CN102268423A - Radioactive rhenium (Re) labeled human plasminogen kringle5 protein and preparation method thereof - Google Patents

Radioactive rhenium (Re) labeled human plasminogen kringle5 protein and preparation method thereof Download PDF

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Publication number
CN102268423A
CN102268423A CN2010101930531A CN201010193053A CN102268423A CN 102268423 A CN102268423 A CN 102268423A CN 2010101930531 A CN2010101930531 A CN 2010101930531A CN 201010193053 A CN201010193053 A CN 201010193053A CN 102268423 A CN102268423 A CN 102268423A
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rhk5
preparation
carboxyl terminal
expression
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李彪
曹本红
张岚
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Shanghai Atom Kexing Pharmaceuticals Co ltd
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Shanghai Institute of Applied Physics of CAS
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Shanghai Atom Kexing Pharmaceuticals Co ltd
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Shanghai Institute of Applied Physics of CAS
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Abstract

The invention discloses a <188>Re-rhk5 (recombinant human plasminogen kringle5) and a preparation method thereof. A tricarbonyl rhenium labeled method adopts histidine as a bifunctional chelating agent, and rhk5 which is indirectly labeled is not reacted with a disulfide bond, so the activity of the rhk5 protein is reserved. The rhk5 which is labeled by a nuclide <188>Re is affected by beta-rays and crossfire of the nuclide, so the antineoplastic blood vessel generation effect is enhanced, the medicament consumption is reduced, and targeting insufficiencies are offset.

