CN102212125A - Recombinant lamprey lysin as well as preparation method and application thereof in preparing antithrombotic medicament - Google Patents

Recombinant lamprey lysin as well as preparation method and application thereof in preparing antithrombotic medicament Download PDF

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CN102212125A
CN102212125A CN201110101421XA CN201110101421A CN102212125A CN 102212125 A CN102212125 A CN 102212125A CN 201110101421X A CN201110101421X A CN 201110101421XA CN 201110101421 A CN201110101421 A CN 201110101421A CN 102212125 A CN102212125 A CN 102212125A
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arg
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李庆伟
王继红
吕莉
张艳
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Liaoning Normal University
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Liaoning Normal University
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Abstract

The invention discloses a recombinant lamprey lysin protein as well as a preparation method and application thereof in preparing an antithrombotic medicament. The recombinant lamprey lysin protein is a product which is obtained through the following steps: constructing a lamprey protein lysin gene on a pET28a (+) vector; and carrying out efficient induction expression on the gene in Escherichia coli. The lamprey lysin cDNA (complementary deoxyribonucleic acid) sequence 351bp is long, and the protein consists of 117 amino acids, can perform specific binding with glycoprotein GpIIb/IIIa on a blood platelet cell membrane, competitively antagonizes the binding of fibrinogen and blood platelets, prevents the activation of the blood platelets and variation of GpIIb/IIIa receptor conformation and blocks the binding of the receptor and various ligand, so as to inhibit the final common passageway for blood platelet aggregation so as to block thrombosis. The recombinant lamprey lysin has an obvious antithrombotic effect.

