CN102121012A - Dog B lymphocyte activating factor cDNA, cloning method and recombination application thereof - Google Patents

Abstract

The invention discloses a dog B lymphocyte activating factor (dog BAFF) cDNA, a cloning method and recombination application thereof, relating to the field of biological genetic engineering. A gene of the dog B lymphocyte activating factor has a sequence shown in SEQ ID NO.1. The cloning method comprises the steps of: extracting total dog spleen RNA by using a TRIzol method and carrying out reverse transcription; analyzing human, rat, chick, duck and goose BAFFcDNA conversed regions through Clustalw software, and designing a degenerate primer for carrying out PCR (Polymerase Chain Reaction) amplification to obtain a 340bp amplification segment P; and designing a specific primer according to the 340bp amplification segment P and cloning a full-length BAFFcDNA through RACEPCR. The dog B lymphocyte activating factor cDNA can be used for producing a recombinant dog BAFF as an immune potentiator for canoidea through the traditional genetic engineering method.

Description

Dog bone-marrow-derived lymphocyte irritation factor CDNA and cloning process thereof and reorganization are used

Technical field

The present invention relates to the biological gene engineering field, be specifically related to dog bone-marrow-derived lymphocyte irritation 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.HBAFF 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, make it to enter the cell cycle circulation though hBAFF can not independently activate, it is by raising inhibitor of apoptosis protein Bcl-2, the Bcl-X of cell surface _LExpression, can 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 hBAFF 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.HBAFF 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 hBAFF has just shown huge potential applicability in clinical practice since being found.In November, 2000, U.S. FDA official approval recombinant human hBAFF 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, the BAFF cDNA of people, mouse, pig, ox, sheep, rabbit, chicken, duck, goose is cloned at present, and in the GenBank database, applied for sequence number respectively, sequence number is respectively AF116456, AF119383, EF041845, EU213012, FJ793192, EF202603, AF506010, DQ445092 and DQ874394.Dog bone-marrow-derived lymphocyte stimulating factor (dBAFF) cDNA is not also come out by the clone, also is in complete space state about the research of dog BAFF gene both at home and abroad whole.

Dog not only provides delicious meat, can also look after the house, herd and as the capable warrior of public security, army to us, and protection country, the people's property safeties are described as " mankind Achates ".Along with the raising of people's living standard, pet dog is also closely bound up with people's life gradually, is the highest pet of raising rate.Therefore, the prevention of the disease of dog and control have become the problem that everybody is concerned about.Because bone-marrow-derived lymphocyte stimulating factor (BAFF) has stronger immunostimulant ability, genetically engineered dog BAFF albumen might be developed to a kind of biotechnological formulation that can strengthen dog para-immunity ability, to strengthen the immunological competence of dog class.In recent years, along with deepening continuously and the development of Protocols in Molecular Biology of pair cell factor research, a large amount of cytokines is cloned and is recombinant expressed, many cytokine gene engineering medicines appear on the market, new means are provided for the treatment disease, created huge economic and social benefit simultaneously, therefore, reorganization dBAFF albumen has potential great market using value.

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 dog is provided, and the cloning process and the recombinant technology of dog bone-marrow-derived lymphocyte irritation factor CDNA are provided.

The bone-marrow-derived lymphocyte irritation factor CDNA is cloned in the present invention from dog, it has the sequence shown in the SEQ ID NO.1.

The cloning process of dog bone-marrow-derived lymphocyte irritation factor CDNA of the present invention is as follows:

(1) extracts total RNA from the dog spleen tissue

(2) utilize the RT-PCR method by homologous clone (homology cloning) strategy and in conjunction with terminal rapid amplifying method (RACE) cloned dog of cDNA total length BAFF cDNA sequence SEQ ID NO.1.

The cloning process concrete operations of above-mentioned dog bone-marrow-derived lymphocyte irritation factor CDNA are as follows:

(1) utilize Trizol reagent single stage method to extract the total RNA of dog spleen tissue

(2) by Clustal w software analysis people, mouse, chicken, duck, goose, pig BAFF cDNA conserved regions, design degenerate primer dBAFF1:5'-TTGTNCCNTGGCTTCTNAGCTTTAA-3'(SEQ ID NO.4); DBAFF2:5'-GCACCAAANAANGTNNCNTCTCC-3'(SEQ ID NO.5) carries out pcr amplification, obtain the fragment P1 of 340bp.

