CN103060284A - Streptococcus agalactiae PGK (Phosphoglycerate Kinase) sub-unit recombinant protein and preparation method thereof - Google Patents
Streptococcus agalactiae PGK (Phosphoglycerate Kinase) sub-unit recombinant protein and preparation method thereof Download PDFInfo
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- CN103060284A CN103060284A CN2013100074689A CN201310007468A CN103060284A CN 103060284 A CN103060284 A CN 103060284A CN 2013100074689 A CN2013100074689 A CN 2013100074689A CN 201310007468 A CN201310007468 A CN 201310007468A CN 103060284 A CN103060284 A CN 103060284A
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Abstract
The invention provides a streptococcus agalactiae PGK (Phosphoglycerate Kinase) sub-unit recombinant protein and a preparation method thereof. The recombinant protein provided by the invention is the protein described by a formula 1 or 2 shown in the specification: (1), a protein composed of amino acid sequence shown in sequence 2 in a sequence table; and (2), a protein which is derived from the sequence 2 by substituting, deleting and/or adding one or more amino acids in/to the amino acid sequence limited in the formula (1) and has the same activity as the protein defined in sequence 2 in the sequence table. The detection by the recombinant protein prepared by the invention is free from dangers of infection and toxicity spreading; and in addition, the preparation method disclosed by the invention is advanced in production technology for preparing an immunomodulator, low in production cost, stable in performance of the prepared immunomodulator, high in product attached value, suitable for factory production and wide in market application prospect.
Description
Technical field
The present invention relates to a kind of recombinant protein, especially a kind of streptococcus agalactiae pgk subunit recombinant protein.
Background technology
Mastadenitis of cow (Dairy cow mastitis) is one of main epidemic disease that affects world's dairy health, steady progression.Its cause of disease has multiple, but streptococcus agalactiae (S.agalactiae) is one of topmost pathogenic bacterium, and its recall rate reaches about 70% in suffering from mazoitis cow's milk, mainly causes milk cow clinical type and subclinical mastitis.The countries in the world scientist has made unremitting effort around the vaccine of anti-mastadenitis of cow processed, the conclusion that finally draws is traditional whole cell split vaccine, inactivated vaccine, capsular polysaccharide mixed vaccine for streptococcus agalactiae property mazoitis all without obvious preventive effect, the research and development of subunit's (comprising single subunit and many subunits) immunological reagent are the preferred approach of seeking effective this disease of prevention and control, and this is the conclusion of whole world association area approval.Streptococcus agalactiae is important people and animals' common sense metachromia pathogenic bacterium, mainly resides in the reproductive tract the women the mankind.Abroad medical field for prevent fetus the Gestation period or in birth process baby's streptococcus agalactiae infect and cause septicemia or pneumonia; the traditional vaccine of development is proven all without positive effect, and the good immanoprotection action of the subunit vaccine of development performance.And milk cow streptococcus agalactiae surface protein phosphoglyceric kinase (Phosphoglycerate kinase is called for short pgk) is the important surface antigen albumen of bovine mastitis streptococcus agalactiae.Therefore research and application ox source streptococcus agalactiae surface protein prokaryotic expression product have important using value.
Summary of the invention
The invention provides a kind of recombinant protein, it is milk cow streptococcus agalactiae pgk subunit recombinant protein, and corresponding, the present invention also provides encoding gene and the application of this recombinant protein.
A kind of recombinant protein provided by the invention is following 1) or 2) described albumen:
1) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
2) with 1) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and have with sequence table in sequence 2 identical activity by the derivative protein of sequence in the sequence table 2.
Second purpose of the present invention provides the encoding gene of above-mentioned recombinant protein.
Preferably, described encoding gene is the nucleotide sequence shown in the sequence 1 in the sequence table.
The 3rd purpose of the present invention provides the method for a kind of streptococcus agalactiae subunit recombinant protein, is that the gene with the coding recombinant protein imports in the intestinal bacteria, expresses obtaining recombinant protein.
Preferably, described intestinal bacteria are the BL21-DE3 intestinal bacteria.
The 4th purpose of the present invention provides the application of described streptococcus agalactiae pgk subunit's recombinant protein in detecting milk cow streptococcus agalactiae property mazoitis infection antibody and immune antibody.
Streptococcus agalactiae of the present invention subunit recombinant protein has following effect:
Description of drawings
Fig. 1 is the streptococcus agalactiae subunit recombinant protein electrophorogram behind the purifying of the present invention;
Fig. 2 is BALB/c mouse immune antibody detection level result.