Description

Reorganization Lampetra japonica (Martens). eel lysin, preparation method and the application in the preparation antithrombotic reagent
Technical field
The present invention relates to a kind of medicine and engineering in medicine field of belonging to, especially a kind of thrombosis capable of blocking and effective dose is low, safety range is big, action intensity is high, untoward reaction is light and the preparation method of the reorganization Lampetra japonica (Martens). eel lysin of low toxicity and the application in preparing antithrombotic reagent.
Background technology
Under the normal circumstances, hemostasis in the body, blood coagulation system and fibrinolytic system are in physiologic equilibrium, keep state of normal blood flow.But under some pathologic conditions, during as tunica intima damage or venous blood flow retardance, because platelet activation and adhere to subendothelial tissue, and then cause a series of hemostasis, coagulation process, formed thrombus at last.The main component of thrombus is made up of activatory thrombocyte and scleroproein.Therefore, the special anti-activated blood platelet and the medicine of antifibrin can be used as the thrombus directed agents.The platelet antibody of being developed at present mainly prepares at two main sites on the activated blood platelet:
Figure 612022DEST_PATH_IMAGE001
GPIIb-IIIa
Figure 469120DEST_PATH_IMAGE002
GMP140.And the most effective antiplatelet drug is exactly a GPIIb-IIIa antagonist, because no matter by what acceptor, and what pathways metabolism, thrombocyte at last must be by the fibrinogen deceptor of GPIIb-IIIa mixture, and " gathering " and formation thrombus could take place.At present, a class medicine that is called " antiplatelet drug " has been used as the effective means of treatment thrombotic diseases and has been used widely clinically, as Asprin etc.Yet platelet aggregation-against when the characteristics of acetylsalicylic acid antiplatelet effects are low dose produces anti thrombotic action; And promote platelet aggregation when heavy dose of, produce and promote the thrombosis effect.In addition, bring " aspirin resistance " and also more and more limit its antithrombotic clinical application along with the acetylsalicylic acid prolonged application.
Jamprey-ell ( Lampetra japonica) the genus Cyclostomata ( Cyclostomata), Peteromyzoni-form, Petromyzonidae, Lampetra, be the most ancient no jaw vertebrates of finding so far.It is the oceanodromous migration fish, and adult is lived in the ocean, is attached on other fish body through sucker commonly used and sucks its blood and meat, and The blood streamed down to make the fish that is adsorbed, and this unique life habit of jamprey-ell is hinting in its oral gland to have potent anticoagulant substances.But, be not raw material also up to now with the Japanese lamprey oral gland, the relevant report of preparation antithrombotic reagent.
Summary of the invention
The present invention is in order to solve the above-mentioned technical problem of existing in prior technology, provides a kind of thrombosis capable of blocking and effective dose is low, safety range is big, action intensity is high, untoward reaction is light and the reorganization eel lysin albumen of low toxicity, preparation method and the application in the preparation antithrombotic reagent.
Technical solution of the present invention is: a kind of reorganization Lampetra japonica (Martens). albumen-eel lysin, it is characterized in that Lampetra japonica (Martens). eel lysin gene constructed in pET28a (+) carrier, and it has been carried out the product of efficient abduction delivering in intestinal bacteria, described eel lysin cDNA sequence 351bp is long, albumen is made up of 117 amino acid, and cDNA and aminoacid sequence are as follows:
1
tca?acg?ttc?atc?aac?gga?acc?cag?gaa?gtg?gat?gcc?att?tgt?cat?aag 48
Ser?Thr?Phe?Ile?Asn?Gly?Thr?Gln?Glu?Val?Asp?Ala?Ile?Cys?His?Lys
1 5 10 15
cag?aat?tat?ccc?atg?ggt?acg?gag?aca?cag?gga?gac?aca?cgt?gga?gac 96
Gln?Asn?Tyr?Pro?Met?Gly?Thr?Glu?Thr?Gln?Gly?Asp?Thr?Arg?Gly?Asp
20 25 30
aca?cgg?aca?cac?acg?gag?aca?caa?gct?gag?gca?cgg?aca?cac?gca?gag 144
Thr?Arg?Thr?His?Thr?Glu?Thr?Gln?Ala?Glu?Ala?Arg?Thr?His?Ala?Glu
35 40 45
acg?cac?gga?gac?aca?cgt?gga?gac?aca?cgg?aga?cac?acg?tgg?aga?cac 192
Thr?His?Gly?Asp?Thr?Arg?Gly?Asp?Thr?Arg?Arg?His?Thr?Trp?Arg?His
50 55 60
acg?cgg?aga?cac?acg?gac?aca?cac?gga?cac?aca?cgg?aga?cac?aag?ctg 240