(3) with above-mentioned homologous clone fragment P1 design Auele Specific Primer (GST2:5'-CGCAAAGCTGGAAGAAGGAGATGAG-3', the SEQ ID NO.6 that have obtained; GST3:5'-GTCCCATGGCAAAG GTGTTATCGGTG-3', SEQ ID NO.7).The 3'RACE amplification obtains BAFF full-length cDNA 3' terminal fragment P2(and comprises 3'UTR), the 5'RACE amplification obtains BAFF full-length cDNA 5' terminal fragment P3(and comprises 5'UTR).Three sequences of P1, P2, P3 that obtain are spliced with BLAST2 software, obtain BAFF full-length cDNA (SEQ ID NO.1).

Institute's calling sequence and GenBank database sequence are carried out similarity searching, discovery and known people, mouse, pig, ox, sheep, rabbit, chicken, duck, goose BAFF sequence similarity rate are all than higher, be respectively 83%, 66%, 62%, 81%, 81%, 80%, 37%, 57%, 57%, can determine that this sequence is the full-length cDNA of dog bone-marrow-derived lymphocyte stimulating factor (dBAFF).

Further research to this gene: quantitative fluorescent PCR is analyzed the expression of dBAFF in each tissue (heart, liver, spleen, lung, kidney) and is shown, dBAFF mainly expresses at immune organ, and as spleen, expression amount is minimum in small intestine; The recombination fusion protein of this gene has the high reactivity function of dBAFF; The albumen of this genes encoding has significantly short survival/increment effect to mouse lymphocyte.

The reorganization of the cDNA of above-mentioned dog bone-marrow-derived lymphocyte stimulating factor (dBAFF) is used, and by existing gene engineering method, produces reorganization dBAFF, as the immunostimulant of dog, strengthens its immunizing power, uses in raising.

Description of drawings

Fig. 1 be dBAFF full-length cDNA base sequence (GenBank NO. FJ793193) and corresponding amino acid sequence () and (★) mark be terminator codon TGA

Fig. 2 is dBAFF and ox, pig, people, rabbit, mouse BAFF full length amino acid sequence homology comparison diagram.Stride diaphragm area with dashed lines mark, square frame and arrow are depicted as the furin protease cutting site, and two-wire mark position is the Flap ring, (★) is three conservative Cys residues, and the line part is the TNF conserved regions.

Fig. 3 is the expression level analysis of dBAFF mRNA in each tissue.GAPDH is as confidential reference items.

Fig. 4 is the prediction space structure (left side) of dBAFF, and the right side is the prediction space structure of human BAFF.

Fig. 5 is that fusion rotein sumo-dsBAFF is at BL21(DE3) in abduction delivering, 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 among the figure; The 2nd, the whole bacterial protein after IPTG induces 4.5 hours; The 3rd, through Ni ⁺Purified target protein; The 4th, cut albumen dsBAFF afterwards with sumo proteolytic enzyme enzyme; The 5th, use mouse-anti His ₆The western-blotting qualification result of monoclonal antibody.

Fig. 6 detects the result of sumo-dsBAFF to the short survival effect of mouse lymphocyte external by the MTT cytotoxicity test.As shown in the figure, PBS is a blank, 10ug/ml SUMO-dsBAFF, the negative contrast of 10ug/ml SUMO+anti-mouse IgM.

Fig. 7 is the short survival effect of Flow cytometry sumo-dsBAFF to mouse lymphocyte.The left side is for only adding the blank of PBS, and the right side is for using the detected result after 16ug/ml SUMO-dsBAFF+anti-mouse IgM handles 48 hours.

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

8 monthly ages China Taihang dog; Available from red sun farm, Nanjing

(1) design of primers: by Clustal w software analysis people, mouse, chicken, duck, goose, pig BAFF cDNA conserved regions, design degenerate primer dBAFF1:5'-TTGTNCCNTGGCTTCTNAGCTTTAA-3'; DBAFF2:5'-GCACCAAANAANGTNNCNTCTCC-3'.

