METHOD FOR EXTRACTION AND PURIFICATION OF HIRUDIN MUTANT AND USE THEREOF

Abstract

Provided are a method for extracting a hirudin mutant, HV2-Lys47, from a fermentation broth of the hirudin mutant HV2-Lys47 produced by the fermentation of Escherichia coli, and purifying same, the use of the hirudin mutant HV2-Lys47 in an anticoagulated blood collection tube, and an anticoagulated blood collection tube containing the hirudin mutant HV2-Lys47. In the purification method, a membrane technology and a primary column chromatography technology are used to obtain the hirudin mutant HV2-Lys47.

Claims

1. A method for extracting and purifying hirudin mutant HV2-Lys47 (SEQ ID NO.: 1) from a fermentation broth of Escherichia coli for fermentative production of hirudin mutant HV2-Lys47, comprising the steps of: treating the fermentation broth of Escherichia coli at an elevated temperature to sterilize the fermentation broth and remove impurity proteins; treating the sterilized fermentation broth by a ceramic membrane or by a centrifuge to remove the bacteria; optionally, treating the resultant fermentation broth from the ceramic membrane or the centrifuge by an ultrafiltration membrane to remove impurity proteins; treating the resultant fermentation broth from the ceramic membrane or the centrifuge, or the resultant fermentation broth from the ultrafiltration membrane by a nanofiltration membrane to obtain a concentrated solution of crudely separated hirudin mutant HV2-Lys47; adding a first salt into the concentrated solution of crudely separated hirudin mutant HV2-Lys47 as an auxiliary material, followed by spray drying to obtain dry powder; dissolving the dry powder in water and filtering out impurities, optionally by filter paper, a sand core funnel or a membrane device; subjecting the filtrate to molecular sieve column chromatography only once, eluting the same with water, and concentrating the collected solution to obtain a crude hirudin mutant HV2-Lys47; and dissolving the crude hirudin mutant HV2-Lys47 in water, adding a second salt or an organic solvent thereto to precipitate the hirudin mutant HV2-Lys47, and drying, optionally vacuum drying, or more optionally vacuum lyophilizing the same to obtain a pure hirudin mutant HV2-Lys47.

2. The method according to claim 1, wherein the treatment at high temperature is 65° C.-67° C. for 5-20 min, optionally for 10 min, and the fermentation broth sterilized at high temperature is treated by a centrifuge, optionally, the centrifuge is a tube centrifuge or a disk centrifuge.

3. (canceled)

4. The method according to claim 1, wherein the first salt or the second salt is selected from one or more of sodium chloride, potassium chloride, ammonium sulfate, sodium sulfate, potassium sulfate, the weight in grams of the first salt added as an auxiliary material is 5% or more of the volume in milliliters of the concentrated solution of crudely separated hirudin mutant HV2-Lys47, the weight in grams of the second salt added is 20%-30% of the volume in milliliters of the aqueous solution in which the crude hirudin mutant HV2-Lys47 is dissolved.

5. The method according to claim 1, wherein the molecular sieve column chromatography is selected from the group consisting of Sephadex G25, Sephadex G50, Sephadex G75 and Sephadex G100.

6. The method according to claim 1, wherein concentrating the collected solution resulting from the molecular sieve column chromatography is conducted by vacuum concentration under reduced pressure, nanofiltration membrane concentration or reverse osmosis membrane concentration.

7. The method according to claim 1, wherein the organic solvent is selected from one or more of ethanol, methanol, acetone, isopropanol or acetonitrile, and the volume of the organic solvent added is 5-9 volumes of the aqueous solution in which crude hirudin mutant HV2-Lys47 is dissolved.

8. (canceled)

9. The method according to claim 1, wherein the volume in milliliters of water for dissolving the dry powder is 10 times or more of the weight in grams of the dry powder.

