CN103031266B - msCT-AcAPc2 fusion protein transgenic engineering strain - Google Patents
msCT-AcAPc2 fusion protein transgenic engineering strain Download PDFInfo
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Abstract
The invention relates to a fusion protein transgenic engineering strain, in particular to an msCT-AcAPc2 (multi-slice spiral CT-ancylostoma caninum anticoagulant peptide c2) fusion protein transgenic engineering strain which solves the defect that a drug that can simultaneously and effectively treat osteoporosis and thrombosis does not exist at present. The msCT-AcAPc2 fusion protein transgenic engineering strain, namely escherichia coli BL21 (DE3-msCT/AcAPc2), is prepared by the following steps: 1, fusion protein DNA (deoxyribonucleic acid) segments are obtained; 2, plasmids are subjected to double enzyme digestion; 3, enzymes are linked; and 4, the escherichia coli BL21 is converted. The fusion protein transgenic engineering strain can be applied in the field of medicine preparation.
Description
Technical field
The present invention relates to a kind of fusion rotein transgenic engineering bacterial strain.
Background technology
Senile osteoporosis sickness rate is higher, and there are 200,000,000 sufferers of osteoporosis face in the whole world, and women is more than the male sex.According to the standard of the World Health Organization (WHO), American National health and nutrition survey (NHANES III, 1988 ~ 1994 years) result shows, osteoporosis has a strong impact on life of elderly person quality, more than 50 years old in crowd, can there is in life osteoporotic fracture theirs in 1/2 women, 1/5 the male sex, once patient experience osteoporotic fracture for the first time, the danger of secondary fracture obviously strengthens.Chinese Aged occupy first place, the world, and existing patients with osteoporosis 9,000 ten thousand, accounts for 7.1% of total population.Along with the process of social senilization, the sickness rate of osteoporosis is in rising trend, expects 2050 and will be increased to 2.21 hundred million, and whole world osteoporotic fracture over half will occur in Asia at that time, and the overwhelming majority is in China.There is scholar, to the year of 1995 ~ 1996 years U.S.'s osteoporosis, myocardial infarction, palsy and mammary cancer, number occurs and investigate demonstration, annual osteoporotic fracture 1,500,000 times, wherein vertebral fracture 700,000 times, the Wrist fracture 200,000 times of occurring, Hip Fracture 300,000 times, other fracture 300,000 times.
Thrombosis is a kind of process of the multifactor variation that relates to many h and E factors interact with each other.And have its singularity for its blood coagulation system of the elderly, the elderly's Fibrinogen (FIB) content, tissue plasminogen activator increase and the increase of Type 1 plasminogen activator inhibitor mixture all causes the elderly's hypercoagulative state, so more easily form thrombus; The incidence of thrombus that wherein women after climacteric is found in research is far above the male sex.
Become common frdquently encountered disease for senile osteoporosis and thrombus, and often existed simultaneously.If patient takes multi-medicament and treats osteoporosis and thrombus simultaneously, easily produce drug antagonism reaction; If stagger, will, by considering the transformation period of medicine, also will consider on the other hand medicine effective concentration, and bring inconvenience to patient medicine time on the one hand.
Lack at present a kind of medicine that can effectively treat osteoporosis and thrombus simultaneously.
Summary of the invention
The present invention will solve and there is no at present simultaneously the effectively defect for the treatment of osteoporosis and thrombus medicine, and a kind of msCT-AcAPc2 fusion rotein transgenic engineering bacterial strain providing.
MsCT-AcAPc2 fusion rotein transgenic engineering bacterial strain is called e. coli bl21 (DE3-msCT/AcAPc2), and e. coli bl21 (DE3-msCT/AcAPc2) is prepared according to the following steps:
One, by the plasmid vector pUC(msCT/AcAPc2 that contains msCT-AcAPc2 antigen-4 fusion protein gene) use BamH I and EcoR I double digestion, obtain the DAN fragment of msCT-AcAPc2 fusion rotein;
Two, with BamHI and EcoR I double digestion plasmid pGEX-6P-1;
Three, the DAN fragment of msCT-AcAPc2 fusion rotein with carry out enzyme through the vector pGEX-6P-1 of double digestion and be connected, then place to connect and spend the night for 16 ℃, obtain vector pGEX-msCT/AcAPc2;
Four, transform e. coli bl21 (DE3) with vector pGEX-msCT/AcAPc2, select positive recombinant, obtain msCT-AcAPc2 fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/AcAPc2).