(2) extract total RNA: the total RNA that extracts the dog spleen cell application RNA extraction agent TRIzol(Invitrogen company) according to its operational manual, and identify its quality and purity by the denaturing formaldehyde agarose gel electrophoresis, ultraviolet spectrophotometer is measured its concentration.

(3) RT-PCR: with above-mentioned dBAFF1 and dBAFF2 is that primer carries out pcr amplification, obtains the fragment P1 of 340bp.

1. reverse transcription

In the 1.5ml Eppendorf pipe that DEPC handles, add total RNA extract 3 μ l successively, Oligo d (T) ₁₈1 μ l, 10mmol/L dNTP 2 μ l, DEPC water 5 μ l place; 65 ° of C, ice bath 5min immediately behind the 15min; In above-mentioned system, add 5 * RT reaction buffer, 4 μ l again, RNase inhibitor 0.5 μ l (20u), AMV ThermoScript II 2 μ l, DEPC water 2.5 μ l to cumulative volume be the reaction solution of 20 μ l, in 42 ° of C reaction 1.5h, 94 ° of C, the 10min termination reaction places on ice;

②PCR

Utilize reverse transcription gained template to carry out PCR, system is 50 μ l, each 2.5 μ l of 10 μ mol/L upstream and downstream primers, 2.5mmol/L dNTP 4 μ l, 25mmol/L MgCl ₂3 μ l, 10 * damping fluid, 5 μ l, cDNA template 5 μ l, r TaqEnzyme 1 μ l adds ddH ₂O 27 μ l.Response procedures is: 94 ° of C 5min, and 94 ° of C 30s, 56 ° of C 30s, 72 ° of C 1min, 30 circulations, last 72 ° of C are hatched 5min.

The reaction finish after with product electrophoretic separation in 1% sepharose, reclaim test kit with glue and reclaim the DNA band that obtains about 340bp, be cloned into the pMD18-T carrier, through containing the agarose plate screening transformant of Amp microbiotic (50mg/ml), choose positive colony and be sent to Shanghai English fine horse order-checking company and carry out base sequence and measure.

(4) terminal rapid amplifying method (RACE) cloned dog of cDNA total length BAFF cDNA sequence.With above-mentioned fragment P1 design Auele Specific Primer (the GST2:5'-CGCAAAGCTGGAAGAAGGAG ATGAG-3 ' that has obtained; GST3:5'-GTCCCATGGCAAAGGTGTTATCGGTG-3').The 3'RACE amplification obtains BAFF full-length cDNA 3' terminal fragment P2(and comprises 3'UTR), the 5'RACE amplification obtains BAFF full-length cDNA 5' terminal fragment P3(and comprises 5'UTR).Three sequences of P1, P2, P3 that obtain are spliced with BLAST2 software, obtain the BAFF full-length cDNA.According to this BAFF full length cDNA sequence, design primer dBAFF3(5'-ATGGATGGCTGCACAGAAGGCCAGC-3' respectively at 5' end and 3' end), dBAFF4(5'-TCA CAGA A GC T TCAATGCACCAAAAAACG-3') coding region sequence (SEQ ID NO.2) of the dBAFF that increases, and amino acid sequence corresponding carried out sequence alignment, phylogenetic relationship and molecular structure are carried out bioinformatic analysis.

(5) homology retrieval: submit to cDNA sequence that the clone obtains at GenBank to http://www.ncbi.nlm.nih.gov/BLAST/ institute's calling sequence, EMBL and DDBJ carry out similarity searching and homology analysis in international three big main nucleic acid sequence data storehouses, as shown in Figure 2, find and known people (GenBank NP-006564), mouse (GenBank NP-296371), ox (GenBank NP-001107978), pig (GenBank NP-001090967), rabbit (GenBank NP-001093434) amino acid sequence similarity is very high, be respectively 80,58,78,82 and 76%, its soluble part amino acid sequence similarity is respectively 91,82,96,95 and 91%, can determine that this sequence is the coding region cDNA SEQ ID NO.2 sequence of dog bone-marrow-derived lymphocyte stimulating factor (dBAFF), the proteic aminoacid sequence of rock sign indicating number is shown in SEQ ID NO.3.

