CN101195656B - Recombined Japanese lamprey glandulae oris excreted L-250 protein anticoagulant efficacy - Google Patents
Recombined Japanese lamprey glandulae oris excreted L-250 protein anticoagulant efficacy Download PDFInfo
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- CN101195656B CN101195656B CN2006101346835A CN200610134683A CN101195656B CN 101195656 B CN101195656 B CN 101195656B CN 2006101346835 A CN2006101346835 A CN 2006101346835A CN 200610134683 A CN200610134683 A CN 200610134683A CN 101195656 B CN101195656 B CN 101195656B
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Abstract
The invention relates to a gene colony and expression technique of TCTP motif protein-L-250, with histamine release factor activity, while the TCTP motif protein-L-250 is secreted from oral gland of Lampetra Japonica, belonging to biological technical field. The invention relates to the cDNA sequence and colony of L-250 protein secreted from oral gland of Lampetra Japonica, relative protein amino acid sequence and expression in bacillus coli or pichia, and the application for using L-250 protein as histamine release factor to excite basophil to release histamine, wherein histamine can improve vascular endothelial surface expression thrombin regulator TM which can be combined with thrombin to reduce thrombin activity and can activate protein C to trigger anti-coagulation cascade reaction. The histamine release factor activity of L-250 protein has potential value for resisting coagulation and treating cardio-cerebrovascular diseases.
Description
Technical field
The gene cloning and expression of the TCTP die body albumen-L-250 of Japanese lamprey oral gland secretion tool histamine release factor active belongs to biological technical field.Relate to Japanese lamprey oral gland secretion proteic cDNA sequence of L-250 and clone; Protein amino acid sequence corresponding and the expression in intestinal bacteria or pichia spp thereof with it; And L-250 albumen stimulates basophilic granulocyte to discharge histamine as the histamine release factor.Histaminergic strengthens vascular endothelial cell surface expression zymoplasm regulin TM, and it can combine with zymoplasm on the one hand, reduces thrombin activity; On the other hand can activated protein C, thus the anticoagulation cascade reaction triggered.This prompting, the proteic histamine release factor active of L-250 has the potential using value aspect anti-freezing and the cardiovascular and cerebrovascular diseases.
Background technology
Jamprey-ell (Lampetra japonica) is Cyclostomata (Cycpostomata), Peteromyzoni-form (Petromyzoniformes), Petromyzonidae (Petromyzontidae), Lampetra (Lethenteron).On evolving, has high researching value because of connecting vertebra and invertebrates.The life of battalion's semiparasitism is adsorbed on other fish bodies with its distinctive oral sucker in seawater, survives by sucking its flesh and blood, and the host is bled incessantly, and there is certain anti-freezing mechanism in prompting in its oral gland.
Mainly there are two kinds of anti-freezing mechanism in blood vessel, is respectively the protease blood coagulation and suppresses mechanism and proteinase inhibitor class inhibition mechanism.In protease blood coagulation inhibition mechanism, PROTEIN C plays a major role.PROTEIN C (PC) is a Tryase, is that it is present in the blood plasma with zymogen forms by a kind of physiological antithrombotics of hepatic secretion, and PC is a non-activity under state of nature, through the activation of zymoplasm, can become activatory PROTEIN C (APC).APC is through making thrombin deactivation such as the activatory VIII factor, the V factor, thus the anticoagulant process.Yet, zymoplasm to the activation of PC be quite slow, zymoplasm has only and the thrombomodulin that is present in the endothelial cell membrane surface, after being called zymoplasm regulin (TM) again and forming mixture, could activate PC effectively.TM is one of thrombin receptor on the endothelial cell membrane, combines with zymoplasm, can reduce thrombin activity, and strengthens the effect of thrombin activation PROTEIN C.Therefore, TM makes zymoplasm suppress composition by the short important vessel intravascular coagulation of anti-freezing that turns to fixed attention.