In the present invention, the gene order of msCT-AcAPc2 fusion rotein is as shown in SEQ ID NO:1.
In the present invention, the aminoacid sequence of msCT-AcAPc2 fusion rotein is as shown in SEQ ID NO:2.
The msCT-AcAPc2 fusion rotein that msCT-AcAPc2 fusion rotein transgenic engineered bacteria e. coli bl21 of the present invention (DE3-msCT/AcAPc2) fermentation produces contains 118 amino acid.The msCT-AcAPc2 fusion rotein that msCT-AcAPc2 fusion rotein transgenic engineered bacteria e. coli bl21 of the present invention (DE3-msCT/AcAPc2) fermentation produces can be used for treating osteoporosis and thrombus simultaneously, is applicable to suffering from patient's use of these two kinds of diseases simultaneously.And msCT-AcAPc2 fusion rotein is microbial preparation, do not produce antagonism reaction, use safety.
The present invention adopts biological gene engineering means to obtain e. coli bl21 (DE3-msCT/AcAPc2).Adopt genetic engineering bacterium e. coli bl21 of the present invention (DE3-msCT/AcAPc2) to produce msCT-AcAPc2 fusion rotein by large scale fermentation, there is wide application prospect.The present invention has established basic substance for treating osteoporosis and thrombus.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment msCT-AcAPc2 fusion rotein transgenic engineering bacterial strain is called e. coli bl21 (DE3-msCT/AcAPc2), and e. coli bl21 (DE3-msCT/AcAPc2) is prepared according to the following steps:
One, by the plasmid vector pUC(msCT/AcAPc2 that contains msCT-AcAPc2 antigen-4 fusion protein gene) use BamH I and EcoR I double digestion, obtain the DAN fragment of msCT-AcAPc2 fusion rotein;
Two, with BamHI and EcoR I double digestion plasmid pGEX-6P-1;
Three, the DAN fragment of msCT-AcAPc2 fusion rotein with carry out enzyme through the vector pGEX-6P-1 of double digestion and be connected, then place to connect and spend the night for 16 ℃, obtain vector pGEX-msCT/AcAPc2;
Four, transform e. coli bl21 (DE3) with vector pGEX-msCT/AcAPc2, select positive recombinant, obtain msCT-AcAPc2 fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/AcAPc2).
Present embodiment step 4 adopts electric shock conversion method to transform e. coli bl21 (DE3).
Embodiment two: the difference of present embodiment and embodiment one is: plasmid vector pUC(msCT/AcAPc2 in step 1) endonuclease reaction system is
Other steps and parameter are identical with embodiment one.
Present embodiment step 1 endonuclease reaction reacts 10h under 37 ℃ of conditions, then 1.5% agarose gel electrophoresis, and object fragment is purified recovery with DNA GEL EXTRACTION KIT.
Embodiment three: the difference of present embodiment and embodiment one or two is: in step 2, plasmid pGEX-6P-1 endonuclease reaction system is
Other steps and parameter are identical with embodiment one or two.
Present embodiment step 2 endonuclease reaction reacts 10h under 37 ℃ of conditions, then 0.6% agarose gel electrophoresis, and object fragment is purified recovery with DNA GEL EXTRACTION KIT.
Embodiment four: the difference of present embodiment and embodiment one, two or three is: in step 3, enzyme ligation system is
Other steps and parameter are identical with embodiment one, two or three.
The ligation of present embodiment step 3 enzyme is carried out under 16 ℃ of conditions.
Embodiment five: the difference of one of present embodiment and embodiment one to four is: in step 1, the gene order of msCT-AcAPc2 fusion rotein is as shown in SEQ ID NO:1.Other steps and parameter are identical with one of embodiment one to four.
The nucleotide sequence of present embodiment msCT-AcAPc2 fusion rotein is synthesized by bio-engineering corporation, and is made up the plasmid vector pUC(msCT/AcAPc2 of the nucleic acid that contains fusion rotein of bio-engineering corporation).
