymphocyte stimulating factor (duBAFF).
The reorganization of the cDNA of above-mentioned duck B lymphocyte stimulating factor (duBAFF) is used, and by existing gene engineering method, produces reorganization duck BAFF, as duck para-immunity toughener.
Said duck B lymphocyte stimulating factor cDNA changes in the duck body by existing gene engineering method, increases its immunizing power, uses in raising.
Description of drawings
Fig. 1 is a duck BAFF Full Length cDNA Cloning process synoptic diagram; Wherein P1 represents the homologous clone fragment; P2 represents 3 '-RACE product; P3 represents 5 '-RACE product; Arrow is represented primer location; Full-length cDNA is 1311bp altogether.
Fig. 2 is duck BAFF full-length cDNA base sequence and infers encoding amino acid sequence.AATAAA represents the polyadenylic acid tailing signal; Unstable sequence is transcribed in the ATTTA representative.
Fig. 3 is duck BAFF and chicken, people, mouse BAFF full length amino acid sequence homology comparison diagram.Wherein shade is represented conservative amino acid; The arrow indication is represented the furin recognition site, and the C end after the cutting is the outer solvable functional zone of the born of the same parents of BAFF.
Fig. 4 is the expression level analysis chart of duck BAFF mRNA in each tissue.β-actin is as confidential reference items.
Fig. 5 is the abduction delivering of fusion rotein NusA-His-dsBAFF in BL21 (DE3), Ni
+The SDS-PAGE evaluation of column purification and the western-blotting qualification result of mouse-anti His6-tag, band 1 is without IPTG inductive whole bacterial protein, the 2nd, the whole bacterial protein after IPTG induces 5 hours, the 3rd, target protein behind the Ni+ column purification among the A figure; B figure is the western-blotting qualification result of mouse-anti His6-tag, specific band occurred in the target protein position.
Fig. 6 is the short survival exercising result synoptic diagram of cloned genes of the present invention to duck B lymphocyte.Figure A is that the NusA-His-dsBAFF of explanation different concns after 48 hours, to the visible significant short survival effect of duck B lymphocyte, and is dose-dependent effect at the effect duck B lymphocyte with respect to contrast NusA-His, His-TRAIL and PBS; Figure B is that the NusA-His-dsBAFF of explanation fixed concentration is acting on the duck B lymphocyte survival quantity comparable situation of duck B lymphocyte after 24/48/72 hour respectively with respect to contrast NusA-His and PBS.
Fig. 7 is that duck BAFF has the short propagation biological function to people and mouse bone-marrow-derived lymphocyte.Figure A is altogether the stimulate proliferation effect of the NusA-His-dsBAFF of explanation different concns to human B lymphocyte; Figure B is altogether the stimulate proliferation effect of the NusA-His-dsBAFF of explanation different concns to the mouse bone-marrow-derived lymphocyte; Anti-IgM is a costimulating factor.
Embodiment
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with concrete preparation embodiment and Application Example, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art.Used primer is all indicated when occurring first, and used thereafter same primers as is all identical with the content of indicating first.
Embodiment 1:
Duck (Anas Platyrhynchos), artificial breeding.
(1) design of primers: use ClustalX software people, mouse, the chicken B lymphocyte stimulating factor full length cDNA sequence that has cloned carried out the homology compare of analysis, select positive MODN degenerate primer du1 (5 '-CTGNNTGCANCTGATTGCNGACAG-3 ' (N=A, T, G, C)) that two sections high conserved region territories are used for designing homologous clone and antisense oligonucleotide degenerate primer du2 (5 '-GCACCAAANAANGTNNCNTCTCC-3 ' (N=A, T, G, C)) meticulously
(2) extract total RNA: use RNA extraction agent TRIzol (Invitrogen company) extracts about 0.5 gram duck spleen cell according to its operational manual total RNA, and identify its quality and purity by the denaturing formaldehyde agarose gel electrophoresis, ultraviolet spectrophotometer is measured its concentration.
(3) using the RT-PCR method, is Auele Specific Primer with above-mentioned du1 and du2, amplifies one section sequence of duck BAFF cDNA, and total length is 408bp, and called after P1 is cloned into the pMD18-T carrier, and its base sequence is measured.The reaction conditions of RT-PCR is: with total RNA is template, in 50 μ l reaction systems, adds 5 * RT-PCR reaction buffer, 10 μ l, 25mM MgSO
4Each 2.5 μ l of the upstream and downstream primer of 2 μ l, 10mM dNTP mixture 1 μ l, 20 μ M, AMV reversed transcriptive enzyme (Takara company) 1 μ l, Tf1 archaeal dna polymerase 1 μ l and RNA template 2 μ l, moisturizing to 50 μ l.The RT-PCR loop parameter is: 48 ℃ of reverse transcription 45min, 94 ℃ of sex change 2min carry out 40 PCR circulations (68 ℃ are extended 2min for 94 ℃ of sex change 30s, 57 ℃ of annealing 1min) again, extend 7min in 68 ℃ at last.The RT-PCR product reclaims the DNA band that test kit reclaims about 408bp with glue after separating with 1.5% agarose gel electrophoresis, puts 4 ℃ of preservations.