Embodiment 2:

Dog 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 the dog heart (heart), liver (liver), spleen (spleen), lung (lung), intestines (intestine) respectively, and become cDNA with the reversed transcriptive enzyme reverse transcription, with GAPDH is confidential reference items, and utilization real time-PCR method is studied at the expression level of each tissue dog BAFF gene.

The amplimer of goal gene is dBAFFr1:5'-TCTTTGGGGATGAACTGA GC-3'; DBAFFr2:5'-CAGAAGCTTCAATGCACCAA-3'; The amplimer of GAPDH is dGAPD H1:5'-AGTCATCCATGACCACTTCGGCAT-3'; DGAPDH2:5 '-AAGCAGGG

ATGATGTTCTGGGCAGC-3'。With DEPC water is blank, and every sample is done three multiple holes.Reaction system is: Mix 12.5 μ l, H ₂O 9.5 μ l, Primer1 1 μ l, Primer2 1 μ l, templete 1 μ l.Response procedures is: 95 ℃/3 min, 40 cycles (95 ℃/30 s, 60 ℃/1 min).Setting fluorescence detection limit value in the appropriate location, exponential growth district at amplification curve of each sample is the intersection point Ct value that threshold value (threshold) will obtain threshold value and amplification curve, the Ct value is meant the circulation number of turns (threshold cycle) when reaching threshold value, the logarithmic value of this value and the starting template number linear relationship that is inversely proportional to.Each goal gene CThe t value is with corresponding GAPDH's CThe difference of t value is a Δ CT, the Δ of each sample CThe Δ of the sample that t and expression amount are minimum CThe difference of t is the Δ Δ CT, the dBAFF relative template number in each sample can be expressed as 2 ^{-Δ Δ Ct}

The result as shown in Figure 3, dBAFF mainly is expressed in immune organ and expresses, as spleen, expression amount is minimum in small intestine.

The structure of dog BAFF recombinant expression vector and the abduction delivering in intestinal bacteria thereof:

According to dog BAFF cDNA sequence and pSUMO StuThe I restriction enzyme site designs a pair of specific PCR primer ds-su1(5 '-TGCCATTCAGGGGCCGGAAGAA-3 ') and ds-su2(5 '-TCA T GGATCCTCA CAGAAGCTTCAATGCA-3 '), and at downstream primer insert BamHThe I restriction enzyme site.The amplified fragments subclone is gone into the pSUMO carrier, be built into recombinant vectors pSUMO-dsBAFF.Change this recombinant vectors over to escherichia coli expression bacterial strain BL21(DE3), but high dissolubility gives expression to target protein SUMO-dsBAFF in the endochylema.Detect (Fig. 5) through SDS-PAGE and show, the size of this fusion rotein is 37kDa approximately.With after the label SUMO excision, obtain the dsBAFF that size is about 17kDa with SUMO proteolytic enzyme.

The Ni+ affinity purification of reorganization target protein:

The IPTG low temperature induction contains the escherichia coli expression bacterial strain BL21(DE3 of recombinant vectors pSUMO-dsBAFF) after 24 hours, receive bacterium for 4 ℃, ultrasonication on ice (160W, work 4s, suspend 8s, 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 Binding Buffer balance Ni ⁺Behind-NTA the post, manually go up the supernatant that sample contains the solubility recombinant protein, successively use the Binding buffer of 10 times of volumes and the Washing buffer(20 mmol/L Imidazole of 6 times of volumes, 0.5 mol/L NaCl, 20 mmol/L Tris HCl, pH 7.9) the flush away foreign protein, use Elute buffer (1 mol/L Imidazole, 0.5 mol/L NaCl at last, 20 mmol/L TrisHCl, pH 7.9) the wash-out target protein, be in charge of collection, the PBS desalination.SDS-PAGE detects each collection tube purity of protein and ultraviolet spectrophotometer is measured protein concentration.Shown in Fig. 5 (band 3), the target protein that expression obtains obtains purity through Ni+ post affinity purification and reaches 95% active target protein.