10. (canceled)

11. The method according to claim 1, wherein the fermentation broth of Escherichia coli is produced by culturing the strain Escherichia coli pBH2 CGMCC No. 0908 with pBH-2 as an expression vector, optionally, the culturing includes culturing under aerobic conditions at temperature of 25° C.-35° C. until the end of the logarithmic growth phase, and then the temperature is elevated to 35° C.-45° C. for a further culturing until the yield of the hirudin mutant HV2-Lys47 reaches a peak.

12. A hirudin mutant HV2-Lys47, produced according to the method of claim 1.

13. The hirudin mutant HV2-Lys47 according to claim 12, wherein the anticoagulant activity of the hirudin mutant HV2-Lys47 is greater than or equal to 18000 ATU/mg.

14. Use of hirudin mutant HV2-Lys47 (SEQ ID NO.: 1) in an anticoagulant blood collection tube.

15. The use according to claim 14, wherein the anticoagulant activity of the hirudin mutant HV2-Lys47 is greater than or equal to 18000 ATU/mg.

16. The use according to claim 14, wherein the hirudin mutant HV2-Lys47 is directly added to the anticoagulant blood collection tube, or is applied to the anticoagulant blood collection tube by being contained in a coating.

17. (canceled)

18. The use according to claim 16, wherein the hirudin mutant HV2-Lys47 is used in an amount of 8.5 μg/ml blood.

19. The use according to claim 16, wherein the hirudin mutant HV2-Lys47 is coated onto the anticoagulant blood collection tube by being contained in a coating, and then the coating is dried by vacuum lyophilization.

20. The use according to claim 14, wherein the anticoagulant blood collection tube is used to contain blood, and the blood is used for one or more tests of routine blood tests, blood biochemistries, electrolytes, tumor markers, homocysteine and five items of HBV.

21. The use according to claim 14, wherein the hirudin mutant HV2-Lys47 is prepared from a fermentation broth of Escherichia coli for fermentative production of hirudin mutant HV2-Lys47 by a method comprising the steps of: treating the fermentation broth of Escherichia coli at an elevated temperature to sterilize the fermentation broth and remove impurity proteins; treating the sterilized fermentation broth by a ceramic membrane or by a centrifuge to remove the bacteria; optionally, treating the resultant fermentation broth from the ceramic membrane or the centrifuge by an ultrafiltration membrane to remove impurity proteins; treating the resultant fermentation broth from the ceramic membrane or the centrifuge, or the resultant fermentation broth from the ultrafiltration membrane by a nanofiltration membrane to obtain a concentrated solution of crudely separated hirudin mutant HV2-Lys47; adding a salt into the concentrated solution of crudely separated hirudin mutant HV2-Lys47 as an auxiliary material, followed by spray drying to obtain dry powder; dissolving the dry powder in water and filtering out impurities, optionally by filter paper, a sand core funnel or a membrane device; subjecting the filtrate to molecular sieve column chromatography only once, eluting the same with water, and concentrating the collected solution to obtain a crude hirudin mutant HV2-Lys47; and dissolving the crude hirudin mutant HV2-Lys47 in water, adding a salt or an organic solvent thereto to precipitate the hirudin mutant HV2-Lys47, and drying, optionally vacuum drying, or more optionally vacuum lyophilizing the same to obtain a pure hirudin mutant HV2-Lys47; or the hirudin mutant HV2-Lys47 is prepared by the method above and the anticoagulant activity of the hirudin mutant HV2-Lys47 is greater than or equal to 18000 ATU/mg.

22. An anticoagulant blood collection tube, wherein the anticoagulant blood collection tube has a hirudin mutant HV2-Lys47 (SEQ ID NO.: 1) directly added or is coated with a coating containing a hirudin mutant HV2-Lys47 (SEQ ID NO.: 1).

23. The anticoagulant blood collection tube according to claim 22, wherein the anticoagulant activity of the hirudin mutant HV2-Lys47 is greater than or equal to 18000 ATU/mg.