Embodiment
One, the intestinal bacteria vivoexpression of recombinant protein
(1), the Auele Specific Primer of design amplification ox source streptococcus agalactiae surface protein subunit gene pgk gene;
Utilize primer-design software to design the primer of 1 pair of amplification pgk gene major antigen Predominance Area sequence.The upstream primer sequence is 5'-GGA
GGATCCCTAATGGCTAAATTG-3', the downstream primer sequence is: 5'-GACT
AAGCTTTTTCAGTCAATGCTG-3', the line part is respectively the restriction enzyme of introducing
BamH 
With
Hind The restriction enzyme site sequence.Expanding fragment length 984bp.See sequence table 1.
(2), obtain the pgk gene fragment by pcr amplification;
The PCR reaction totally is 50 μ L, wherein the super purified water (ddH of sterilization
2O) 14 μ L, 10 * PCR buffer(contain Mg
2+) 5 μ L, dNTP mixture 4 μ L, upstream primer (10pmol/ μ L) 4 μ L, downstream primer (10pmol/ μ L) 4 μ L, template DNA 4 μ L, Taq DNA enzyme (5U/ μ l) 1 μ L, the super purified water (ddH of sterilization
2O) 14 μ L.Reaction conditions is 95 ℃ of 5min of denaturation; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations; Last 72 ℃ are extended 10min.The PCR product after 1% agarose gel electrophoresis is identified, is indicated according to relative molecular mass Marker, downcut the gel that contains the purpose fragment from sepharose, press DNA fast purifying glue and reclaim the test kit operation steps, reclaim amplified production.It is the TAKARA product that glue reclaims test kit.
(3), to the evaluation of cloning, check order of pgk gene;
The target DNA fragment that reclaims is connected in 16 ℃ with the pMD18-T cloning vector spends the night, will connect product and be converted into the JM109 competent cell, screen extraction recombinant plasmid pgk-pMD18-T, usefulness with the LB agar plate that contains Amp
BamH 
With
Hind 
Carry out that single, double enzyme is cut and PCR identifies.The JM109 competent cell that changes recombinant plasmid pgk-pMD18-T over to is delivered to the Huada Gene Research Center, Beijing check order, sequencing result is analyzed with the DNAStar biosoftware.
(4), with the directed subclone of pgk gene to pET-30a(+) prokaryotic expression carrier;
Recombinant cloning vector pgk-pMD-18 and expression vector pET30a (+) that order-checking is correct use
BamH
,
Hind 
Glue reclaims after carrying out double digestion, and structure recombinant expression vector pgk-pET30a (+) cuts enzyme again and PCR identifies that correct recombinant plasmid pgk-pET30a (+) is converted into Host Strains e. coli bl21 (DE3) according to a conventional method, obtains recombinant bacterium.
(5), the pgk gene is at colibacillary abduction delivering:
Recombinant bacterium in the LB liquid nutrient medium, is cultivated 3h, to OD under 37 ℃
600nmWhen reaching 0.65 left and right sides, add the IPTG of concentration 1.00 mM/L, induce 5h under 37 ℃, its expression amount is maximum.Its expressing protein accounts for 43.2% of bacterial protein, and the solubility expression amount accounts for 46.5% of thalline supernatant total protein.
Two, the affinitive layer purification of recombinant protein
(1) preparation of sample to be purified
Bacterium is expressed in centrifugal collection: the induction expression protein centrifugal 10min under 10000 rpm conditions with obtaining in the above-mentioned steps one, get precipitation, use PBS(1mol/L, pH7.4) wash 3 times, bacterial precipitation is dissolved by PBS solution.
The cracking thalline obtains the supernatant soluble proteins: adding N,O-Diacetylmuramidase to final concentration is 1% (1mg/ml), and then ice bath 30min carries out supersound process, is tending towards transparent, inhales when blowing without obvious grumeleuse as degree with liquid-transfering gun take suspension.Centrifuging and taking supernatant (being soluble proteins) also adds 1% TritionX-100(Triton X-100) and the DTT(dithiothreitol (DTT) of 1mmol/L) to final concentration be in-20 ℃ of preservations, in order to purifying behind the 1mmol/L.
(2) the affinity chromatography purification flow process of expressing protein
With reference to Ni-NTA His band Resin(His Trap
TMFF crude, GE Heathcare product) schedule of operation is carried out, and then carries out SDS-PAGE and Western Blot, detects purity of protein, and as shown in Figure 1, after testing, purity is more than 95%.