Thr?Arg?Arg?His?Thr?Asp?Thr?His?Gly?His?Thr?Arg?Arg?His?Lys?Leu
65 70 75 80
agg?cac?gaa?cac?acg?cag?aga?cac?acg?ggg?gcc?cgc?gga?gac?gca?cgg 288
Arg?His?Glu?His?Thr?Gln?Arg?His?Thr?Gly?Ala?Arg?Gly?Asp?Ala?Arg
85 90 95
aga?cac?gga?cac?aac?aaa?cat?tta?cac?aga?atg?agt?gca?gcg?gtg?agt 336
Arg?His?Gly?His?Asn?Lys?His?Leu?His?Arg?Met?Ser?Ala?Ala?Val?Ser
100 105 110
gaa?tgt?gtt?ggg?gag
Glu?Cys?Val?Gly?Glu
115
The proteic preparation method of a kind of above-mentioned reorganization eel lysin is characterized in that carrying out as follows successively:
A. prepare eel lysin gene:
(1) getting Japanese lamprey oral gland places liquid nitrogen to preserve rapidly;
(2) take by weighing the 0.2g lamprey oral gland, add the homogenate of 1ml TRIzol reagent preparation poison gland, hatch 5min for 4 ℃;
(3) add the 0.2ml chloroform, the tight lid back jolting of lid 15sec places 5min on ice then;
(4) 4 ℃, 12000xg, centrifugal 15min;
(5) upper water is moved into another centrifuge tube mutually, add the 0.5ml Virahol, and hatch 10min on ice;
(6) 4 ℃, 12000xg, centrifugal 15min;
(7) abandon supernatant, in precipitation, add 1ml 75% washing with alcohol, the vortex mixing;
(8) 4 ℃, the centrifugal 5min of 10000xg, the RNA that obtains the eel lysin precipitates;
(9) after the dry air, with TE or not have RNase water dissolution RNA standby;
B. carry out the RT-PCR amplification, obtain purpose cDNA;
The concrete operations condition of RT-PCR amplification is 42 ℃ of 20min, 99 ℃ of 5min, and 5 ℃ of 5min carry out reverse transcription reaction;
Primer sequence is as follows: P1:5 '-XXcatatgtcaacgttcatcaacggaacc-3 ';
P2:?5’-XXaagcttctccccaacacattcactcac-3’；
C. the purpose cDNA that obtains is connected with pET-28a (+) carrier, is transformed into the Screening and Identification of carrying out positive transformant behind the clone bacterium DH5a;
Concrete operations are as follows:
(1) recovery of goal gene: the recovery of goal gene adopts TaKaRa PCR Fragment Recovery Kit to carry out;
(2) extraction of plasmid: adopt plasmid extraction kit to carry out;
(3) goal gene dna fragmentation and carrier pET-28a (+) with NdeI and HinD III double digestion is with T 4DNA Ligase carries out 16 ℃ of connections of spending the night, and goal gene is linked to each other with pET-28a (+);
(4) will connect product C aCl 2Method is converted into clone bacterium E. ColiAmong the DH5a;
(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;
D. with the positive recombinant CaCl that identifies 2Method is converted into expresses bacterium E. ColiAmong the BL21, carrying out final concentration is the IPTG abduction delivering of 1mmol/L, and the abduction delivering condition is that 30 ℃ of low temperature spend the night and induce expression product called after eel lysin.
The application of a kind of above-mentioned reorganization eel lysin in the preparation antithrombotic reagent.
Reorganization eel lysin albumen of the present invention can combine with glycoprotein Gp II b/ III a specificity on the platelet cell film and the competitive antagonism Fibrinogen combines with hematoblastic, prevent the change of platelet activation and GPIIb/IIIa receptor conformation, the blocking-up acceptor combines with multiple part, thereby the final co-channel of anticoagulant has remarkable anti-bolt effect with the blocking-up thrombosis.Effective dose of the present invention is low, and only for rat, the effective dose of acetylsalicylic acid is 2 mgkg -1, and effective dose of the present invention reaches the Gamma Magnitude level, its toxicity is slight, nontoxicity in the antithrombotic agent weight range.The present invention simultaneously is good dose-effect relationship in effective dosage ranges, promptly increase with dosage, and its anti-bolt effect also strengthens.Have that effective dose is low, safety range is big, action intensity is high, untoward reaction is light and advantage such as low toxicity.
Description of drawings
Fig. 1 is that the embodiment of the invention is through the Tricine of affinitive layer purification SDS-PAGE electrophorogram.
Fig. 2 is that the intravenous injection embodiment of the invention is to the thrombotic dose-effect relationship figure that influences of rat carotid artery.
Fig. 3 is the intravenous injection embodiment of the invention 50.0 μ gkg -1To rat carotid artery thrombosis influence the time-the effect graphic representation.