Description

A kind of radioactive rhenium marked human plasminogen kringle5 albumen and preparation method thereof
[technical field]
The present invention relates to the biological medicine technology field, more particularly, relate to a kind of radioactive marker protein and preparation method thereof and this radioactive marker protein's application.
[background technology]
The medicine that at present with the tumor vessel is target spot in the world is existing multiple, has manyly entered the clinical trial or the clinical application that goes through.Entered clinical in 2005 by the FDA approval as Endostatin (Endostatin); Angiostatin (angiostatin) has also entered the clinical trial I phase.
Cao etc. have found that (Recombinant HumanPlasminogen Kringle5, rhk5) as angiogenesis inhibitor, each side such as activity are better than said medicine to recombinant human plasminogen Kringle 5, have the potential using value.
The isotope labeling angiogenesis inhibitor can reach the effect that strengthens neoplasm targeted therapy by the β ray, reduces the consumption of angiogenesis inhibitor, reduces cost.External at present existing similar research is as uses such as Yang 99mThe experimental study of Tc mark Endostatin shows that tumour can clear video picture; Uses such as Lee 123The Agiostatin of I mark has obtained same effect.
Have not yet to see and adopt radionuclide with good physico-chemical property 188The Re mark has the research report of the research of the inhibiting rhk5 of stronger tumor vessel, particularly treatment.Endostatin and Angiostatin etc. are if will reach result of treatment need continue medication, and the expense costliness. 99mThe experimental study of Tc mark Endostatin shows that tumour can clear video picture, but can not be used for the treatment of; Uses such as Lee 123The Agiostatin of I mark has obtained same effect, but the mark instability.The direct labelled protein of nucleic regular meeting causes the damage of protein-active.
[summary of the invention]
First purpose of the present invention provides a kind of antitumor drug efficiently 188Re-rhk5, i.e. nucleic 188The Re mark be recombined into plasminogen Kringle 5 albumen.
Second purpose of the present invention provides 188The preparation method of Re-rhk5.
The 3rd purpose of the present invention provides 188The application of Re-rhk5 in suppressing tumor growth.
For achieving the above object, the present invention discloses following technical scheme:
A kind of isotope labeling rhk5 albumen is characterized in that described nucleic is 188Re.
188The preparation method of Re-rhk5 comprises step (1) and step (2):
The proteic expression of step (1) rhk5 forms the fusion rotein that carboxyl terminal connects 6 * HisTag, can preferably obtain by following dual mode:
With Kringle 5 gene fragments be built into prokaryotic expression carrier plasmid pET-22b (+) → aminoterminal add signal peptide sequence → carboxyl terminal add 6 * His sequence → when pET-22b (+)-k5 after the BL21-CodonPlus competence bacteria is induced, in rhk5 aminoterminal expression signal peptide section, and under well-oxygenated environment, form disulfide linkage, excise signal peptide simultaneously, form the fusion rotein that carboxyl terminal connects 6 * HisTag.
Perhaps, Kringle 5 gene fragments are built into eukaryotic expression plasmid pFastbacl → carboxyl terminal add 6 * His sequence → after pFastbacl-rhk5 is hatched in the insect sf9 of 27 ℃ of cultivations cell, express rhk5 albumen in the solubility mode, and under well-oxygenated environment, form disulfide linkage, form the fusion rotein that carboxyl terminal connects 6 * HisTag.
The core procedure of step (2) three rhenium carbonyl mark rhk5:
1) gets 5mg BH 3NH 3Put into dry cillin bottle, seal, the logical about 20min of CO gas.
2) at 1mL 188ReO 4-The physiological saline leacheate in, add the dense H of 7 μ L 3PO 4
3) be expelled in the cillin bottle of the CO that had friendly relations 70 ℃ of heating in water bath 15min behind the mixing.
4) the intermediate fac-[that generates behind the assaying reaction 188Re[(CO) 3(H 2O) 3] +Chelation percent.
5) three rhenium carbonyl mark rhk5, the flag condition after the optimization is: rhk520 μ g adds 0.1mL 188ReO 4-Leacheate, the pH value of regulator solution is 5.0, fully behind the mixing, puts incubation 1h in 50 ℃ of water-baths.
The intermediate fac-[that generates behind the assaying reaction in the step (2) 188Re[(CO) 3(H 2O) 3] +Chelation percent, be stationary phase with the GF254 silica gel thin sheet, V (methyl alcohol): V (concentrated hydrochloric acid)=99: 1 is a moving phase, measures on the radioactivity thin layer chromatography scanner.
Using the pH value of phosphoric acid buffer regulator solution in the step (2) is 5.0.
Biodistribution research shows tumor tissues in the tumor bearing nude mice body 188The Re-rhk5 picked-up prolongs in time and increases gradually, and is the highest when 2h; Rhk5 can gather at the new vessel dense part, and it is feasible carrying out tumor imaging with radioisotope labeling rhk5; Preliminary treatment experiment shows single intratumor injection 37MBq 188Re-rhk5 has the radiotherapy effect to tumour and tumor vessel, and inhibitory rate 37.2% after 18 days.
Positively effect of the present invention: (1) three rhenium carbonyl labelling method adopts Histidine as bifunctional chelating agent, and indirect labelling rhk5 with the disulfide linkage effect, has not kept the rhk5 protein-active; (2) nucleic 188Re mark rhk5, β ray and cross fire effect by nucleic not only strengthen the antineoplastic vascular nucleus formation, have reduced drug dose, and have remedied the deficiency of target; (3) 188Re-rhk5 can carry out video picture, monitoring therapeuticing effect with the gamma-rays of its emission when using beta ray therapy.
[description of drawings]
Fig. 1 intermediate fac-[ 188Re (CO) 3(H 2O) 3] +Synthetic and with radiation thin plate chromatography (RTLC) evaluation;
Marked product RTLC analytical results in 9g/L physiological saline expansion system before Fig. 2 purifying;