Western Blot identifies that the primary antibodie of using is the anti-bovine mastitis streptococcus agalactiae of rabbit antibody, and two anti-are goat anti-rabbit igg HRP traget antibody.
(3) mensuration of expressing protein content
Adopt the uItraviolet absorption methods quantitative protein.After the suitable dilution of sample to be checked, measure simultaneously this protein soln in the OD value at λ 260nm and λ 280nm place, use simultaneously diluent as blank, calculate protein content by following formula:
(1.46 * A280-0.74 * A260) * extension rate=mg/ml protein content
The mass concentration of the pgk albumen of purifying is 〉=5.03mg/mL.
Use recombinant protein of the present invention as follows as the experiment effect that detectable antigens detects the immune mouse immune antibody:
Recombinant protein and Freund's complete adjuvant (Freund complete adjuvant with preparation in the embodiment of the invention 1, FCA) with Freund's incomplete adjuvant (Freund incomplete adjuvant, FIA) carry out emulsification by the 1:1 equal-volume, emulsification is got antigen after the dilution with disposable sterilized injector, another syringe is got Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant, connect (emulsion tube and syringe slippage when preventing that pressure is excessive with sterile milk sebific duct jointing behind the air in the Inside Syringe, tighten joint with thin wire before the emulsification), at first will dilute antigen and push fast adjuvant, form water-in-oil, then Quick push/pull mixed approximately 10～30 minutes repeatedly.Water and milk does not separate mutually in the distilled water, finish emulsification when the centrifugal 5min of 5000rpm/min is not stratified when emulsifying agent is creamy white, puts, and prepares the immune vaccine of bovine mastitis streptococcus agalactiae.Utilize the streptococcus agalactiae pgk recombinant protein antigen of preparation to detect the antibody situation that this immune vaccine immunity produces.
It is as follows that pgk recombinant protein antigen of the present invention detects immune vaccine antibody effect:
With this immune vaccine to BALB/c mouse immunity three times (interval 7d) after, for the first time immunity is with Fu Shi Freund's complete adjuvant emulsification recombinant antigen, dosage be 1mL/ only, back 3 inoculations in subcutaneous minute; With freund 's incomplete adjuvant emulsification recombinant antigen, dosage is 1mL/, back 3 inoculations in subcutaneous minute for the second time; For the third time immunity is carried out immunity with non-emulsification recombinant protein antigen, and dosage is 1mL/, back 3 inoculations in subcutaneous minute.Its antibody titers reached 1:4000 in immune rear 35 days for the third time.See Fig. 2 (the horizontal effect of antibody test).
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment the present invention is had been described in detail, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Sequence table
＜110〉National University of the Inner Mongol
＜120〉a kind of streptococcus agalactiae pgk subunit recombinant protein and preparation method thereof
<160> 2
<210> 1
<211> 984
<212> DNA
＜213〉artificial recombination gene
<400> 1
atg gct aaa ttg act gtt aaa gac gtt gat ttg aaa ggt aaa aaa gtc 48
Met Ala Lys Leu Thr Val Lys Asp Val Asp Leu Lys Gly Lys Lys Val
1 5 10 15
ctc gtt cgt gtt gac ttt aat gtg cct ttg aaa gac ggc gtt atc act 96
Leu Val Arg Val Asp Phe Asn Val Pro Leu Lys Asp Gly Val Ile Thr
20 25 30
aac gac aac cgt atc act gcg gct ctt cca aca atc aag tat atc atc 144