Fig. 4 is the influence synoptic diagram of the intravenous injection various dose embodiment of the invention to rat postcava thrombosis ratio.
Fig. 5 is that the intravenous injection various dose embodiment of the invention influences synoptic diagram to rat postcava formation thrombus dry weight.
Embodiment
A. prepare eel lysin gene:
(1) getting Japanese lamprey oral gland places liquid nitrogen to preserve rapidly;
(2) take by weighing the 0.2g lamprey oral gland, add the homogenate of 1ml TRIzol reagent preparation poison gland, hatch 5min for 4 ℃;
(3) add the 0.2ml chloroform, the tight lid back jolting of lid 15sec places 5min on ice then;
(4) 4 ℃, 12000xg, centrifugal 15min;
(5) upper water is moved into another centrifuge tube mutually, add the 0.5ml Virahol, and hatch 10min on ice;
(6) 4 ℃, 12000xg, centrifugal 15min;
(7) abandon supernatant, in precipitation, add 1ml 75% washing with alcohol, the vortex mixing;
(8) 4 ℃, the centrifugal 5min of 10000xg, the RNA that obtains the eel lysin precipitates;
(9) after the dry air, with TE or not have RNase water dissolution RNA standby;
B. carry out the RT-PCR amplification, obtain purpose cDNA;
The concrete operations condition of RT-PCR amplification is 42 ℃ of 20min, 99 ℃ of 5min, and 5 ℃ of 5min carry out reverse transcription reaction;
Primer sequence is as follows: P1:5 '-XXcatatgtcaacgttcatcaacggaacc-3 ';
P2:?5’-XXaagcttctccccaacacattcactcac-3’；
C. the purpose cDNA that obtains is connected with pET-28a (+) carrier, is transformed into the Screening and Identification of carrying out positive transformant behind the clone bacterium DH5a;
Concrete operations are as follows:
(1) recovery of goal gene: the recovery of goal gene adopts TaKaRa PCR Fragment Recovery Kit to carry out;
(2) extraction of plasmid: adopt plasmid extraction kit to carry out;
(3) goal gene dna fragmentation and carrier pET-28a (+) with NdeI and HinD III double digestion is with T 4DNA Ligase carries out 16 ℃ of connections of spending the night, and goal gene is linked to each other with pET-28a (+);
(4) will connect product C aCl 2Method is converted into clone bacterium E. ColiAmong the DH5a;
(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;
D. with the positive recombinant CaCl that identifies 2Method is converted into expresses bacterium E. ColiAmong the BL21, carrying out final concentration is the IPTG abduction delivering of 1mmol/L, expression product called after eel lysin;
E. the recombinant protein of expressing is carried out the Histidine affinitive layer purification.
The purified proteic Tricine SDS-of eel lysin PAGE electrophorogram as shown in Figure 1.1. Rainbow among the figure TMColoured Low Molecular Weight Markers; 2. Peptide Marker; 3. lamprey oral gland is secreted native protein; 4. the eel lysin 15kDa albumen of purifying.
The reorganization eel lysin effect of the embodiment of the invention is as follows:
1. the dose-effect relationship of the anti-arterial thrombus effect of intravenous injection eel lysin
Get 60 of male SD rats, divide equally 6 groups of (10 every group): NS groups at random, eel lysin 12.5 μ gkg -1, 25 μ gkg -1, 50 μ gkg -1With 100 μ gkg -1Dosage group, and positive control medicine di-lysine-aspirin (LAS) 18.0 mgkg -1Group.Rats by intraperitoneal injection urethane 1 gkg -1Anesthesia, separate a side arteria carotis communis, the stimulating electrode that thrombus in vivo is formed instrument places the arteria carotis communis proximal part, distal end is put a temperature electrode, rat sublingual vein injection difference is subjected to reagent thing (the NS group waits capacity physiological saline) afterwards during the effect peak value in the 10 min(time-effect relationships), with intensity is 1.6mA direct current continued stimulus arteria carotis communis 7 min, instrument is reported to the police when the thrombosis block blood flow, record begins to the temperature decrease required time from electricity irritation, claim blood flow (Occlusion Time blocking time, OT), be thrombus formation time.With the NS group is benchmark, calculates different dosing group OT and prolongs percentage.