(Translationally controlled tumor protein TCTP) is called the histamine release factor again to translation control oncoprotein, is considered in the chronic anaphylaxis disease, play an important role.This family protein has 2 feature structure territories; Be TCTP-1 ([IFAE]-[GA]-[GAS]-N-[PAK]-S-[GTA]-E-[GDEV]-[PAGEQV]-[DEQGAV] and TCTP-2 ([FLIV]-x (4)-[FLVH]-[FY]-[MIVCT]-G-E-x (4; 7)-[DENP]-and [GAST]-x-[LIVM]-[GAVI]-x (3)-[FYWQ]), these 2 zones have the sequence homology of height between different plant species.Nineteen ninety-five MacDonald etc. has found the extracellular biological function of this protein family first; Through in the allergic disease people body fluid with hereditary anaphylaxy children's lymphocyte in the Ig-E dependency histamine release factor that exists carry out the amino-terminal end sequential analysis; The translation control oncoprotein of finding the histamine release factor and mouse and people has homology; TCTP can stimulate people's basophilic granulocyte to discharge histamine; Multiple parasites such as people schistosomicide and plasmodium can secrete TCTP to host's blood, and the stimulation of host cell discharges histamine, thereby influences host immune response.In addition, histamine can also cause hemangiectasis effect, strengthens regional flow; The expression of vascular endothelial cell surface zymoplasm regulin TM is increased, and it can combine with zymoplasm on the one hand, reduces its activity; On the other hand can activated protein C; The activatory PROTEIN C can hinder factor Xa and combine with hematoblastic; And stimulate the release of plasminogen activator and promote fibrinolysis; Can also be under the effect of its cofactor Protein S deactivation prothrombinase and VIIIa, these three paths finally can both cause the generation of anticoagulation reaction.The present invention has found to be present in the new gene of TCTP die body albumen in the Japanese lamprey oral gland first; And with its called after L-250; And TCTP die body albumen-L250 has the histamine release factor active in the Japanese lamprey oral gland of this project; Can stimulate basophilic granulocyte to discharge histamine, thereby cause the generation of a series of anticoagulation cascade reactions, prompting L250 albumen will have using value aspect the clinical anticoagulant therapy.
Summary of the invention
This invention has been found the proteic ORFs of L-250 in Japanese lamprey oral gland cDNA library, through angling of its mRNA total length being got, check order, reach the cDNA clone, its recombinant protein is carried out efficient induction express in intestinal bacteria.L-250 recombinant protein BA relates to as the histamine release factor, stimulates basophilic granulocyte to discharge histamine.
The present invention relates to Japanese lamprey oral gland secretion proteic cDNA sequence of L-250 and clone, the protein sequence and the expression in intestinal bacteria or pichia spp thereof of correspondence with it; And the BA of stimulation basophilic granulocyte release histamine, and the application of anticoagulation aspect.
The present invention is separation and Extraction mRNA from Japanese lamprey oral gland; Biological information that obtains in the cDNA library of utilization structure in early stage, the EST storehouse and mRNA total length angle the sequencing result of getting to choose ORFs design primer; Adopt the RT-PCR method to obtain proteic cDNA of L-250 and sequence thereof; The present invention also provides the albumen and the sequence thereof of L-250 albumen successful expression in e. coli bl21; L-250 albumen has the BA of the histamine release factor, and there is huge application potential the anticoagulation aspect clinically.
L-250 albumen cDNA sequence is following with the protein amino acid sequence of its derivation:
L-250 albumen cDNA sequence 519bp, its protein is made up of 172 amino acid, contains a TCTP die body, and molecular weight of albumen is 22,408kDa.