The Western marking is analyzed:

Used one anti-is mouse-anti His among the Western-blot ₆Monoclonal antibody, two anti-be the sheep anti-mouse igg of horseradish peroxidase mark.Its concise and to the point step is: sample is carried out SGS-PAGE earlier, then with the immerseable method with the albumen electrotransfer to nitrocellulose filter (NC film), with each band of marking pen labelled protein marker, yet successively through 5% skim-milk room temperature sealing 1h, with an anti-1h of hatching, hatch 1h with two anti-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 (band 5) target protein position that is shown in.

The prediction of dsBAFF space structure:

As shown in Figure 4, dsBAFF space structure predictive model and hsBAFF space-filling model relatively find out that thus its space structure and people BAFF known structure are closely similar.

Dog BAFF urgees survival effect experiment to the dog lymphocyte:

（1）MTT

Mouse spleen is shredded in the little plate that fills PBS solution, fully grind the back and cross 200 order mesh screens, wash for several times, remove and add lymphocyte separation medium behind the red corpuscle and separate and obtain mouse lymphocyte with PBS.The mouse lymphocyte that separation is obtained contains 10%FCS with RPMI 1640(, 100U/ml penicillin/strep tomycin) nutrient solution adjusts cell concn to about 5 * 10 ⁶Individual/ml, every hole adds 50 μ l cells in 96 orifice plates, 150 μ l RPMI RPMI-1640s.Grouping adds recombinant protein successively again, and the final concentration of recombinant protein is respectively 2,4,6,8,10,12 μ g/ml, and the anti-IgM final concentration is 2 μ g/ml; With PBS is blank, 6 μ g/ml SUMO-dsBAFF, the 2 μ g/ml SUMO+negative contrast of anti-mouse IgM.At 37 ° of C, 5%CO ₂Every hole adds 50 μ l, 2 mg/mL MTT, 37 ° of C, 5%CO after cultivating 48h under the concentration conditions ₂Continue to cultivate 4h, centrifugal, remove supernatant, add the concussion of 150 μ l DMSO microwell plates and go up concussion 10 minutes, make the crystallisate dissolving, measure each hole OD ₄₉₀Value.Three multiple holes are established in experiment, get its mean value, and the base of calculation error.The result as shown in Figure 6, the SUMO-dsBAFF that this experiment clone obtains has significantly short survival effect to mouse lymphocyte, and presents dose-dependent effect.

(2) flow cytometry FCM

Mouse spleen is shredded in the little plate that fills PBS solution, fully grind the back and cross 200 order mesh screens, wash for several times, remove and add lymphocyte separation medium behind the red corpuscle and separate and obtain mouse lymphocyte with PBS.The mouse lymphocyte (5 * 10 that separation is obtained ⁵) with 10 μ g/ml SUMO-dsBAFF+anti-mouse IgM at 37 ° of C, 5% CO ₂Cultivate behind the 48h with PBS and give a baby a bath on the third day after its birth time, give a baby a bath on the third day after its birth time with PBS after 37 ° of C dye 30min with the PI of 10 μ g/ml again.Detect with flow cytometer (BD bioseicnces).To add PBS as blank.The result as shown in Figure 7, compared with the control, under the common hormesis of anti-mouse IgM, fusion rotein SUMO-dsBAFF can promote the survival of mouse lymphocyte.

Embodiment 3:

The dog bone-marrow-derived lymphocyte irritation factor CDNA that embodiment 1 is obtained passes through existing gene engineering method, produces reorganization dog BAFF, as dog para-immunity toughener.

Embodiment 4:

The dog bone-marrow-derived lymphocyte irritation factor CDNA that embodiment 1 is obtained passes through existing gene engineering method, changes in the animal body, increases its immunizing power, uses in raising.