The result is as shown in Figure 2: compare intravenous injection eel lysin 12.5 μ gkg with physiological saline (NS) group -1, 25.0 μ gkg -1, 50.0 μ gkg -1And 100.0 μ gkg -1Back blood flow blocking time (Occlusion Time OT) prolongs percentage and is respectively 11.98 %, 23.83 %, 37.53 % and 60.52 %(p＜0.01) illustrates that the eel lysin has tangible anti-arterial thrombus effect, and is dose-dependently.Positive control drug di-lysine-aspirin (LAS) 18.0 mgkg -1OT prolongation percentage is 26.20 % after the intravenous injection.* p＜0.01 is compared with the NS control group; N=10; 10 min measure behind the iv medicine.
2. the time-effect relationship of the anti-arterial thrombus effect of intravenous injection eel lysin
Get 55 of male SD rats, be divided into 11 groups (5 every group) at random: NS control group, 5 min, 10 min, 15 min, 20 min, 30 min, 60 min, 90 min, 120 min, 180 min and 240min group behind the medicine.Administration group rat sublingual vein injection eel lysin 50.0 μ gkg -1Back different time is measured the OT value, and the NS group waits capacity physiological saline.
Statistical method adopts analysis of variance.
The result is as shown in Figure 3: intravenous injection eel lysin 50.0 μ gkg -1Suppress rat carotid artery and can reach 180min thrombotic action time.Intravenous injection eel lysin 50.0 μ gkg -1Back 5,10,15,20,30,60,90,120,180 and 240 min, OT prolong percentage and are respectively 41.09 %, 65.44 %, 48.89 %, 41.51 %, 39.97 %, 20.89 %, 35.85 %, 51.95 %, 21.37 % and 10.27 %.Compare with the NS group, significant difference is all arranged the 240min behind medicine, the illustration time can reach 180 min.* p＜0.01, △ P〉0.05 compare with the NS control group; N=5.
3. intravenous injection eel lysin is to the thrombotic influence of rat vein
Get 60 of male SD rats, be divided into 6 groups at random: NS control group, eel lysin 25.0 μ gkg -1, 12.5 μ gkg -1, 6.25 μ gkg -1With 3.13 μ gkg -1Group, Heparin 400.0 IUkg -1Group.Rats by intraperitoneal injection urethane 1 gkg -1Anesthesia, the separation postcava of cutting open the belly is put a silk thread in the left renal vein below and is equipped with ligation blood vessel usefulness, is subjected to the reagent thing from tongue intravenous injection difference, the ligation postcava causes extravasated blood behind 5 min, close the abdominal cavity then, opened the abdominal cavity once more in 4 hours after the ligation, folder closes blood vessel in 2 cm places, ligation below, blood vessel is cut in stringer open, removal of thromboses claims the thrombus dry weight after 55 ℃ of 30 min drying, record has or not thrombosis and thrombus dry weight.With the NS group is that benchmark calculates each administration group thrombosis ratio and the thrombus dry weight suppresses percentage.
Statistical method adopts the test of difference of variance analysis, rate.
Result such as Fig. 4, Fig. 5 compare eel lysin 3.13,6.25,12.5 and 25.0 μ gkg with the NS group as can be known -1Behind the iv thrombus dry weight inhibiting rate be respectively 36.1%, 54.0%, 82.0% and 88.6%(p＜0.01), the thrombosis inhibiting rate is respectively 10%, 20%, 40% (p〉0.05) and 70% (p＜0.01).Illustrate that iv eel lysin has tangible anti-phlebothrombosis effect, and be dose-dependently.Positive control drug heparin 400.0 IUkg -1Thrombus dry weight inhibiting rate and thrombosis inhibiting rate behind the iv are respectively 84.0%(p＜0.01) and 60 %(p＜0.05).
4. toxicity research-the LD of eel lysin 50Measure
Get 50 of 18 ~ 22g Kunming mouses, male and female half and half are divided into 5 groups of (10 every group): 27.468mgkg at random -1, 24.721mgkg -1, 22.249 mgkg -1, 20.024 mgkg -1With 18.022 mgkg -1Group.Mouse tail vein is at the uniform velocity injected different concns eel lysin, and the administration capacity is a 0.1ml/ 10g body weight, has annotated in 30 seconds, and whether observe behind the medicine in 7 days mouse dead.Use the Bliss method and calculate the LD of mouse mainline eel lysin 50
The result shows: the LD of mouse mainline eel lysin 50Be 25.3160 mgkg -1, and its anti-freezing medicine for treating thrombus effect effective dose only is the Gamma Magnitude level, maximal dose is 2.0 mgkg in its treatment window -1, only be LD 501/12.7, its safety is described: as can be seen from Table 1, cause mouse death rate from low dosage to high dosage eel lysin and be respectively 10 %, 20 %, 30 %, 50 % and 60 %.Per cent death loss is converted into probit (Y) carries out linear regression, get regression equation Y=8.5232 X-6.9614(r=0.9956 with log10 dose (X)), calculate LD 50Be 25.3160 mgkg -1
The LD of table 1. mouse mainline eel lysin 50Mensuration
Figure 225723DEST_PATH_IMAGE003
Sequence table
 