(4) use terminal rapid amplifying (RACE) method of cDNA, adopt SMART RACE Kit (Clontech company) according to acquired P1 base sequence design sense primer GSP2 and antisense primer GSP1 respectively in order to amplify duck BAFFcDNA 3 '-and 5 '-total length stub area, amplified fragments is called after P2 (642bp) and P3 (697bp) respectively, be cloned into the pMD18-T carrier respectively, its base sequence is measured.Operation requires to carry out according to the test kit operational manual.
(5) as shown in Figure 1, the full length sequence that splices duck BAFF cDNA according to the overlapping region of P1, P2, P3, and design head and the tail primers F L1 and one step of FL2 amplify full length cDNA sequence, be cloned into the pMD18-T carrier, through containing the agarose plate screening transformant of Amp microbiotic (50mg/ml), picking goes out 8 positive colonies and is sent to Shanghai English fine horse order-checking company and carries out base sequence and measure, and obtains duck BAFF full-length cDNA base sequence as shown in Figure 2 and infers encoding amino acid sequence.
(6) homology retrieval: with institute's calling sequence to
Http:// www.ncbi.nlm.nih.gov/BLAST/The cDNA sequence of submitting to the clone to obtain is carried out similarity searching and homology analysis at GenBank, EMBL and DDBJ international three big main nucleic acid sequence data storehouses, as shown in Figure 3, find and known chicken (GenBank accession number AF506010), people (GenBank accession number AF116456), mouse (GenBank accession number AF119383) BAFF amino acid sequence similarity the highest (the predicted amino acid sequence that does not comprise other kinds BAFF), be respectively 91%, 53%, 50%, can determine that this sequence is the full-length cDNA of duck B lymphocyte stimulating factor (duBAFF).And its open reading frame has SEQ ID NO.1 sequence, and the proteic aminoacid sequence of rock sign indicating number is shown in SEQ ID NO.2.
Embodiment 2:
Duck BAFF gene is in the expression level analysis of each tissue
Adopt the method for the extraction RNA among the embodiment 1, extracted total RNA of duck liver (liver), kidney (kidney), thymus gland (thymus), cloacal bursa (bursa), spleen (spleen), the heart (heat) and brain (brain) respectively, the utilization semi-quantitative RT-PCR is studied at the expression level of each tissue duck BAFF gene, the result as shown in Figure 4, duck BAFF gene mainly is expressed in the duck para-immunity organ, comprises bursa of Fabricius, spleen and thymus gland; Wherein the bursa of Fabricius expression level is the highest, and spleen takes second place, and thymus gland is minimum.Duck β-actin is as confidential reference items in the test, and the primer of employing is ch β 1 (5 '-ATGGCTCCGGTATGTGCAAGG-3 ') and ch β 2 (5 '-AGCTTCTCCTTGATGICACGC-3 ').RT-PCR system such as embodiment 1.
The structure of solubility duck BAFF recombinant vectors and the abduction delivering in intestinal bacteria thereof
The duck BAFF full-length cDNA that obtains with embodiment 1 is a template, design primer sdu1 (5 '-TCAGGACCGCGGGTTCTGCCGTCCACACAGAAG-3 ' (SacII)) and sdu2 (5 '-ACTGAGCCCGGGTCAGAAGAGTCTGACGGCACCA-3 ' (SmaI)) amplify the outer solvable section (dsBAFF) of duck BAFFcDNA born of the same parents, primer is introduced restriction enzyme site Sac II and Sma I respectively, subclone is gone into the pET43.1a carrier, is built into recombinant vectors pET43.1a-dsBAFF.Change this recombinant vectors over to escherichia coli expression bacterial strain BL21 (DE3), but high dissolubility gives expression to target protein NusA-His-dsBAFF in the endochylema, this fusion rotein is through short survival and proliferation test (Fig. 7), the active detection confirms to have the high reactivity function of dsBAFF, and the NusA-His label does not influence the target protein activity.