Can modify dog bone-marrow-derived 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〉dog bone-marrow-derived lymphocyte irritation factor CDNA and cloning process thereof and reorganization are used

 

<130>

 

<160> 7

 

<170> PatentIn?version?3.3

 

<210> 1

<211> 1935

<212> DNA

<213> Canis?familiaris

 

<400> 1

attgggcaat?cgaatagaag?ggccagtgcc?aggaagacct?atcataccgg?gggggccccg 60

 

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ccaggccatg?tagtccgtgt?aggacatgag?caagcacagc?tcacaggaaa?tgatccggtc 180

 

cctgcgctca?cttactcgaa?aggtccaaac?cttcaaagtt?gcgggagtgc?catggatggc 240

 

tgcacagaag?gccagcagtc?acgcctttct?ccttgccttg?agaggggaga?agaaatgaaa 300

 

ctgaaggagt?gtgtctccat?cctccccctg?aaggaaagcc?cctctgtccc?atgctccaaa 360

 

gatggaacgc?tgctggccgt?gaccctgcta?ctggccctga?cggcctgctg?cctctccgtg 420

 

gtgtctttcc?acagggcggc?tgccttgcaa?gcggagctgg?ggagcctccg?ggcggagctg 480

 

cgggaacagc?acgctgagcc?tgagcggctg?ccaggccctc?cgcaggccag?agccagggcc 540

 

cccagggcag?ccatgcggga?gaccccagct?gtcacctcag?ctctgaacgg?aatcttggca 600

 

ccatccgctg?caggagaagg?caactccagc?caaagcagca?gaaacaagcg?tgccattcag 660

 

gggccggaag?aaacagtcac?tcaggactgc?ttgcaactga?ttgcagacag?tgacacacct 720

 

actatacgaa?aaggagctta?cacatttgtt?ccatggctgc?tcagctttaa?aaggggaaga 780

 

gctctggagg?agaaagaaaa?taaaatactt?gtaaaagaag?ccggttactt?tttcatatac 840

 

agtcaggttt?tatacaccga?taacaccttt?gccatgggac?acctaataca?gaggaaaaaa 900

 

gtccatgtct?ttggggatga?actgagcctg?gtgactttgt?tccgatgtat?tcaaaatatg 960

 

cctgaaacac?tacccaataa?ttcctgttat?tcagctggca?tcgcaaagct?ggaagaagga 1020

 

gatgagctcc?agcttgcaat?accgcgtgag?gacgctaaaa?tatcacgaga?tggggacggc 1080

 

acgttttttg?gtgcattgaa?gcttctgtga?cctacttaca?cctcgtttgt?ggctgtatcc 1140

 

cttcttttct?ctgtacctct?aaagagaagg?ctcttaactg?gaaacaccaa?aaggaagaaa 1200

 

aacagtagtt?accgtagcct?tctctgtgag?ctctttgttt?tgattcgcta?aaactagacc 1260

 

aaaacaggaa?atttaacaga?caaccacagc?caaagggtgt?catgtgaatt?atgagaaata 1320

 

gggcccattt?aaggaaagat?agaagtagcc?ttctccctat?aatgccatgt?taaacaattt 1380

 

agtcatagtt?tcttgacctg?gaggaagcac?aggctgcaaa?agagcaagaa?tcatttcttc 1440

 

caaaacggca?ttccatcact?gctgcacacc?aactgccggc?gtccacagga?ctgtaggaca 1500

 

tgtgggaact?ctcaggtcca?tcctctctct?tcgtgtgcaa?tctgccagtc?tctttcagtt 1560

 

aatattgtag?taagtaagaa?aaacaaacaa?atcttgcact?taacacatga?aaaaaggaat 1620

 

taaaagctta?catttaaata?tattaagcag?ttggacttca?aattcaggta?ctatttaaat 1680

 

tcgtctggtt?tacttcaagt?gtgcagtttt?tacctgtaat?attttgtatc?acctgatgga 1740

 

gcaccagata?tttttcacag?gagggaggta?attctctcgt?gtatctgcac?ctgatgtatt 1800

 

ttttcgtagc?taatttcatt?cacttcagaa?attttactgt?ggggtaattc?cagttattac 1860

 

gcatacgagc?attaggtgga?aaaaaaaaaa?aaaaaagggg?acggcacgtt?ttttggtgca 1920

 

ttgaagcttc?tgtga 1935

 

 

<210> 2

<211> 879

<212> DNA

<213> Canis?familiaris

 