24. The anticoagulant blood collection tube according to claim 22, wherein the hirudin mutant HV2-Lys47 is a hirudin mutant HV2-Lys47 produced from a fermentation broth of Escherichia coli for fermentative production of hirudin mutant HV2-Lys47 by a method comprising the steps of: treating the fermentation broth of Escherichia coli at an elevated temperature to sterilize the fermentation broth and remove impurity proteins; treating the sterilized fermentation broth by a ceramic membrane or by a centrifuge to remove the bacteria; optionally, treating the resultant fermentation broth from the ceramic membrane or the centrifuge by an ultrafiltration membrane to remove impurity proteins; treating the resultant fermentation broth from the ceramic membrane or the centrifuge, or the resultant fermentation broth from the ultrafiltration membrane by a nanofiltration membrane to obtain a concentrated solution of crudely separated hirudin mutant HV2-Lys47; adding a salt into the concentrated solution of crudely separated hirudin mutant HV2-Lys47 as an auxiliary material, followed by spray drying to obtain dry powder; dissolving the dry powder in water and filtering out impurities, optionally by filter paper, a sand core funnel or a membrane device; subjecting the filtrate to molecular sieve column chromatography only once, eluting the same with water, and concentrating the collected solution to obtain a crude hirudin mutant HV2-Lys47; and dissolving the crude hirudin mutant HV2-Lys47 in water, adding a salt or an organic solvent thereto to precipitate the hirudin mutant HV2-Lys47, and drying, optionally vacuum drying, or more optionally vacuum lyophilizing the same to obtain a pure hirudin mutant HV2-Lys47; or the hirudin mutant HV2-Lys47 is prepared by the method above and the anticoagulant activity of the hirudin mutant HV2-Lys47 is greater than or equal to 18000 ATU/mg.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0062] The accompanying drawings are used to provide a further understanding of the technical solutions of the present application and form a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solutions of the present application and do not constitute a limitation on the technical solutions of the present application.

[0063] FIG. 1 is an electropherogram of hirudin mutant HV2-Lys47 prepared in Example 1.

[0064] FIG. 2 is an HPLC chromatogram of the hirudin mutant HV2-Lys47 prepared in Example 1, in which the peak with retention time of 18.991 min is the corresponding peak of the hirudin mutant HV2-Lys47.

[0065] FIG. 3 is an amino acid residue sequence diagram of the hirudin mutant HV2-Lys47.

[0066] FIG. 4 is the three-dimensional structural diagram of hirudin mutant HV2-Lys47 simulated on the biomolecular database webpage (https://www.rcsb.org/).

DETAILED DESCRIPTION

[0067] In order to make the objects, technical solutions and advantages of the present application clearer, the embodiments of the present application will be described below in detail with reference to the accompanying drawings. It should be noted that the embodiments in this application and the features in the embodiments may be combined with each other arbitrarily unless there is a conflict.

Example 1

[0068] Preparation of a Fermentation Broth:

[0069] Glycerol stocks of pBH-2 engineering bacteria (Deposit Number: CGMCC No. 0908) were inoculated into a fermentation medium at an inoculation amount of 5%, with a volume of 10 ml/100 ml Erlenmeyer flask, a culturing temperature of 30° C., and a rotation speed of 270 rpm. After inoculation, fermentation was carried out under aerobic conditions until the measured OD value of the fermentation broth tended to be stable, that is, the end of the logarithmic growth phase, which took 3.5 h; then the temperature was raised to 40° C. within 5 min and culturing was continued until a lighter color is shown when the bacteria was stained with crystal violet under microscope, which took 11.5 h, giving a fermentation period of 15 h.

[0070] As to the specific operations, see Example 2 of the application No. 201711262810.4. The medium was composed of the followings and had a pH of 7.2: glucose 10 g/L, sucrose 10 g/L, yeast powder 10 g/L, tryptone 10 g/L, ammonium chloride 0.5 g/L, magnesium sulfate 0.9 g/L, dipotassium hydrogen phosphate 1 g/L, sodium sulfate 5.0 g/L, sodium citrate 0.87 g/L, sodium chloride 16 g/L, vitamin B1 0.05 mg/L, trace elements-ferrous sulfate 40 g/L, aluminum sulfate 28 mg/L, manganese sulfate 6.1 mg/L, cobalt chloride 4 mg/L, zinc chloride 0.95 g/L, sodium molybdate 2.16 g/L, boric acid 0.5 mg/L, copper sulfate 2.93 g/L and nickel nitrate 32 g/L. Antibiotic substance AMP 0.5 g/L was added during inoculation. In this example and the following examples, L in g/L was the volume of the final fermentation medium.