atg?atc?atc?tac?aag?gac?atc?ctc?acc?ggg?gac?gag?atg?ttc?tcg 45
Met?Ile?Ile?Tyr?Lys?Asp?Ile?Leu?Thr?Gly?Asp?Glu?Met?Phe?Ser
gac?atc?tac?aag?atc?aag?gag?acg?gcg?gac?ggc?gcc?tgc?ttc?gag 90
Asp?Ile?Tyr?Lys?Ile?Lys?Glu?Thr?Ala?Asp?Gly?Ala?Cys?Phe?Glu
gtg?gaa?ggc?acg?atg?gtg?tcc?cgg?gtg?gaa?ggc?gac?atc?gac?gac 135
Val?Glu?Gly?Thr?Met?Val?Ser?Arg?Val?Glu?Gly?Asp?Ile?Asp?Asp
gcg?gtc?ttt?ggc?gga?aac?gct?tcg?gca?gag?ggg?gga?gaa?ttt?gat 180
Ala?Val?Phe?Gly?Gly?Asn?Ala?Ser?Ala?Glu?Gly?Gly?Glu?Phe?Asp
tcg?tcg?gag?tcg?tct?aca?gtg?tcc?ggc?gtg?gac?atc?gtg?ctc?aac 225
Ser?Ser?Glu?Ser?Ser?Thr?Val?Ser?Gly?Val?Asp?Ile?Val?Leu?Asn
cac?aaa?ctg?cag?gag?acc?agc?ttc?agc?aag?gag?aag?tac?att?gcc 270
His?Lys?Leu?Gln?Glu?Thr?Ser?Phe?Ser?Lys?Glu?Lys?Tyr?Ile?Ala
tac?atc?aag?gcc?tac?atg?aag?acg?att?aag?gag?aac?ctg?atg?gag 315
Tyr?Ile?Lys?Ala?Tyr?Met?Lys?Thr?Ile?Lys?Glu?Asn?Leu?Met?Glu
aaa?cac?ccg?gag?aag?gtg?gaa?ccc?ttc?atg?act?gct?gct?gcc?aag 360
Lys?His?Pro?Glu?Lys?Val?Glu?Pro?Phe?Met?Thr?Ala?Ala?Ala?Lys
aag?gtt?aaa?gat?gtc?tta?aag?caa?ttc?aag?gat?ttc?cag?ttt?ttc 405
Lys?Val?Lys?Asp?Val?Leu?Lys?Gln?Phe?Lys?Asp?Phe?Gln?Phe?Phe
aca?ggt?gag?agc?atg?aac?ccc?gac?ggc?atg?gtc?gct?ctt?ctt?aac 450
Thr?Gly?Glu?Ser?Met?Gln?Pro?Asp?Gly?Met?Val?Ala?Leu?Leu?Gln
ttc?cgc?gag?gac?gga?atc?acg?ccc?tac?atg?ctt?ttc?ttc?aag?gat 495
Phe?Arg?Glu?Asp?Gly?Ile?Thr?Pro?Tyr?Met?Leu?Phe?Phe?Lys?Asp
ggc?ctg?gaa?att?gag?aaa?tgc?taa 519
Gly?Leu?Glu?Ile?Glu?Lys?Cys?*
It is following that L-250 albumen stimulates basophilic granulocyte to discharge the histamine BA:
It is that RBL-2H3 estimates the ability that L-250 albumen discharges histamine that the present invention adopts rat basophilic leukemia cell, and the enzyme immunoassay test kit of employing r-biopharm company detects the concentration of histamine in the nutrient solution supernatant.With 1 * 10
5The RBL-2H3 cell is hatched in 96 orifice plates, converges until cell, and L-250 is with different concentration gradient (0.3; 4.8; 10 μ g/ml), add in 96 orifice plates and hatch, do not add proteic plate hole as contrast; Collect supernatant, the enzyme immunoassay test kit of employing r-biopharm company detects the concentration of histamine in the nutrient solution supernatant.The basis of measuring is antigen antibody reaction.The result shows, when L-250 concentration was 0.3 μ g/ml, the amount of histamine was 2.2 times of control group in the cell culture fluid; Afterwards along with the enhancing of L-250 concentration; The amount of histamine also increases thereupon, explains that L-250 albumen can stimulate the RLB-2H3 cell to discharge histamine, and under trace, just can work.The proteic histamine release factor active of L250 makes it have potential anti-freezing pharmaceutical use.
Description of drawings
Fig. 1, experiment flow of the present invention
Fig. 2, pET23b plasmid map
Fig. 3, reorganization pET23b-L251 double digestion are identified
Fig. 4, L-250 purifying protein SDS-PAGE
Fig. 5, L-250 albumen stimulate the RBL-2H3 cell to discharge the histamine experimental result
Embodiment
1. the extraction of total RNA: the Trizol reagent that adopts GIBCO BRL.Specific as follows:
(1) getting Japanese lamprey oral gland places liquid nitrogen to grind rapidly;
(2) take by weighing 0.2g gland tissue and secretory product, add the homogenate of 1ml Trizol reagent preparation, hatch 5min for 4 ℃;
(3) add the 0.2ml chloroform, the tight lid of lid back is jolting 15sec firmly, places 5min on ice then;
(4) 4 ℃, 12000g, centrifugal 15min;
(5) upper water is moved into another centrifuge tube mutually, add the 0.5ml Virahol, and hatch 10min on ice;
(6) 4 ℃, 12000g, centrifugal 15min;
(7) abandon supernatant, in deposition (containing RNA), add 1ml 75% washing with alcohol, the vortex mixing;
(8) 4 ℃, 10000g, centrifugal 5min obtains the RNA deposition;
(9) after the dry air, with an amount of TE or not have the RNase water dissolution subsequent use.