Asn Asp Asn Arg Ile Thr Ala Ala Leu Pro Thr Ile Lys Tyr Ile Ile
35 40 45
gaa caa ggt ggt cgt gct atc ctc ttc tct cac ctt gga cgt gtt aaa 192
Glu Gln Gly Gly Arg Ala Ile Leu Phe Ser His Leu Gly Arg Val Lys
50 55 60
gaa gaa gct gac aaa gaa gga aaa tca ctt gca ccg gta gct gct gat 240
Glu Glu Ala Asp Lys Glu Gly Lys Ser Leu Ala Pro Val Ala Ala Asp
65 70 75 80
tta gct gct aaa ctt ggt caa gat gtt gta ttc cca ggt gtt act cgt 288
Leu Ala Ala Lys Leu Gly Gln Asp Val Val Phe Pro Gly Val Thr Arg
85 90 95
ggt gca aaa tta gaa gaa gta atc aat gct ttg gaa gat gga caa gtt 336
Gly Ala Lys Leu Glu Glu Val Ile Asn Ala Leu Glu Asp Gly Gln Val
100 105 110
ctt ttg gtt gaa aac act cgt ttt gaa gat gtt gac ggt aag aaa gaa 384
Leu Leu Val Glu Asn Thr Arg Phe Glu Asp Val Asp Gly Lys Lys Glu
115 120 125
tct aag aat gac gaa gaa ctt ggt aaa tac tgg gct tca ctt gga gat 432
Ser Lys Asn Asp Glu Glu Leu Gly Lys Tyr Trp Ala Ser Leu Gly Asp
130 135 140
gga atc ttc gtt aac gat gca ttt ggt aca gca cac cgt gct cat gca 480
Gly Ile Phe Val Asn Asp Ala Phe Gly Thr Ala His Arg Ala His Ala
145 150 155 160
tca aac gta ggt att tca gca aac gtt gaa aaa gct gta gct ggt ttc 528
Ser Asn Val Gly Ile Ser Ala Asn Val Glu Lys Ala Val Ala Gly Phe
165 170 175
ctt ctt gaa aac gaa att gct tac atc caa gaa gca gtt gaa act cca 576
Leu Leu Glu Asn Glu Ile Ala Tyr Ile Gln Glu Ala Val Glu Thr Pro
180 185 190
gaa cgc cca ttc gta gct att ctt ggt ggc tca aaa gtt tct gat aag 624
Glu Arg Pro Phe Val Ala Ile Leu Gly Gly Ser Lys Val Ser Asp Lys
195 200 205
att ggt gtt atc gaa aac ctt ctt gaa aaa gct gat aaa gtt ctt atc 672
Ile Gly Val Ile Glu Asn Leu Leu Glu Lys Ala Asp Lys Val Leu Ile
210 215 220
ggt ggt ggt atg act tac aca ttc tac aaa gct caa ggt atc gag atc 720
Gly Gly Gly Met Thr Tyr Thr Phe Tyr Lys Ala Gln Gly Ile Glu Ile
225 230 235 240
ggt aac tca ctt gta gaa gaa gac aaa ttg gat gtt gct aaa gac ctc 768
Gly Asn Ser Leu Val Glu Glu Asp Lys Leu Asp Val Ala Lys Asp Leu
245 250 255
ctt gaa aaa tca aac ggt aaa ttg atc ttg cca gtt gac tca aaa gaa 816
Leu Glu Lys Ser Asn Gly Lys Leu Ile Leu Pro Val Asp Ser Lys Glu
260 265 270
gca aac gca ttt gct ggt tat act gaa gtt cgc gac act gaa ggt gaa 864
Ala Asn Ala Phe Ala Gly Tyr Thr Glu Val Arg Asp Thr Glu Gly Glu
275 280 285
gca gtt tca gaa ggg ttc ctt ggt ctt gac atc ggt cct aaa tca atc 912
Ala Val Ser Glu Gly Phe Leu Gly Leu Asp Ile Gly Pro Lys Ser Ile
290 295 300
gct aaa ttt gat gaa gca ctt act ggt gct aaa aca gtt gta tgg aac 960
Ala Lys Phe Asp Glu Ala Leu Thr Gly Ala Lys Thr Val Val Trp Asn
305 310 315 320
gga cct atg ggt gtc ttt gaa aac 984
Gly Pro Met Gly Val Phe Glu Asn
325
<210> 2
<211> 328
<212> PRT
＜213〉artificial recombination albumen
<400> 2
Met Ala Lys Leu Thr Val Lys Asp Val Asp Leu Lys Gly Lys Lys Val
1 5 10 15
Leu Val Arg Val Asp Phe Asn Val Pro Leu Lys Asp Gly Val Ile Thr
20 25 30
Asn Asp Asn Arg Ile Thr Ala Ala Leu Pro Thr Ile Lys Tyr Ile Ile
35 40 45
Glu Gln Gly Gly Arg Ala Ile Leu Phe Ser His Leu Gly Arg Val Lys
50 55 60
Glu Glu Ala Asp Lys Glu Gly Lys Ser Leu Ala Pro Val Ala Ala Asp
65 70 75 80
Leu Ala Ala Lys Leu Gly Gln Asp Val Val Phe Pro Gly Val Thr Arg
85 90 95
Gly Ala Lys Leu Glu Glu Val Ile Asn Ala Leu Glu Asp Gly Gln Val
100 105 110
Leu Leu Val Glu Asn Thr Arg Phe Glu Asp Val Asp Gly Lys Lys Glu
115 120 125
Ser Lys Asn Asp Glu Glu Leu Gly Lys Tyr Trp Ala Ser Leu Gly Asp
130 135 140
Gly Ile Phe Val Asn Asp Ala Phe Gly Thr Ala His Arg Ala His Ala
145 150 155 160
Ser Asn Val Gly Ile Ser Ala Asn Val Glu Lys Ala Val Ala Gly Phe
165 170 175
Leu Leu Glu Asn Glu Ile Ala Tyr Ile Gln Glu Ala Val Glu Thr Pro