Marked product RTLC analytical results in 9g/L physiological saline expansion system behind Fig. 3 purifying;
Marked product is at ethanol behind Fig. 4 purifying: ammonia: water (2: 1: RTLC analytical results in 5V/V/V) the expansion system;
Fig. 5 188Re-rhk5 is in the intravital tissue distribution of lung cancer transplanted tumor nude mice (n=3);
Fig. 6 188Histology before and after Re-rhk5 blocks in lung cancer transplanted tumor nude mouse distributes (2h) (n=3);
Fig. 7: 188Re-rhk5 is at the intravital video picture figure of lung cancer transplanted tumor nude mice: A, B are respectively the video picture figure of each time point; A, 0.5h; B, 2h; Arrow indication position is a tumour;
Fig. 8 188Re-rhk5 treatment back tumor growth suppresses curve: 188Re-rhk5 (37MBq), 188Re (37MBq), rhk5 (15mg/kg) and 0.9% physiological saline are treated the change of tumour size in back 18 days, and 6 every group, * represents P＜0.05, * * represent P＜0.01 ( 188Re-rhk5 treatment group and all the other each groups are relatively).
[embodiment]
Below in conjunction with accompanying drawing the present invention is elaborated.
Preparation embodiment 1
(1) the proteic expression of rhk5: with Kringle 5 gene fragments be built into prokaryotic expression carrier plasmid pET-22b (+) → aminoterminal add signal peptide sequence → carboxyl terminal add 6 * Hi s sequence → when pET-22b (+)-k5 after the BL21-CodonPlus competence bacteria is induced, can be in rhk5 aminoterminal expression signal peptide section, protein product can be secreted into the inside and outside intermembranous outer pericentral siphon of intestinal bacteria by signal peptide, and under well-oxygenated environment, form disulfide linkage, formation has the soluble fusion protein of function, excise signal peptide simultaneously, make it near native protein.Carboxyl terminal connects the fusion rotein of 6 * HisTag, utilizes HisTag not only to can be used to carry out ni-sepharose purification, and is that proteinic western blot identifies and follow-up nucleic three rhenium carbonyl indirect method mark rhk5 lay a good foundation.
(2) three rhenium carbonyl mark rhk5: get 5mg BH 3NH 3Put into dry cillin bottle, seal, the logical about 20min of CO gas → at 1mL 188ReO 4-The physiological saline leacheate in, add the dense H of 7 μ L 3PO 4Be expelled to behind → the mixing in the cillin bottle of the CO that had friendly relations, the intermediate fac-[that generates behind thin plate chromatography (RTLC) assaying reaction is radiated in 70 ℃ of heating in water bath 15min → employings 188Re[(CO) 3(H 2O) 3] +Chelation percent, be stationary phase with the GF254 silica gel thin sheet, V (methyl alcohol): V (concentrated hydrochloric acid)=99: 1 is a moving phase, measures on the radioactivity thin layer chromatography scanner.As shown in Figure 1, radiation thin plate chromatography is identified and is shown intermediate fac-[ 188Re (CO) 3(H 2O) 3] +Synthetic back purity very high (peak of front), inclusion-free almost can directly be used for carrying out next step experiment.→ adopting the flag condition after orthogonal experiment draws optimization: rhk520 μ g adds 0.1mL 188ReO 4-Leacheate (37-370MBq) is 5.0 with the pH value of phosphoric acid buffer regulator solution, fully behind the mixing, puts incubation 1h in 50 ℃ of water-baths.Marked product through the RTLC analytical results as shown in Figure 1, mark rate can reach more than 65%, radiochemical purity can reach more than 95%, as shown in Figure 3, Figure 4.
Preparation embodiment 2
(1) the proteic expression of rhk5: Kringle 5 gene fragments are built into eukaryotic expression plasmid pFastbacl → carboxyl terminal add 6 * His sequence → after pFastbacl-rhk5 is hatched in the insect sf9 of 27 ℃ of cultivations cell, can express rhk5 albumen in the solubility mode, and under well-oxygenated environment, form disulfide linkage, formation has the soluble fusion protein of function, carboxyl terminal connects the fusion rotein of 6 * HisTag, utilize HisTag not only to can be used to carry out ni-sepharose purification, and be that proteinic western blot identifies and follow-up nucleic three rhenium carbonyl indirect method mark rhk5 lay a good foundation.The rhk5 protein-active of eukaryotic expression is higher, but output is lower.Still select for use the method for prokaryotic expression to be more suitable for during mass preparation.
The method of (2) three rhenium carbonyl mark rhk5 is with embodiment 1.
Application Example 1
Biodistribution research shows tumor tissues in the tumor bearing nude mice body 188The Re-rhk5 picked-up prolongs in time and increases gradually, and is the highest when 2h; Rhk5 can gather at the new vessel dense part, and it is feasible carrying out tumor imaging with radioisotope labeling rhk5; Preliminary treatment experiment shows single intratumor injection 37MBq 188Re-rhk5 has the radiotherapy effect to tumour and tumor vessel, and inhibitory rate 37.2% after 18 days.
188Re-rhk5 after the single dose intravenous injection, as shown in Figure 2, 188Re-rhk5 removes in blood comparatively fast.Kidney is compared with other organ-tissues, in different time sections the picked-up of tangible high radioactivity is arranged, and is because due to marker mainly drains by urinary system.All the other internal organs radioactive uptakes are relatively low, prolong in time and absorb gradually and to lower.Tumor group is woven with high relatively picked-up, and opposite with other internal organs, in 0-2h, prolongs in time and absorbs and increase gradually, and the residence time is longer in tumour.In the experiment of subsequently specific inhibition in 18830min intravenous injection 100 μ g rhk5 before the Re-rhk5 video picture, 2h puts to death nude mice after injection, the measure of spread result of each internal organs as shown in Figure 3: tumor locus 188Re-rhk5 absorbs decline.Confirmed 188Re-rhk5 and tumour bonded specificity.All the other internal organs are not seen considerable change before and after being distributed in blocking-up.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (7)