2. carry out the RT-PCR amplification with designed primer voluntarily, carry out reverse transcription reaction to obtain the proteic cDNA of Japanese lamprey oral gland L-250 by following condition: 42 ℃ of 20min, 99 ℃ of 5min, 5 ℃ of 5min
Primer sequence is following:
P1：5’-XXcatatgatcatctacaaggacatcctc-3’
P2：5’-XXaagcttgcatttctcaatttccaggcc-3’
3. the purpose cDNA that obtains is connected with the pET23b carrier, is transformed into the Screening and Identification of carrying out positive transformant behind the clone bacterium DH5 α; For generation has histidine-tagged fusion rotein, select pET-23b (+) as gene cloning carrier, clone's concrete operations are following:
(1) recovery of goal gene and order-checking: the recovery of goal gene adopts TaKaRa PCR Fragment Recovery Kit to carry out; Check order to reclaiming DNA, this work is accomplished by precious biotechnology (Dalian) ltd;
(2) extraction of plasmid: adopt precious biological plasmid extraction kit to carry out;
(3) being connected of goal gene dna fragmentation and carrier pET23b: because institute's designed primer has Nde I and Hind III restriction enzyme site respectively; And these two MCSs that restriction enzyme site also is pET23b, this makes the possibility that is connected to become of goal gene dna fragmentation and carrier pET23b;
(4) will connect product C aCl
2Method is converted among the clone bacterium E.coli BL21;
(5) Screening and Identification of positive transformant: utilize T
7Universal primer method and double digestion method are carried out the Screening and Identification of positive transformant.
4. positive recombinant is carried out the IPTG abduction delivering that final concentration is 0.5mmol/L.The abduction delivering condition is 28 ℃, and low temperature spends the night and induces.
5. the recombinant protein of expressing is carried out the Histidine affinitive layer purification.
(1) the centrifugal 15min of 7000g collects thalline, abandons supernatant, and raffinate is flowed out as far as possible.The ratio re-suspended cell that adds the ice-cold 1 * Binding Buffer of 4ml with the original fluid of every 100ml;
(2) above-mentioned sample is placed ultrasonic degradation cell on ice, until solution thickness no longer;
(3) 4 ℃, 14000g, centrifugal 20min is to remove cell debris;
(4) supernatant is with the membrane filtration of 0.45 μ m;
(5) the storage liquid of chamber on the His.Bind Column is removed in suction, and opens the following mouth of pipe;
(6) with 10ml 1 * Binding Buffer pillar is carried out balance;
(7) will filter appearance on the good supernatant;
(8) wash post with 10ml 1 * Binding Buffer;
(9) wash post with 10ml 1 * Wash Buffer;
(10) with 5ml 1 * Elute Buffer wash-out target protein.
6. recombinant protein stimulates basophilic granulocyte to discharge the histamine activity identification: adopting rat basophilic leukemia cell is the histamine release ability that RBL-2H3 estimates recombinant protein, and the enzyme immunoassay test kit of employing r-biopharm company detects the concentration of histamine in the nutrient solution supernatant.Specific as follows
(1) 1 * 10
5Individual RBL-2H3 cell is hatched in 96 orifice plates, converges until cell;
(2) with recombinant protein with the different concentration gradient, be respectively 0.3,4.8,10 μ g/ml and add in the plate and hatch, not add any proteic plate hole as contrast;
(3) collecting cell nutrient solution supernatant, and with 10 times of its dilutions;
(4) get 100 μ l standards, Quality Control and sample add in the acidylate plate, in plate hole, add 25 μ l acylating reagents afterwards;
(5) add 200 μ l acidylate damping fluids to the acidylate plate, gently rocker and mix incubated at room 15min;
(6) get the standard of 25 μ l acidylates, Quality Control and sample are in the micropore that is coated with histamine;
(7) add 100 μ l histamine antibody in each micropore, mixing, incubated at room 40min;
(8) pour out liquid in the hole, charge in the hole, outwell liquid in the micropore once more, repeat aforesaid operations again twice with 250 μ l lavation buffer solutions;
(9) add 100 μ l enzyme labelling things in each micropore, thorough mixing is also at room temperature hatched 20min;
(10) wash plate.Repeat the operation of (8);
(11) add 100 μ l substrate/chromogenic reagents in each micropore, thorough mixing, 15min is hatched in the room temperature dark place;
(12) add 100 μ l reaction terminating liquids in each micropore, mix the back gently and measure light absorption value in the 450nm place.