180 185 190
Glu Arg Pro Phe Val Ala Ile Leu Gly Gly Ser Lys Val Ser Asp Lys
195 200 205
Ile Gly Val Ile Glu Asn Leu Leu Glu Lys Ala Asp Lys Val Leu Ile
210 215 220
Gly Gly Gly Met Thr Tyr Thr Phe Tyr Lys Ala Gln Gly Ile Glu Ile
225 230 235 240
Gly Asn Ser Leu Val Glu Glu Asp Lys Leu Asp Val Ala Lys Asp Leu
245 250 255
Leu Glu Lys Ser Asn Gly Lys Leu Ile Leu Pro Val Asp Ser Lys Glu
260 265 270
Ala Asn Ala Phe Ala Gly Tyr Thr Glu Val Arg Asp Thr Glu Gly Glu
275 280 285
Ala Val Ser Glu Gly Phe Leu Gly Leu Asp Ile Gly Pro Lys Ser Ile
290 295 300
Ala Lys Phe Asp Glu Ala Leu Thr Gly Ala Lys Thr Val Val Trp Asn
305 310 315 320
Gly Pro Met Gly Val Phe Glu Asn
325
Claims (6)
1. streptococcus agalactiae pgk subunit recombinant protein, it is characterized in that: described recombinant protein is following 1) or 2) described albumen:
1) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
2) with 1) aminoacid sequence that limits through replacement and/or disappearance and/or add one or several amino acid and have with sequence table in sequence 2 identical activity by the derivative protein of sequence in the sequence table 2.
2. the encoding gene of streptococcus agalactiae pgk claimed in claim 1 subunit recombinant protein.
3. the encoding gene of described streptococcus agalactiae pgk subunit recombinant protein according to claim 2, it is characterized in that: described encoding gene is the nucleotide sequence shown in the sequence 1 in the sequence table.
4. the recombinant bacterium that contains claim 2 or 3 described encoding genes.
5. the method for the one kind high described streptococcus agalactiae pgk of efficient expression claim 1 subunit recombinant protein is that the gene with the coding recombinant protein imports in the intestinal bacteria, expresses obtaining recombinant protein.
6. the application of streptococcus agalactiae pgk subunit's recombinant protein claimed in claim 1 in detecting mastadenitis of cow streptococcus agalactiae infection antibody and vaccine immunity antibody.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2013100074689A CN103060284A (en) | 2012-10-31 | 2013-01-09 | Streptococcus agalactiae PGK (Phosphoglycerate Kinase) sub-unit recombinant protein and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210427252.3 | 2012-10-31 | ||
| CN201210427252 | 2012-10-31 | ||
| CN2013100074689A CN103060284A (en) | 2012-10-31 | 2013-01-09 | Streptococcus agalactiae PGK (Phosphoglycerate Kinase) sub-unit recombinant protein and preparation method thereof |
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| CN103060284A true CN103060284A (en) | 2013-04-24 |
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|---|---|---|---|
| CN2013100074689A Pending CN103060284A (en) | 2012-10-31 | 2013-01-09 | Streptococcus agalactiae PGK (Phosphoglycerate Kinase) sub-unit recombinant protein and preparation method thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357045A (en) * | 1998-12-22 | 2002-07-03 | 微科学有限公司 | Outer surface proteins, their genes, and their use |
-
2013
- 2013-01-09 CN CN2013100074689A patent/CN103060284A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357045A (en) * | 1998-12-22 | 2002-07-03 | 微科学有限公司 | Outer surface proteins, their genes, and their use |
Non-Patent Citations (3)
| Title |
|---|
| 《GENBANK》 20081024 Tettelin H "登录号：AAN00629.1" , * |
| TETTELIN H: ""登录号：AAN00629.1"", 《GENBANK》 * |
| 张海宝 等: "《牛乳腺炎无乳链球菌Pgk基因抗原优势区原核表达产物的抗原性分析》", 《中国动物传染病学报》 * |
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Application publication date: 20130424 |