[0071] The sources of the materials were as shown in the table below:

TABLE-US-00002 Material name Manufacturer Grade Glycerol Guangzhou Baojili Chemical Co., Ltd. Food grade Glucose Wujiang Jiancheng Fine Chemical Food grade Co., Ltd. Ammonium Guangzhou Chemical Reagent Factory AR grade nitrate Sucrose Guangzhou Chemical Reagent Factory AR Peptone OXOID AR Yeast powder OXOID AR Sodium Guangdong Chemical Reagent Factory AR chloride Magnesium Guangdong chemical reagent factory AR sulfate Ammonium Xilong Scientific Co., Ltd. AR chloride Disodium Guangdong Chemical Reagent Factory AR hydrogen phosphate Potassium Guangdong Chemical Reagent Factory AR dihydrogen phosphate Sodium Sinopharm Chemical Reagent Co. Ltd. AR sulfate Sodium Shanghai Lingfeng Chemical Reagent AR citrate Co., Ltd. Vitamin B1 Shanghai Aibo Trading Co., Ltd. BR Ferrous Guangdong Chemical Reagent Factory AR sulfate Aluminum Sinopharm Chemical Reagent Co. Ltd. AR sulfate Manganesium Guangdong Taishan Chemical Plant AR sulfate Cobalt Xilong Scientific Co., Ltd. AR chloride Zinc chloride Xilong Chemical Co., Ltd. AR Sodium Guangdong Chemical Reagent Factory AR molybdate Boric acid Guangdong Chemical Reagent Factory AR Copper Xilong Scientific Co., Ltd. AR sulfate Nickel nitrate Xilong Chemical Co., Ltd. AR AMP CSPC Zhongnuo Pharmacy Prescription (Shijiazhuang) Co., Ltd. drug of ampicillin sodium for injection

[0072] The hirudin mutant HV2-Lys47 was extracted and purified from the fermentation broth by the following steps:

[0073] 1) sterilizing the fermentation broth at a high temperature of 65° C. for 5 min to remove impurities;

[0074] 2) centrifuging the sterilized fermentation broth in step 1) by a tube centrifuge (Liaoyang Zhenxing Vacuum Equipment Factory GQB-770) to remove Escherichia coli;

[0075] 3) passing the supernatant of centrifugation in step 2) through an ultrafiltration membrane (Suntar Membrane Technology (Xiamen) Co., Ltd., Model: NFM-84S-6/3) to remove impurity proteins;

[0076] 4) concentrating the ultrafiltration dialysate in step 3) by passing through a nanofiltration membrane (Suntar Membrane Technology (Xiamen) Co., Ltd., Model: UFM-84S-2) to obtain a concentrated solution of crudely separated hirudin mutant HV2-Lys47;

[0077] 5) adding sodium chloride as an auxiliary material into the concentrated solution of crudely separated hirudin mutant HV2-Lys47 obtained in step 4), and spray drying the same to obtain dry powder, wherein the weight in grams of the salt added as an auxiliary material was 10% of the volume in milliliters of the concentrated solution of crudely separated hirudin mutant HV2-Lys47;

[0078] 6) dissolving the dry powder obtained in step 5) in water and filtering out impurities to obtain a filtrate, wherein the volume in milliliters of water for dissolving the dry powder was 10 times the weight in grams of the dry powder;

[0079] 7) purifying the filtrate in step 6) by molecular sieve column chromatography (Sephadex G50, manufacturer: General Electric Company (GE)) once, eluting the same with pure water, and vacuum concentrating the pooled eluants under reduced pressure; and

[0080] 8) adding 9 volumes of ethanol into the concentrated eluants obtained in step 7) to precipitate the hirudin mutant HV2-Lys47, and vacuum drying the precipitates under reduced pressure.