Embodiment six: the difference of one of present embodiment and embodiment one to five is: in step 1, the aminoacid sequence of msCT-AcAPc2 fusion rotein is as shown in SEQ ID NO:2.Other steps and parameter are identical with one of embodiment one to five.
Embodiment seven: present embodiment msCT-AcAPc2 fusion rotein transgenic engineering bacterial strain is called e. coli bl21 (DE3-msCT/AcAPc2), and e. coli bl21 (DE3-msCT/AcAPc2) is prepared according to the following steps:
One, by the plasmid vector pUC(msCT/AcAPc2 that contains msCT-AcAPc2 antigen-4 fusion protein gene) use BamH I and EcoR I double digestion, obtain the DAN fragment of msCT-AcAPc2 fusion rotein;
Two, with BamHI and EcoR I double digestion plasmid pGEX-6P-1;
Three, the DAN fragment of msCT-AcAPc2 fusion rotein with carry out enzyme through the vector pGEX-6P-1 of double digestion and be connected, then place to connect and spend the night for 16 ℃, obtain vector pGEX-msCT/AcAPc2;
Four, transform e. coli bl21 (DE3) with vector pGEX-msCT/AcAPc2, select positive recombinant, obtain msCT-AcAPc2 fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/AcAPc2);
Wherein plasmid vector pUC(msCT/AcAPc2 in step 1) endonuclease reaction system is:
Wherein in step 2, plasmid pGEX-6P-1 endonuclease reaction system is:
Wherein in step 3, enzyme ligation system is:
Wherein in step 1 the gene order of msCT-AcAPc2 fusion rotein as shown in SEQ ID NO:1; In step 1, the aminoacid sequence of msCT-AcAPc2 fusion rotein is as shown in SEQ ID NO:2.
In present embodiment, the nucleotide sequence of msCT-AcAPc2 fusion rotein is synthesized by bio-engineering corporation, and is made up the plasmid vector pUC(msCT/AcAPc2 of the nucleic acid that contains fusion rotein of bio-engineering corporation).Present embodiment has been removed the original signal peptide of AcAPc2 gene in the time building msCT-AcAPc2 fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/AcAPc2), and between msCT and Ancylostoma caninum anticoagulant hemepeptide, insert Kpn I restriction enzyme site, both can improve the stability of coded fusion rotein, also change the secondary structure of fusion rotein, its biological property is improved simultaneously.And add respectively BamHI and EcoR I restriction enzyme site in antigen-4 fusion protein gene both sides, and according to the feature of intestinal bacteria preference codon, redesign fusion gene coding base sequence.
On vector pGEX-6P-1, do not contain Kpn I restriction enzyme site, the synthetic pGEX-msCT/AcAPc2 of present embodiment all can open for Kpn I single endonuclease digestion, illustrates that present embodiment msCT-AcAPc2 fusion rotein successfully imports in plasmid pGEX-6P-1.Then plasmid pGEX-msCT/AcAPc2 is imported in e. coli bl21 (DE3), choose positive colony.
Random 37 ℃ of incubated overnight of picking positive colony e. coli bl21 (DE3-msCT/AcAPc2) bacterium colony, extract matter DNA, carry out single endonuclease digestion by Kpn I, and with corresponding blank plasmid pGEX-6P-1 in contrast, the plasmid that contains Kpn I restriction enzyme site is carried out to gene sequencing.Examining order entrusts biotech firm to carry out, and e. coli bl21 (DE3-msCT/AcAPc2) contains DNA shown in SEQ ID NO:1.
E. coli bl21 (DE3-msCT/AcAPc2) is placed under 28 ℃ of envrionment conditionss of LB substratum and cultivates 15h, then adopt GST tag fusion protein purification process to carry out the separation and purification of albumen, the purity that present embodiment is used for the treatment of the fusion rotein of osteoporosis and thrombus is 98%, and the expression amount of fusion rotein is 38.4%.