<400> 2

atggatggct?gcacagaagg?ccagcagtca?cgcctttctc?cttgccttga?gaggggagaa 60

 

gaaatgaaac?tgaaggagtg?tgtctccatc?ctccccctga?aggaaagccc?ctctgtccca 120

 

tgctccaaag?atggaacgct?gctggccgtg?accctgctac?tggccctgac?ggcctgctgc 180

 

ctctccgtgg?tgtctttcca?cagggcggct?gccttgcaag?cggagctggg?gagcctccgg 240

 

gcggagctgc?gggaacagca?cgctgagcct?gagcggctgc?caggccctcc?gcaggccaga 300

 

gccagggccc?ccagggcagc?catgcgggag?accccagctg?tcacctcagc?tctgaacgga 360

 

atcttggcac?catccgctgc?aggagaaggc?aactccagcc?aaagcagcag?aaacaagcgt 420

 

gccattcagg?ggccggaaga?aacagtcact?caggactgct?tgcaactgat?tgcagacagt 480

 

gacacaccta?ctatacgaaa?aggagcttac?acatttgttc?catggctgct?cagctttaaa 540

 

aggggaagag?ctctggagga?gaaagaaaat?aaaatacttg?taaaagaagc?cggttacttt 600

 

ttcatataca?gtcaggtttt?atacaccgat?aacacctttg?ccatgggaca?cctaatacag 660

 

aggaaaaaag?tccatgtctt?tggggatgaa?ctgagcctgg?tgactttgtt?ccgatgtatt 720

 

caaaatatgc?ctgaaacact?acccaataat?tcctgttatt?cagctggcat?cgcaaagctg 780

 

gaagaaggag?atgagctcca?gcttgcaata?ccgcgtgagg?acgctaaaat?atcacgagat 840

 

ggggacggca?cgttttttgg?tgcattgaag?cttctgtga 879

 

 

<210> 3

<211> 292

<212> PRT

<213> Canis?familiaris

 

<400> 3

 

Met?Asp?Gly?Cys?Thr?Glu?Gly?Gln?Gln?Ser?Arg?Leu?Ser?Pro?Cys?Leu

1 5 10 15

 

 

Glu?Arg?Gly?Glu?Glu?Met?Lys?Leu?Lys?Glu?Cys?Val?Ser?Ile?Leu?Pro

20 25 30

 

 

Leu?Lys?Glu?Ser?Pro?Ser?Val?Pro?Cys?Ser?Lys?Asp?Gly?Thr?Leu?Leu

35 40 45

 

 

Ala?Val?Thr?Leu?Leu?Leu?Ala?Leu?Thr?Ala?Cys?Cys?Leu?Ser?Val?Val

50 55 60

 

 

Ser?Phe?His?Arg?Ala?Ala?Ala?Leu?Gln?Ala?Glu?Leu?Gly?Ser?Leu?Arg

65 70 75 80

 

 

Ala?Glu?Leu?Arg?Glu?Gln?His?Ala?Glu?Pro?Glu?Arg?Leu?Pro?Gly?Pro

85 90 95

 

 

Pro?Gln?Ala?Arg?Ala?Arg?Ala?Pro?Arg?Ala?Ala?Met?Arg?Glu?Thr?Pro

100 105 110

 

 

Ala?Val?Thr?Ser?Ala?Leu?Asn?Gly?Ile?Leu?Ala?Pro?Ser?Ala?Ala?Gly

115 120 125

 

 

Glu?Gly?Asn?Ser?Ser?Gln?Ser?Ser?Arg?Asn?Lys?Arg?Ala?Ile?Gln?Gly

130 135 140

 

 

Pro?Glu?Glu?Thr?Val?Thr?Gln?Asp?Cys?Leu?Gln?Leu?Ile?Ala?Asp?Ser

145 150 155 160

 

 

Asp?Thr?Pro?Thr?Ile?Arg?Lys?Gly?Ala?Tyr?Thr?Phe?Val?Pro?Trp?Leu

165 170 175

 

 

Leu?Ser?Phe?Lys?Arg?Gly?Arg?Ala?Leu?Glu?Glu?Lys?Glu?Asn?Lys?Ile

180 185 190

 

 