[0081] The hirudin mutant HV2-Lys47 prepared above was subjected to electrophoretic analysis. As shown in FIG. 1, a single band appeared in the resultant electropherogram (see the band corresponding to hirudin 1 in FIG. 1, wherein hirudin 1 represents hirudin mutant HV2-Lys47 prepared in the present application), and the molecular weight of the purified recombinant hirudin was demonstrated by electrophoresis to be consistent with the theoretical value.

[0082] The operating conditions of electrophoresis were: stacking gel 3%, separating gel 15%, loading volume of 30 uL (10,000 ATU/ml), running on gels first at a voltage of 100V for 1 h, then at a voltage of 200V for 2.2 h.

[0083] The specific operations of electrophoresis were as follows:

[0084] 1. installing a precast gel in a min electrophoresis apparatus, adding SDS-PAGE electrophoresis buffer to cover the top of the gel, and pulling the comb teeth out of sample wells;

[0085] 2. taking 40 uL of the sample, adding 10 uL of 5× protein loading buffer, mixing, then subjecting the sample in a water bath at 100° C. for 5 min, and taking 40 uL thereof and adding the same to the sample well on the gel;

[0086] 3. after the completion of sample loading, powering on the electrophoresis apparatus, setting a constant voltage of 150V for about 50 min, and ending the electrophoresis run when the bromophenol blue line reached the bottom of the gel; and

[0087] 4. fixing for 20 min, staining for 20 min, decolorizing for 2 h, and then observing the result.

[0088] According to the analysis of anticoagulant activity by biochemical method determination, the anticoagulant activity of the prepared hirudin mutant HV2-Lys47 was calculated to be 17000 ATU/mg. For the specific determination of anticoagulant activity, reference may be made to the left column of page 84 of the Chinese Pharmacopoeia (2005 edition). Moreover, the purity of the hirudin mutant HV2-Lys47 calculated according to the proportion of color bands in gel electrophoresis had reached to 95%.

[0089] In addition, it was demonstrated through HPLC determination that, there was indeed a significantly large peak of the hirudin mutant HV2-Lys47 in the prepared hirudin mutant HV2-Lys47, as shown in FIG. 2.

Example 2

[0090] Hirudin mutant HV2-Lys47 was prepared and purified by the method of Example 1, except that in step 2), Escherichia coli was removed by a disk centrifuge (Alfa laval stainless products skogstop: 881095-05-S/01), and that in step 5), the weight in grams of the salt added as an auxiliary material was 8% of the volume in milliliters of the concentrated solution of crudely separated hirudin mutant HV2-Lys47.

[0091] The resultant hirudin mutant HV2-Lys47 was subjected to electrophoretic analysis, and the results were the same as those shown in FIG. 1, in which there was only shown a single band in the electropherogram.

[0092] The anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was tested to be 15800 ATU/mg, and the purity was about 93%.

Example 3

[0093] Hirudin mutant HV2-Lys47 was prepared and purified by the method of Example 1, except that in step 2), Escherichia coli was removed by a ceramic membrane (Suntar Membrane Technology (Xiamen) Co., Ltd.).

[0094] The resultant hirudin mutant HV2-Lys47 was subjected to electrophoretic analysis, and the results were the same as those shown in FIG. 1, in which there was only shown a single band in the electropherogram.

[0095] The anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was tested to be 16800 ATU/mg, and the purity was 93% or more.

Example 4

[0096] Recombinant hirudin was extracted and purified by the method of Example 1, except that in step 2), Escherichia coli was removed by a hollow fiber membrane (Suntar Membrane Technology (Xiamen) Co., Ltd.).

[0097] The resultant hirudin mutant HV2-Lys47 was subjected to electrophoretic analysis, and the results were the same as those shown in FIG. 1, in which there was only shown a single band in the electropherogram.