The msCT-AcAPc2 fusion rotein that utilizes present embodiment e. coli bl21 (DE3-msCT/AcAPc2) fermentation to obtain is tested:
The experiment of msCT-AcAPc2 fusion rotein treatment osteoporosis
Get female sd inbred rats and extract bilateral ovaries under aseptic condition, after 12 weeks, get 32 of survival healthy rats, be divided at random 4 groups (positive controls 1, positive controls 2, fusion rotein experimental group and negative control group), 8 every group.Separately get female sd inbred rats excision bilateral one fritter fat, after 12 weeks, get at random 8 of survival healthy rats as Sham-operated control group.Amount to 5 groups, respectively oral following medicine:
Sham-operated control group: the CMC-Na solution that mass concentration is 0.5%, gavage dosage is 5ml/kg;
Negative control group: the CMC-Na solution that mass concentration is 0.5%, gavage dosage is 5ml/kg;
Fusion rotein experimental group: the msCT-AcAPc2 fusion rotein solution that mass concentration is 0.5%, gavage dosage is 5ml/kg; (the msCT-AcAPc2 fusion rotein sterilized water that is used for the treatment of osteoporosis and thrombus of acquisition dissolves)
Positive controls 1: alendronate sodium (Alen) 5mg/kg;
Positive controls 2: the msCT protein solution that mass concentration is 0.5%, gavage dosage is 5ml/kg.
Successive administration three months, gets rat femur head after execution, immerse in 4% glutaraldehyde fixing, femoral head sagittal plane is cut with dentistry diamond saw, get its a slice, through cleaning, 10% clorox soaks 6h, ultrasonic cleaning 15min, Gradient elution using ethanol, ether soaks, dry, ion sputtering film coating, SX-40 scanning electron microscopic observation, acceleration voltage 20kV.
Observe osteoporosis therapy contrast and experiment, experimental result is in table 1, and result shows that fusion rotein experimental group, positive controls 1 and positive controls 2 all have the osteoporotic effect for the treatment of, and the effect of fusion rotein experimental group is best.
The comparison of table 1 bone trabecula width and surface of bone amass (X ± SD)
| Group | n | Dosage | Bone trabecula width X ± SD | Bone trabecula area X ± SD |
| Sham-operated control group | 8 | 5ml/kg | 112.48±15.20 | 0.6901±0.0531 |
| Negative control group | 8 | 5ml/kg | 50.30±16.8 | 0.5649±0.0564 |
| Positive controls 1 | 8 | 5ml/kg | 112.35±14.7 | 0.7078±0.0510 |
| Positive controls 2 | 8 | 5ml/kg | 116.24±14.3 | 0.7121±0.0457 |
| Fusion rotein experimental group | 8 | 5ml/kg | 125.95±13.7 | 0.7385±0.0429 |
It is better that fusion rotein (msCT-AcAPc2) is treated osteoporosis effect by intravenous administration.
The anti-rat suppository experiment of msCT-AcAPc2 fusion rotein:
Get 48 of SD rats, be divided at random 6 groups, 8 every group, blank group, control group 1 (positive drug), control group 2 (AcAPc2 polypeptide) and the basic, normal, high dosage group of msCT-AcAPc2 fusion rotein.Control group 1 is selected heparin sodium injection (dosage is 1650 U/kg), control group 2 (dosage is 200 μ g/kg), the dosage of the basic, normal, high dosage group of msCT-AcAPc2 fusion rotein (dosage 5 times increase progressively) is respectively 40 μ g/kg, 200 μ g/kg, 1 mg/kg, and blank group gives the physiological saline of same volume.All, by intravenous administration, after intravenous administration, immediately the carotid artery of separator well is placed in the detection joint of YLS-14B instrument for generating thrombus in small animal, starts thrombus instrument for generating, with constant current galvanic current stimulation (1 mA).Record probe is measured the circulation of blood and is conversed blood vessel blockage degree (timed interval of measurement is 4 s, is expressed as a percentage data, and all processes continues 5 min) by infrared scan.
Observe antithrombotic therapy experimental result, experimental result is in table 2, and result shows that the carotid artery chocking-up degree of blank group all use obvious difference with other five groups.