Leu?Val?Lys?Glu?Ala?Gly?Tyr?Phe?Phe?Ile?Tyr?Ser?Gln?Val?Leu?Tyr

195 200 205

 

 

Thr?Asp?Asn?Thr?Phe?Ala?Met?Gly?His?Leu?Ile?Gln?Arg?Lys?Lys?Val

210 215 220

 

 

His?Val?Phe?Gly?Asp?Glu?Leu?Ser?Leu?Val?Thr?Leu?Phe?Arg?Cys?Ile

225 230 235 240

 

 

Gln?Asn?Met?Pro?Glu?Thr?Leu?Pro?Asn?Asn?Ser?Cys?Tyr?Ser?Ala?Gly

245 250 255

 

 

Ile?Ala?Lys?Leu?Glu?Glu?Gly?Asp?Glu?Leu?Gln?Leu?Ala?Ile?Pro?Arg

260 265 270

 

 

Glu?Asp?Ala?Lys?Ile?Ser?Arg?Asp?Gly?Asp?Gly?Thr?Phe?Phe?Gly?Ala

275 280 285

 

 

Leu?Lys?Leu?Leu

290

 

 

<210> 4

<211> 25

<212> DNA

＜213〉artificial sequence

 

 

<220>

<221> misc_feature

<222> (5)..(5)

<223> n?is?a,?c,?g,?or?t

 

<220>

<221> misc_feature

<222> (8)..(8)

<223> n?is?a,?c,?g,?or?t

 

<220>

<221> misc_feature

<222> (17)..(17)

<223> n?is?a,?c,?g,?or?t

 

<400> 4

ttgtnccntg?gcttctnagc?tttaa 25

 

 

<210> 5

<211> 23

<212> DNA

＜213〉artificial sequence

 

 

<220>

<221> misc_feature

<222> (9)..(9)

<223> n?is?a,?c,?g,?or?t

 

<220>

<221> misc_feature

<222> (12)..(12)

<223> n?is?a,?c,?g,?or?t

 

<220>

<221> misc_feature

<222> (15)..(16)

<223> n?is?a,?c,?g,?or?t

 

<220>

<221> misc_feature

<222> (18)..(18)

<223> n?is?a,?c,?g,?or?t

 

<400> 5

gcaccaaana?angtnncntc?tcc 23

 

 

<210> 6

<211> 25

<212> DNA

＜213〉artificial sequence

 

<400> 6

cgcaaagctg?gaagaaggag?atgag 25

 

 

<210> 7

<211> 26

<212> DNA

＜213〉artificial sequence

 

<400> 7

gtcccatggc?aaaggtgtta?tcggtg 26

 

 

Claims (4)

1. dog bone-marrow-derived lymphocyte irritation factor CDNA is characterized in that, has the sequence shown in the SEQ ID NO.1.

2. the cloning process of the described dog bone-marrow-derived lymphocyte of claim 1 irritation factor CDNA is as follows:

(1) the TRIzol method is extracted the total RNA of dog spleen;

(2) design degenerate primer dBAFF1:5'-TTGTNCCNTGGCTTCTNAGCTTTAA-3';

DBAFF2:5'-GCACCAAANAANGTNNCNTCTCC-3'; Carry out pcr amplification, obtain the fragment P1 of 340bp;

(3) with the above-mentioned homologous clone fragment P1 design Auele Specific Primer GST2:5'-CGCAAAGCTGGAAGAAGGAGATGAG-3' that obtains; GST3:5'-GTCCCATGGCAAAG GTGTTATCGGTG-3'; The 3'RACE amplification obtains BAFF full-length cDNA 3' terminal fragment P2; The 5'RACE amplification obtains BAFF full-length cDNA 5' terminal fragment P3; Three sequences of P1, P2, P3 that obtain are spliced with BLAST2 software, obtain the BAFF full-length cDNA.

3. the reorganization of the described dog bone-marrow-derived lymphocyte of claim 1 irritation factor CDNA is used, and is by existing gene engineering method, produces reorganization dog bone-marrow-derived lymphocyte stimulating factor, as dog para-immunity toughener.

4. dog bone-marrow-derived lymphocyte stimulating factor is characterized in that, has the sequence shown in the SEQ ID NO.2.

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