[0098] The anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was tested to be 16000 ATU/mg, and the purity was 93% or more.

Example 5

[0099] Recombinant hirudin was purified by the method of Example 1, except that in step 8), 8 volumes of methanol was added for precipitation.

[0100] The resultant hirudin mutant HV2-Lys47 was subjected to electrophoretic analysis, and the results were the same as those shown in FIG. 1, in which the hirudin mutant HV2-Lys47 was shown as a single band in the electropherogram.

[0101] The anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was tested to be 15800 ATU/mg, and the purity was about 93%.

Example 6

[0102] Recombinant hirudin was extracted and purified by the method of Example 1, and in step 8), 5 volumes of acetone was added for precipitation.

[0103] The resultant hirudin mutant HV2-Lys47 was subjected to electrophoretic analysis, and the results were the same as those shown in FIG. 1, in which the hirudin mutant HV2-Lys47 was shown as a single band in the electropherogram.

[0104] The anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was tested to be 17000 ATU/mg, and the purity was 95%

Example 7

[0105] Recombinant hirudin was extracted and purified by the method of Example 1, except that in step 8), ammonium sulfate with saturation of 75% was used for precipitation, wherein the weight in grams of ammonium sulfate was 20% of the volume in milliliters of the aqueous solution in which the crude hirudin mutant HV2-Lys47 was dissolved.

[0106] The resultant hirudin mutant HV2-Lys47 was subjected to electrophoretic analysis, and the results were the same as those shown in FIG. 1, in which there was only shown a single band in the electropherogram.

[0107] The anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was tested to be 15000 ATU/mg.

Example 8

[0108] Recombinant hirudin was extracted and purified by the method of Example 1, except that the sterilized fermentation broth was directly subjected to separation by a ceramic membrane to remove Escherichia coli from the fermentation broth, and the dialysate of the ceramic membrane was then treated by a nanofiltration membrane once, and that in step 8), sodium chloride was used for precipitation, wherein the weight in grams of sodium chloride was 30% of the volume in milliliters of the aqueous solution in which the crude hirudin mutant HV2-Lys47 was dissolved.

[0109] The resultant hirudin mutant HV2-Lys47 was subjected to electrophoretic analysis, and the results were the same as those shown in FIG. 1, in which there was only shown a single band in the electropherogram.

[0110] The anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was tested to be 14500 ATU/mg.

Example 9

[0111] Recombinant hirudin was extracted and purified by the method of Example 1, except that in step 8), 9 times volume of isopropanol was used to precipitate hirudin mutant HV2-Lys47.

[0112] The resultant hirudin mutant HV2-Lys47 was subjected to electrophoretic analysis, and the results were the same as those shown in FIG. 1, in which there was only shown a single band in the electropherogram.

[0113] The anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was tested to be 18000 ATU/mg, and the purity was 98%.

Example 10

[0114] Effects of drying process on the activity of hirudin mutant HV2-Lys47 in anticoagulant blood collection tubes were studied.

[0115] The hirudin prepared in Example 9 was dissolved in sterile pure water with a dissolution concentration of 3.4 g/100 ml pure water. 2 ml blood collection tubes were used, and each of them was sprayed with the solution of the prepared hirudin mutant HV2-Lys47 by needle spray, and then dried.

[0116] The common drying process in the art is high temperature drying: drying three times at 45° C., 50° C. and 70° C., three times for each temperature, and 60 seconds each time. The drying method in the present application is: drying the sprayed blood collection tubes in a vacuum lyophilizer at a temperature of −50° C. and a vacuum degree of −4 Mpa.

[0117] The effects of high temperature drying and vacuum lyophilization methods on the activity of hirudin mutant HV2-Lys47 were as shown in Table 1 below.