The reorganization target protein the Ni+ affinity purification
IPTG induces the escherichia coli expression bacterial strain BL21 (DE3) that contains recombinant vectors pET43.1a-dsBAFF after 5 hours, receives bacterium for 4 ℃, and (work 10s suspends 10s in ultrasonication on ice, totally 99 times) the expression thalline, 4 ℃, 13,000 * g, behind the centrifugal ultrasonication liquid of 20min, abandon precipitation, stay supernatant to cross Ni
+-NTA post.Concise and to the point process is: 3 volume BindingBuffer balance Ni
+Behind-NTA the post, manually go up the supernatant that sample contains the solubility recombinant protein, 6 volume Wash Buffer (60mM imidazoles, 0.5M NaCl, 20mM Tris-HCl pH7.9) flush away is not in conjunction with last Ni
+The foreign protein of-NTA post, last 6 volume Elute Buffer (1M imidazoles, 0.5M NaCl, 20mM Tris-HCl pH7.9) wash-out target protein, the PBS desalination of dialysing is with SDS-PAGE and capillary electrophoresis analysis purity and with the UV spectrophotometer measuring protein concentration, as Fig. 5 (A, band 3) shown in, the target protein that expression obtains obtains purity through Ni+ post affinity purification and reaches 95% active target protein.
The western engram analysis
Used one anti-is goat-anti His among the Western-blot
6, two anti-be the mouse-anti sheep IgG of horseradish peroxidase mark.Its concise and to the point step is: with the inductive product SDS-PAGE that goes ahead of the rest, then with the immerseable method with the albumen electrotransfer to nitrocellulose filter (NC film), with each band of marking pen labelled protein marker, however successively through 5% skim-milk room temperature sealing 1h, with anti-His
6Monoclonal antibody is hatched 1h, hatches 1h with the mouse-anti sheep IgG of HRP mark, per step finish after all strictness wash film, add the colour developing of TMB lucifuge at last.Specific band has appearred in result such as Fig. 5 (B) target protein position that is shown in.
Duck BAFF is to the short survival effect experiment of duck bursa of Fabricius bone-marrow-derived lymphocyte
Adopt the conventional aseptic separation duck bursa of Fabricius lymphocyte (about 98% is bone-marrow-derived lymphocyte) of lymphocyte separation medium, regulate cell density to 5 * 10 with RPMI1640
7Individual/mL adds 96 well culture plates with the B cell suspension, every hole 100 μ l.The NusA-His-dsBAFF/NusA-His that in every hole, adds different concns
6/ His-TRAIL, 37 ℃, 5%CO
2Every hole adds 10 μ l 5mg/mL MTT, 37 ℃, 5%CO after cultivating 72h
2Continue to cultivate 5h, add 100 μ l SDS-HCl dissolution precipitation things, 37 ℃ are spent the night, and measure each hole OD
570Three multiple holes are established in the value experiment, get its mean value, and the base of calculation error.Detect the short survival effect of duck BAFF external by the MTT cytotoxicity test to duck B lymphocyte, as shown in Figure 6, the result shows that the present invention clones the duck BAFF that obtains duck B lymphocyte is had significantly short survival effect, identical with the biological function of chicken, people, mouse BAFF, and present dose-dependent effect, promptly the short proliferation function of duck BAFF is the strongest when NusA-His-dsBAFF is 8 μ g/ml.Simultaneously, the NusA-His-dsBAFF of fixed concentration is acting on duck B lymphocyte after 24/48/72 hour respectively with respect to contrast NusA-His and PBS, and the short survival effect to duck B lymphocyte when can be observed 72 hours is the most obvious.
Duck BAFF and anti-IgM stimulate people mouse bone-marrow-derived lymphocyte propagation altogether
Adopt antibody magnetic bead aseptic separation healthy human peripheral blood bone-marrow-derived lymphocyte and BALB/c mouse spleen bone-marrow-derived lymphocyte, detect duck BAFF to the short proliferation function of human peripheral blood B lymphocyte (anti-people IgM stimulates the back in advance) and to the short proliferation function of mouse spleen bone-marrow-derived lymphocyte (anti-mouse IgM stimulates the back in advance) external by the MTT cytotoxicity test, method is the same, the result as shown in Figure 7, show that the present invention clones the duck BAFF that obtains has short propagation to the bone-marrow-derived lymphocyte of people and mouse biological function, and present dose-dependent effect, saturation concentration is respectively 12 μ g/ml and 14 μ g/ml, has further confirmed the BAFF conservative property of biological function in vivo.
Embodiment 3:
The duck B lymphocyte stimulating factor cDNA that embodiment 1 is obtained passes through existing gene engineering method, produces reorganization duckBAFF, as duck para-immunity toughener.