The effect of table 2 msCT-AcAPc2 fusion rotein Chinese People's Anti-Japanese Military and Political College Rat common carotid thrombus [n, (± s) %]
| Group | n | Dosage | Carotid chocking-up degree (%) |
| Blank group | 8 | 0μg/kg | 100.00±0.00 |
| Control group 1 | 8 | 1650U/kg | 48.47±42.65 |
| Control group 2 | 8 | 200μg/kg | 32.99±22.07 |
| Fusion rotein low dose group | 8 | 40μg/kg | 39.29±42.13 |
| Dosage group in fusion rotein | 8 | 200μg/kg | 25.39±40.06 |
| Fusion rotein high dose group | 8 | 1mg/kg | 11.88±12.21 |
MsCT-AcAPc2 fusion rotein toxicity test:
Harbin Medical University's Experimental Animal Center provides SPF level kunming mice.Get mouse that 60 6 week age, male and female half and half, weights are 18 ± 2g as experimental subjects.Adopt maximum tolerance administration (according to clinical Interferon, rabbit consumption 50ug/kg).Experimental group injection total dose 1mg/kg, once daily 0.02mL.Control group mice injection Isodose physiological saline.
Administration process small mouse is acted normally, feed, drinking-water is normal, urine excrement is normal, hair color along white and glossy, activity freely, between reactive good, mouse without mutually baiting phenomenon.Raise 14 days and 30 days continuously, mouse does not all occur dead.
The administration of msCT-AcAPc2 fusion rotein is after 14 days, 30 days, put to death mouse, pluck eyeball and get blood, 3000r/min, centrifugal 5 min, draw serum, use Beckman automatic clinical chemistry analyzer to detect Main Biochemical: aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), blood urea nitrogen (BUN), uric acid (URCA), total bilirubin (TBIL), TOTAL BILE ACID (TBA).Blood biochemistry index is as shown in table 3.
Table 3
The administration of msCT-AcAPc2 fusion rotein is put to death mouse after 14 days, 30 days, cores, liver, spleen, lung, kidney, observes internal organs color, form, calculates each organ coefficient.Estimate according to statistics sample, in every group, randomly draw 7 mouse, core, liver, spleen, lung, kidney HE dyeing does histopathologic examination; Get myeloid tissue Wright's staining observation of cell and have or not oedema, sex change, necrosis etc.The pathological observation result of main organs is as shown in table 4.
Table 4
| Group | Number of days | The heart | Liver | Spleen | Lung | Kidney |
| Control group | 14 | 3.57±0.64 | 32.52±2.30 | 4.45±0.70 | 2.75±0.71 | 7.73±0.86 |
| Experimental group | 14 | 3.51±0.62 | 30.98±3.25 | 4.56±0.62 | 2.93±0.45 | 7.75±0.84 |
| Control group | 30 | 3.62±0.60 | 32.87±2.23 | 4.57±0.58 | 2.90±0.60 | 7.86±0.79 |
| Experimental group | 30 | 3.53±0.63 | 31.23±3.16 | 4.48±0.63 | 2.93±0.71 | 7.85±0.77 |
The HE dyeing of experimental group and the control group main organs heart, liver, spleen, lung, kidney and marrow Wright dyeing paired observation are found: respectively organize the heart, liver, spleen, lung, kidney and the marrow of mouse all without abnormal changes such as oedema, sex change, necrosis.
Pathological section and the bone marrow smear of this experiment to the heart, spleen, lung, kidney observed, and is showed no obvious damaging change.The organ coefficient statistical analysis of 5 kinds of internal organs is shown to each group difference is without significance.All illustrate that msCT-AcAPc2 fusion rotein and meta-bolites thereof do not produce organic lesion to internal organs.The each group difference of Biochemistry test is without significantly illustrating that msCT-AcAPc2 fusion rotein is to liver, the infringement of kidney non-functional.Table msCT-AcAPc2 fusion rotein msCT-AcAPc2 fusion rotein good biocompatibility, to mouse without acute toxicity, long term toxicity.
What Ancylostoma caninum anticoagulant hemepeptide (anticoagulant peptide, AcAPs) was applied at present has AcAP5, AcAP6 and tri-kinds of recombinant proteins of AcAPc2.Present embodiment is selected AcAPc2 and has been removed its original signal peptide sequence, formed by 83 amino acid, AcAPc2 is the efficient special inhibitor of Xa factor, its antithrombotic effect is obviously better than thrombin inhibitors, AcAPc2 is very little on platelet aggregation impact as the efficient special inhibitor of Xa factor, causes hemorrhage danger little during for antithrombotic therapy.