＜110〉Liaoning demonstration university
＜120〉reorganization Lampetra japonica (Martens). eel lysin, preparation method and the application in the preparation antithrombotic reagent
 
<160>3
 
<210> 1
<211> 27
<212> DNA
<213> Lampetra?japonica
 
<220>
<221> primer_bind
<222> (1)..(27)
 
<400> catatgtcaa?cgttcatcaa?cggaacc
 
<210> 2
<211> 27
<212> DNA
<213> Lampetra?japonica
 
 
<220>
<221> primer_bind
<222> (1)..(27)
 
<400> aagcttctcc?ccaacacatt?cactcac
 
<210> 3
<211> 351
<212> DNA
<213> Lampetra?japonica
<220>
<221> exon
<222> (1)..(351)
 
<400>
tca?acg?ttc?atc?aac?gga?acc?cag?gaa?gtg?gat?gcc?att?tgt?cat?aag 48
Ser?Thr?Phe?Ile?Asn?Gly?Thr?Gln?Glu?Val?Asp?Ala?Ile?Cys?His?Lys
1 5 10 15
cag?aat?tat?ccc?atg?ggt?acg?gag?aca?cag?gga?gac?aca?cgt?gga?gac 96
Gln?Asn?Tyr?Pro?Met?Gly?Thr?Glu?Thr?Gln?Gly?Asp?Thr?Arg?Gly?Asp
20 25 30
aca?cgg?aca?cac?acg?gag?aca?caa?gct?gag?gca?cgg?aca?cac?gca?gag 144
Thr?Arg?Thr?His?Thr?Glu?Thr?Gln?Ala?Glu?Ala?Arg?Thr?His?Ala?Glu
35 40 45
acg?cac?gga?gac?aca?cgt?gga?gac?aca?cgg?aga?cac?acg?tgg?aga?cac 192
Thr?His?Gly?Asp?Thr?Arg?Gly?Asp?Thr?Arg?Arg?His?Thr?Trp?Arg?His
50 55 60
acg?cgg?aga?cac?acg?gac?aca?cac?gga?cac?aca?cgg?aga?cac?aag?ctg 240
Thr?Arg?Arg?His?Thr?Asp?Thr?His?Gly?His?Thr?Arg?Arg?His?Lys?Leu
65 70 75 80
agg?cac?gaa?cac?acg?cag?aga?cac?acg?ggg?gcc?cgc?gga?gac?gca?cgg 288
Arg?His?Glu?His?Thr?Gln?Arg?His?Thr?Gly?Ala?Arg?Gly?Asp?Ala?Arg
85 90 95
aga?cac?gga?cac?aac?aaa?cat?tta?cac?aga?atg?agt?gca?gcg?gtg?agt 336
Arg?His?Gly?His?Asn?Lys?His?Leu?His?Arg?Met?Ser?Ala?Ala?Val?Ser
100 105 110
gaa?tgt?gtt?ggg?gag 351
Glu?Cys?Val?Gly?Glu
115

Claims (3)