Embodiment 4:
The duck B lymphocyte stimulating factor cDNA that embodiment 1 is obtained passes through existing gene engineering method, changes in the duck body, increases its immunizing power, uses in raising.
Can modify the duck B lymphocyte stimulating factor gene and be used for described research and production by existing genetic engineering technique according to method of the present invention.
SEQUENCE?LISTING
＜110〉Nanjing Normal University
＜120〉duck B lymphocyte stimulating factor cDNA and cloning process thereof and reorganization are used
<160>2
<210>1
<211>867
<212>cDNA
＜213〉duck (Anas Platyrhynchos)
<400>1
atgaaatccg?tggactgtgt?gcacgtcatc?caacagaagg?ataccgcctc?ctctccctct 60
gccccccctg?gtgctgcttc?aggcaccacg?ggactttttc?ctgtcacatt?cctgtggctt 120
gcaatgctcc?tgtcctcttg?tcttgcagca?gtgtctcttt?accatgttct?caccctgaaa 180
acagagctag?aagctctgcg?ccacgagctg?atctacaggg?tccaggcaag?gtctccacta 240
gagcggcgag?ccgtctcccc?tgatgacgag?aaagcaggtg?cccctgtttc?ttccttcctg 300
caagcctctg?cagctggtgc?caggcaggag?aacgagcttc?ctggccctag?cccagctgaa 360
agtttccaaa?aggaaatctc?gaacgggagc?agaaacaggg?ggagaaggtc?tgccgtccac 420
acagaagaaa?cagtgctaca?ggcctgcttg?caactcatcg?ctgacagcaa?aagtgatatc 480
caacagaaag?atgattcaag?cattgtccct?tggcttctga?gctttaaacg?tggaacagct 540
ctggaagaac?atggaaataa?aatagtgatc?aaagaaacag?gatatttttt?catatatggc 600
caggttttat?atacagatac?aacatttgct?atgggacatc?taatacagag?gaagaaggcc 660
cacgtgtttg?gtgatgatct?gagcttggtg?acattatttc?gctgcatcca?aaatatgcca 720
cagtcttatc?cgaataattc?ttgctatacc?gctggcattg?caaaattaga?agaaggggac 780
gaacttcaac?ttacaatacc?acgaagacga?gccaaaatat?ccttggatgg?cgatggtact 840
ttttttggtg?ccgtcagact?cttctga 867
<210>2
<211>288
<212>PRT
＜213〉duck (Anas Platyrhynchos)
<400>2
Met?Lys?Ser?Val?Asp?Cys?Val?His?Val?Ile?Gln?Gln?Lys?Asp?Thr
1 5 10 15
Ala?Ser?Ser?Pro?Ser?Ala?Pro?Pro?Gly?Ala?Ala?Ser?Gly?Thr?Thr
20 25 30
Gly?Leu?Phe?Pro?Val?Thr?Phe?Leu?Trp?Leu?Ala?Met?Leu?Leu?Ser
35 40 45
Ser?Cys?Leu?Ala?Ala?Val?Ser?Leu?Tyr?His?Val?Leu?Thr?Leu?Lys
50 55 60
Thr?Glu?Leu?Glu?Ala?Leu?Arg?His?Glu?Leu?Ile?Tyr?Arg?Val?Gln
65 70 75
Ala?Arg?Ser?Pro?Leu?Glu?Arg?Arg?Ala?Val?Ser?Pro?Asp?Asp?Gln
80 85 90
Lys?Ala?Gly?Ala?Pro?Val?Ser?Ser?Phe?Leu?Gln?Ala?Ser?Ala?Ala
95 100 105
Gly?Ala?Arg?Gln?Glu?Asn?Glu?Leu?Pro?Gly?Pro?Ser?Pro?Ala?Glu
110 1l5 120
Ser?Phe?Gln?Lys?Glu?Ile?Ser?Asn?Gly?Ser?Arg?Asn?Arg?Gly?Arg
125 130 135
Arg?Ser?Ala?Val?His?Thr?Glu?Glu?Thr?Val?Leu?Gln?Ala?Cys?Leu
140 145 150
Gln?Leu?Ile?Ala?Asp?Ser?Lys?Ser?Asp?Ile?Gln?Gln?Lys?Asp?Asp
155 160 165
Ser?Ser?Ile?Val?Pro?Trp?Leu?Leu?Ser?Phe?Lys?Arg?Gly?Thr?Ala
170 175 180
Leu?Glu?Glu?His?Gly?Asn?Lys?Ile?Val?Ile?Lys?Glu?Thr?Gly?Tyr
185 190 195
Phe?Phe?Ile?Tyr?Gly?Gln?Val?Leu?Tyr?Thr?Asp?Thr?Thr?Phe?Ala
200 205 210
Met?Gly?His?Leu?Ile?Gln?Arg?Lys?Lys?Ala?His?Val?Phe?Gly?Asp
215 220 225
Asp?Leu?Ser?Leu?Val?Thr?Leu?Phe?Arg?Cys?Ile?Gln?Asn?Met?Pro
230 235 240
Gln?Ser?Tyr?Pro?Asn?Asn?Ser?Cys?Thr?Thr?Ala?Gly?Ile?Ala?Lys
245 250 255
Leu?GIu?Glu?Gly?Asp?Glu?Leu?Gln?Leu?Thr?Ile?Pro?Arg?Arg?Arg
260 265 270
Ala?Lys?Ile?Ser?Leu?Asp?Gly?Asp?Gly?Thr?Phe?Phe?Gly?Ala?Val
275 280 285
Arg?Leu?Phe
288
Claims (4)
1. duck B lymphocyte stimulating factor cDNA is characterized in that, has the sequence shown in the SEQ ID NO.1.