7. use special-purpose RIDA
the SOFT Win software for calculation of r-biopharm company that the result is assessed
Claims (9)
1. a Japanese lamprey oral gland secretory product L-250 albumen is characterized in that this proteic cDNA sequence 519bp is long., this protein is made up of 172 amino acid, contains a TCTP die body, and molecular weight of albumen is 22,408kDa, its aminoacid sequence is:
M I I Y K D I L T G D E M F S
D I Y K I K E T A D G A C F E
V E G T M V S R V E G D I D D
A V F G G N A S A E G G E F D
S S E S S T V S G V D I V L N
H K L Q E T S F S K E K Y I A
Y I K A Y M K T I K E N L M E
K H P E K V E P F M T A A A K
K V K D V L K Q F K D F Q F F
T G E S M N P D G M V A L L N
F R E D G I T P Y M L F F K D
G L E I E K C 。
2. the proteic gene of Japanese lamprey oral gland L-250 according to claim 1 of encoding.
3. gene according to claim 2 is characterized in that its nucleotide sequence is following:
1 atgatcatctacaaggacatcctcaccggggacgagatgttctcg
46 gacatctacaagatcaaggagacggcggacggcgcctgcttcgag
91 gtggaaggcacgatggtgtcccgggtggaaggcgacatcgacgac
136?gcggtctttggcggaaacgcttcggcagaggggggagaatttgat
181?tcgtcggagtcgtctacagtgtccggcgtggacatcgtgctcaac
226?cacaaactgcaggagaccagcttcagcaaggagaagtacattgcc
271?tacatcaaggcctacatgaagacgattaaggagaacctgatggag
319?aaacacccggagaaggtggaacccttcatgactgctgctgccaag
361?aaggttaaagatgtcttaaagcaattcaaggatttccagtttttc
406?acaggtgagagcatgaaccccgacggcatggtcgctcttcttaac
451?ttccgcgaggacggaatcacgccctacatgcttttcttcaaggat
496?ggcctggaaattgagaaatgctaa?519。
4. recombinant plasmid is characterized in that it contains the gene of claim 2 or 3.
5. recombinant plasmid according to claim 4 is characterized in that said gene is connected on the pET23b carrier.
6. a genetic engineering bacterium is characterized in that its quilt is according to claim 4 or 5 described recombinant plasmid transformed.
7. require described genetic engineering bacterium according to right 6, it is characterized in that its host cell is E.coli BL21.
8. the application of Japanese lamprey oral gland L-250 albumen according to claim 1 aspect anticoagulation.
9. application according to claim 8, the wherein said medicine that is used for the anticoagulation aspect is an anticoagulant.
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| CN102732524B (en) * | 2011-04-11 | 2014-02-26 | 辽宁师范大学 | Use of histidine-rich glycoprotein (HRG)-like lampetra japonica Lj-RGD3 all RGD deletion mutant Lj-112 in antitumor drug |
| CN106589100B (en) * | 2016-12-14 | 2020-04-21 | 辽宁师范大学 | Anti-angiogenesis lamprey recombinant PR-1 protein and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1757727A (en) * | 2005-07-13 | 2006-04-12 | 辽宁师范大学 | Gene clone of and expression Japan lamprey oral cavity gland KGD model protein possessing anti thrombotic action |
| CN1760362A (en) * | 2005-07-13 | 2006-04-19 | 辽宁师范大学 | Gene clone and expression of RGD die body protein of oral gland in Japan lamprey possessing function for anti tumour |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1757727A (en) * | 2005-07-13 | 2006-04-12 | 辽宁师范大学 | Gene clone of and expression Japan lamprey oral cavity gland KGD model protein possessing anti thrombotic action |
| CN1760362A (en) * | 2005-07-13 | 2006-04-19 | 辽宁师范大学 | Gene clone and expression of RGD die body protein of oral gland in Japan lamprey possessing function for anti tumour |
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