1. an isotope labeling rhk5 albumen is characterized in that described nucleic is 188Re.
2. as claimed in claim 1 188The preparation method of Re-rhk5 is characterized in that, comprises the steps:
(1) the proteic expression of rhk5 forms the fusion rotein that carboxyl terminal connects 6 * HisTag;
(2) three rhenium carbonyl mark rhk5:
1) gets 5mg BH 3NH 3Put into dry cillin bottle, seal, the logical about 20min of CO gas;
2) at 1mL 188ReO 4-The physiological saline leacheate in, add the dense H of 7 μ L 3PO 4
3) be expelled in the cillin bottle of the CO that had friendly relations 70 ℃ of heating in water bath 15min behind the mixing;
4) the intermediate fac-[that generates behind the assaying reaction 188Re[(CO) 3(H 2O) 3] +Chelation percent;
5) three rhenium carbonyl mark rhk5, the flag condition after the optimization is: rhk520 μ g adds 0.1mL 188ReO 4-Leacheate, the pH value of regulator solution is 5.0, fully behind the mixing, puts incubation 1h in 50 ℃ of water-baths.
3. according to claim 2 188The preparation method of Re-rhk5 is characterized in that, the proteic expression of step (1) rhk5 comprises the steps:
With Kringle 5 gene fragments be built into prokaryotic expression carrier plasmid pET-22b (+) → aminoterminal add signal peptide sequence → carboxyl terminal add 6 * His sequence → when pET-22b (+)-k5 after the BL21-CodonPlus competence bacteria is induced, in rhk5 aminoterminal expression signal peptide section, and under well-oxygenated environment, form disulfide linkage, excise signal peptide simultaneously, form the fusion rotein that carboxyl terminal connects 6 * HisTag.
4. according to claim 2 188The preparation method of Re-rhk5 is characterized in that, the proteic expression of step (1) rhk5 comprises the steps:
Kringle 5 gene fragments are built into eukaryotic expression plasmid pFastbacl → carboxyl terminal add 6 * His sequence → after pFastbacl-rhk5 is hatched in the insect sf9 of 27 ℃ of cultivations cell, express rhk5 albumen in the solubility mode, and under well-oxygenated environment, form disulfide linkage, form the fusion rotein that carboxyl terminal connects 6 * HisTag.
5. according to claim 2 188The preparation method of Re-rhk5 is characterized in that, the intermediate fac-[that generates behind the assaying reaction in the step (2) 188Re[(CO) 3(H 2O) 3] +Chelation percent, be stationary phase with the GF254 silica gel thin sheet, V Methyl alcohol: V Concentrated hydrochloric acidBe moving phase at=99: 1, measures on the radioactivity thin layer chromatography scanner.
6. according to claim 2 188The preparation method of Re-rhk5 is characterized in that, using the pH value of phosphoric acid buffer regulator solution in the step (2) is 5.0.
7. as claimed in claim 1 188The application of Re-rhk5 in suppressing tumor growth.
CN2010101930531A 2010-06-04 2010-06-04 Radioactive rhenium (Re) labeled human plasminogen kringle5 protein and preparation method thereof Pending CN102268423A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114206398A (en) * 2020-04-30 2022-03-18 霍伯生物技术公司 Visualization of HER2 expression in human patients

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CN101423546A (en) * 2008-09-09 2009-05-06 中国科学院上海应用物理研究所 Radioactive rhenium marked polypeptide containing RGD sequence as well as preparation method and application thereof

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Title
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114206398A (en) * 2020-04-30 2022-03-18 霍伯生物技术公司 Visualization of HER2 expression in human patients

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Application publication date: 20111207