2. the cloning process of the described duck B lymphocyte stimulating factor cDNA of claim 1, as follows:
(1) conserved sequence according to the bone-marrow-derived lymphocyte stimulating factor designs primer:
Positive MODN degenerate primer du1:5 '-CTGNNTGCANCTGATTGCNGACAG-3 ', antisense oligonucleotide degenerate primer du2:5 '-GCACCAAANAANGTNNCNTCTCC-3 ', wherein N is A, T, G or C;
(2) extract total RNA from the duck spleen,
(3) by the RT-PCR method, be Auele Specific Primer with above-mentioned primer du1 and du2, amplify one section cDNA sequence, called after P1 is cloned into the pMD18-T carrier, and its base sequence is measured,
(4) use the terminal rapid amplifying method of cDNA, according to acquired P1 base sequence design sense primer GSP25 '-GAAGGCCCACGTGTTTGGTGATGATCTG-3 ' and antisense primer GSP1 5 '-CAGATCATCACCAAACACGTGGGCCTTC-3 ' respectively in order to amplify above-mentioned cDNA 3 '-and 5 '-total length stub area, amplified fragments is called after P2 and P3 respectively, be cloned into the pMD18-T carrier respectively, its base sequence is measured;
(5) splice the full length sequence of cDNA according to the overlapping region of P1, P2, P3, and design head and the tail primers F L15 '-GGAGGAAAGAGCAGCGATGAAATCC-3 ' and FL2 5 '-GGATGGAACAAAATTAAAATGCACT-3 ' step amplify full length cDNA sequence, be cloned into the pMD18-T carrier, the picking positive colony carries out base sequence and measures.
3. the reorganization of the described duck B lymphocyte stimulating factor cDNA of claim 1 is used, and is by gene engineering method, produces the reorganization duck B lymphocyte stimulating factor, as duck para-immunity toughener.
4. duck B lymphocyte stimulating factor is characterized in that, has the sequence shown in the SEQ ID NO.2.
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| CN102121012A (en) * | 2010-12-21 | 2011-07-13 | 南京师范大学 | Dog B lymphocyte activating factor cDNA, cloning method and recombination application thereof |
| CN102115748A (en) * | 2010-12-21 | 2011-07-06 | 南京师范大学 | Danio rerio B-lymphocyte activating factor (zBAFF) cDNA (complementary deoxyribonucleic acid), and cloning method and application thereof in recombination |
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| WO2001004317A1 (en) * | 1999-07-14 | 2001-01-18 | Institute Of Molecular Agrobiology | Ompa gene for an outer membrane protein of riemerella anatipestifer and methods of use |
| CN1308127A (en) * | 2000-11-02 | 2001-08-15 | 南京师范大学 | Expression carrier for recombinant human B lymphocyte stimulating factor and its construction process |
| CN1660902A (en) * | 2004-12-20 | 2005-08-31 | 南京师范大学 | Segregative technique for purifying stimulating factor of recombined human beta lymphocyte |
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| WO2001004317A1 (en) * | 1999-07-14 | 2001-01-18 | Institute Of Molecular Agrobiology | Ompa gene for an outer membrane protein of riemerella anatipestifer and methods of use |
| CN1308127A (en) * | 2000-11-02 | 2001-08-15 | 南京师范大学 | Expression carrier for recombinant human B lymphocyte stimulating factor and its construction process |
| CN1660902A (en) * | 2004-12-20 | 2005-08-31 | 南京师范大学 | Segregative technique for purifying stimulating factor of recombined human beta lymphocyte |
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