Present embodiment is transformed natural salmon calcitonin see calcimar (salmon calcitonin, sCT), improved msCT ［ Gly
8, Ala
16, del-Tyr
22(α-amino-isovaleric acid that sCT is the 8th becomes glycine, and the leucine of the 16th is replaced with L-Ala, deletes the tyrosine of the 22nd), the biological activity of improved msCT can reach 8600IU/mg.
The msCT-AcAPc2 fusion rotein that present embodiment obtains inserts GlyThr between msCT and Ancylostoma caninum anticoagulant hemepeptide, has changed the secondary structure of polypeptide, but has not only made AcAPc2 and msCT loss of biological activity, has improved on the contrary its biological activity.The expression amount of the interior msCT-AcAPc2 fusion rotein of present embodiment e. coli bl21 (DE3-msCT/AcAPc2) is also high than single AcAPc2 or the expression amount of msCT in intestinal bacteria.
Present embodiment fusion rotein can carry out administration by injection or oral mode, does not have first pass effect.
While building present embodiment e. coli bl21 (DE3-msCT/AcAPc2), remove the original signal peptide of AcAPc2 gene, change the secondary structure of original natural salmon calcitonin see calcimar (sCT) and Ancylostoma caninum anticoagulant hemepeptide AcAPc2, this change does not produce toxicity in vivo, and the fusion rotein that fermentation produces has security; And the change of secondary structure do not affect chromatography and purifying, utilize present embodiment e. coli bl21 (DE3-msCT/AcAPc2) fermentation msCT-AcAPc2 fusion rotein to there is separation and purification and be easy to feature.
Medicine, reagent, enzyme, competent cell and the plasmids etc. that use in present embodiment are all bought acquisition, if without particular requirement concentration be product annotation concentration.
AcAPc2 is by the fVIIa/TF mixture performance anticoagulation of anticoagulant approach, present embodiment e. coli bl21 (DE3-msCT/AcAPc2) the fusion rotein long half time (being greater than 50h) in human body prepared that ferments, and be combined with high-affinity with people fX or fXa, form stable mixture (Kd=640pM), particularly thereby msCT-AcAPc2 fusion rotein can be combined with the proenzyme fX of fXa and be made more than transformation period of AcAPc2 reaches 50 h in human body, can for a long time effectively bring into play anti thrombotic action.Simultaneously present embodiment e. coli bl21 (DE3-msCT/AcAPc2) the msCT-AcAPc2 fusion rotein of preparing that ferments also can be used for treating tumour, septicemia, Ebola virus hemorrhagic fever etc.
Claims (2)
1.msCT-AcAPc2 fusion rotein transgenic engineering bacterial strain, it is characterized in that msCT-AcAPc2 fusion rotein transgenic engineering bacterial strain is called e. coli bl21 (DE3)-msCT/AcAPc2, e. coli bl21 (DE3)-msCT/AcAPc2 is prepared according to the following steps:
One,, by BamH I and EcoR I double digestion for the plasmid vector pUC-msCT/AcAPc2 that contains msCT-AcAPc2 antigen-4 fusion protein gene, obtain the DNA fragmentation of msCT-AcAPc2 fusion rotein;
Two, with BamHI and EcoR I double digestion plasmid pGEX-6P-1;
Three, the DNA fragmentation of msCT-AcAPc2 fusion rotein with carry out enzyme through the vector pGEX-6P-1 of double digestion and be connected, then place to connect and spend the night for 16 ℃, obtain vector pGEX-msCT/AcAPc2;
Four, transform e. coli bl21 (DE3) with vector pGEX-msCT/AcAPc2, select positive recombinant, obtain msCT-AcAPc2 fusion rotein transgenic engineered bacteria e. coli bl21 (DE3)-msCT/AcAPc2;
Wherein, in step 1 the gene order of msCT-AcAPc2 fusion rotein as shown in SEQ ID NO:1.
2. msCT-AcAPc2 fusion rotein transgenic engineering bacterial strain according to claim 1, is characterized in that the aminoacid sequence of msCT-AcAPc2 fusion rotein in step 1 is as shown in SEQ ID NO:2.
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