1. reorganization Lampetra japonica (Martens). eel lysin albumen, it is characterized in that it being that Lampetra japonica (Martens). eel lysin is gene constructed in pET28a (+) carrier, and it has been carried out the product of efficient abduction delivering in intestinal bacteria, described eel lysin cDNA sequence 351bp is long, albumen is made up of 117 amino acid, and cDNA and aminoacid sequence are as follows:
1
tca?acg?ttc?atc?aac?gga?acc?cag?gaa?gtg?gat?gcc?att?tgt?cat?aag 48
Ser?Thr?Phe?Ile?Asn?Gly?Thr?Gln?Glu?Val?Asp?Ala?Ile?Cys?His?Lys
1 5 10 15
cag?aat?tat?ccc?atg?ggt?acg?gag?aca?cag?gga?gac?aca?cgt?gga?gac 96
Gln?Asn?Tyr?Pro?Met?Gly?Thr?Glu?Thr?Gln?Gly?Asp?Thr?Arg?Gly?Asp
20 25 30
aca?cgg?aca?cac?acg?gag?aca?caa?gct?gag?gca?cgg?aca?cac?gca?gag 144
Thr?Arg?Thr?His?Thr?Glu?Thr?Gln?Ala?Glu?Ala?Arg?Thr?His?Ala?Glu
35 40 45
acg?cac?gga?gac?aca?cgt?gga?gac?aca?cgg?aga?cac?acg?tgg?aga?cac 192
Thr?His?Gly?Asp?Thr?Arg?Gly?Asp?Thr?Arg?Arg?His?Thr?Trp?Arg?His
50 55 60
acg?cgg?aga?cac?acg?gac?aca?cac?gga?cac?aca?cgg?aga?cac?aag?ctg 240
Thr?Arg?Arg?His?Thr?Asp?Thr?His?Gly?His?Thr?Arg?Arg?His?Lys?Leu
65 70 75 80
agg?cac?gaa?cac?acg?cag?aga?cac?acg?ggg?gcc?cgc?gga?gac?gca?cgg 288
Arg?His?Glu?His?Thr?Gln?Arg?His?Thr?Gly?Ala?Arg?Gly?Asp?Ala?Arg
85 90 95
aga?cac?gga?cac?aac?aaa?cat?tta?cac?aga?atg?agt?gca?gcg?gtg?agt 336
Arg?His?Gly?His?Asn?Lys?His?Leu?His?Arg?Met?Ser?Ala?Ala?Val?Ser
100 105 110
gaa?tgt?gtt?ggg?gag 351
Glu?Cys?Val?Gly?Glu
115
。
2. preparation method of Lampetra japonica (Martens). eel lysin that recombinates according to claim 1 is characterized in that carrying out as follows successively:
A. prepare eel lysin gene:
(1) getting Japanese lamprey oral gland places liquid nitrogen to preserve rapidly;
(2) take by weighing the 0.2g lamprey oral gland, add the homogenate of 1ml TRIzol reagent preparation poison gland, hatch 5min for 4 ℃;
(3) add the 0.2ml chloroform, the tight lid back jolting of lid 15sec places 5min on ice then;
(4) 4 ℃, 12000xg, centrifugal 15min;
(5) upper water is moved into another centrifuge tube mutually, add the 0.5ml Virahol, and hatch 10min on ice;
(6) 4 ℃, 12000xg, centrifugal 15min;
(7) abandon supernatant, in precipitation, add 1ml 75% washing with alcohol, the vortex mixing;
(8) 4 ℃, the centrifugal 5min of 10000xg, the RNA that obtains Lampetra japonica (Martens). albumen eel lysin precipitates;
(9) after the dry air, with TE or not have RNase water dissolution RNA standby;
B. carry out the RT-PCR amplification, obtain purpose cDNA;
The concrete operations condition of RT-PCR amplification is 42 ℃ of 20min, 99 ℃ of 5min, and 5 ℃ of 5min carry out reverse transcription reaction;
Primer sequence is as follows: P1:5 '-XXcatatgtcaacgttcatcaacggaacc-3 ';
P2:?5’-XXaagcttctccccaacacattcactcac-3’；
C. the purpose cDNA that obtains is connected with pET-28a (+) carrier, is transformed into the Screening and Identification of carrying out positive transformant behind the clone bacterium DH5a;
Concrete operations are as follows:
(1) recovery of goal gene: the recovery of goal gene adopts TaKaRa PCR Fragment Recovery Kit to carry out;
(2) extraction of plasmid: adopt plasmid extraction kit to carry out;
(3) goal gene dna fragmentation and carrier pET-28a (+) with NdeI and HinD III double digestion is with T 4DNA Ligase carries out 16 ℃ of connections of spending the night, and goal gene is linked to each other with pET-28a (+);
(4) will connect product C aCl 2Method is converted into clone bacterium E. ColiAmong the DH5a;
(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;
D. with the positive recombinant CaCl that identifies 2Method is converted into expresses bacterium E. ColiAmong the BL21, carrying out final concentration is the IPTG abduction delivering of 1mmol/L, and the abduction delivering condition is that 30 ℃ of low temperature spend the night and induce, and expression product is reorganization Lampetra japonica (Martens). RGD toxin protein, called after eel lysin.
3. the application of eel lysin in the preparation antithrombotic reagent of recombinating according to claim 1.
CN201110101421XA 2011-04-22 2011-04-22 Recombinant lamprey lysin as well as preparation method and application thereof in preparing antithrombotic medicament Pending CN102212125A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103969228A (en) * 2013-02-04 2014-08-06 百奥泰生物科技(广州)有限公司 Biological effect detection method for integrin GP IIb/IIIa specific binding molecule Batifiban
CN106589100A (en) * 2016-12-14 2017-04-26 辽宁师范大学 Angiogenesis-resistance lampetra japonica recombinant protein PR-1 and preparation method thereof
CN107320713A (en) * 2016-04-28 2017-11-07 辽宁师范大学 Applications of the recombinant protein rLj-RGD4 in antithrombotic reagent is prepared
CN108187030A (en) * 2018-01-31 2018-06-22 辽宁师范大学 Recombinant peptide rLj-RGD3 is preparing the application in preventing and treating acute myocardial infarction AMI drug

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JIHONG WANG ET AL: "A novel RGD-toxin protein, Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities", 《BIOCHIMIE》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103969228A (en) * 2013-02-04 2014-08-06 百奥泰生物科技(广州)有限公司 Biological effect detection method for integrin GP IIb/IIIa specific binding molecule Batifiban
CN107320713A (en) * 2016-04-28 2017-11-07 辽宁师范大学 Applications of the recombinant protein rLj-RGD4 in antithrombotic reagent is prepared
CN106589100A (en) * 2016-12-14 2017-04-26 辽宁师范大学 Angiogenesis-resistance lampetra japonica recombinant protein PR-1 and preparation method thereof
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
CN108187030A (en) * 2018-01-31 2018-06-22 辽宁师范大学 Recombinant peptide rLj-RGD3 is preparing the application in preventing and treating acute myocardial infarction AMI drug

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