TABLE-US-00003 TABLE 1 Content of Activity hirudin mutant after Activity HV2-Lys47 Drying method drying loss 306ATU/tube Drying three times at 45° C., 276  9.8% 60 seconds each time Drying three times at 50° C., 245 19.9% 60 seconds each time Drying three times at 70° C., 160 47.7% 60 seconds each time Drying for 40 min at −50° C. 304 0.65% and with a vacuum degree of −4 Mpa

[0118] It can be seen from the table that, when dried by vacuum lyophilization, the activity loss of the coated hirudin mutant HV2-Lys47 was the smallest.

Example 11

[0119] Effects of the amount of the hirudin mutant HV2-Lys47, which was prepared and vacuum-lyophilized in Example 9, on blood coagulation were as shown in Table 2 below:

TABLE-US-00004 TABLE 2 Amount of hirudin mutant Blood collection volume Coagulation HV2-Lys47 (mg) (ml) 0.001 2 Quick coagulation 0.005 2 No coagulation 0.008 2 No coagulation 0.02 2 No coagulation 0.08 2 No coagulation 0.16 2 No coagulation 0.5 2 No coagulation 1 2 No coagulation 5 2 No coagulation 10 2 No coagulation 15 2 No coagulation 20 2 No coagulation

[0120] It can be seen from the above table that anticoagulant effect can be achieved when the amount of the hirudin mutant HV2-Lys47 added in the blood collection tube is 0.005 mg or more.

Example 12

[0121] Effects of the hirudin mutant HV2-Lys47 (hereinafter referred to as hirudin) prepared in Example 9 used in anticoagulant blood collection tubes on routine blood tests and blood biochemical indexes were as follows, wherein the hirudin mutant HV2-Lys47 was applied to vacuum blood collection tubes by spraying.

[0122] (1) Routine Blood Tests

[0123] Routine blood testing indexes for hirudin anticoagulation vacuum blood collection tubes and K2.EDTA anticoagulation vacuum blood collection tubes were as shown in Table 3 below. Among them, 20 indexes of the two types of vacuum blood collection tubes were highly correlated (r value >0.8), and 4 indexes were moderately correlated (0.5<r value <0.8).

TABLE-US-00005 TABLE 3 Comparison of routine blood tests between hirudin anticoagulation and K2 .Math. EDTA anticoagulation Testing Regression indexes K2 .Math. EDTA Hirudin r equation White blood  5.51 ± 1.38  5.36 ± 1.31 0.9611 y = 0.9148x + cell count 0.3116 Large 30.60 ± 8.55 28.60 ± 7.84 0.9533 y = 0.8743x + platelet ratio 1.8428 Percentage  6.31 ± 1.54  5.20 ± 1.25 0.8332 y = 0.6766x + of monocyte 0.9319 count Absolute  0.34 ± 0.09  0.28 ± 0.09 0.8307 y = 0.8406x − value of 0.0103 monocyte count Hematocrit 39.72 ± 3.26 38.93 ± 3.03 0.9055 y = 0.8436x + 5.424 Red blood 12.43 ± 0.86 12.37 ± 0.86 0.9953 y = 0.9909x + cell 0.0547 distribution width Red blood 38.81 ± 1.66 37.86 ± 1.58 0.9580 y = 0.9095x + cell volume 2.5645 distribution width Percentage 34.06 ± 8.14 34.83 ± 8.69 0.9901 y = 1.0564x − of 1.154 lymphocyte count Absolute  1.80 ± 0.29  1.79 ± 0.32 0.8877 y = 0.9958x − value of 0.0011 lymphocyte count Mean red 339.29 ± 10.89 345.71 ± 10.57 0.9306 y = 0.9033x + blood cell 39.229 protein concentration Mean red 86.16 ± 3.66 84.52 ± 3.66 0.9947 y = 0.9958x − blood cell 1.274 volume Mean red 29.26 ± 1.93 29.24 ± 1.85 0.9881 y = 0.9451x + blood cell 1.5826 hemoglobin content Mean 12.61 ± 2.24 12.27 ± 2.35 0.9287 y = 0.9744x − platelet 0.0188 distribution width Mean 10.71 ± 1.05 10.47 ± 0.98 0.9603 y = 0.8933x + platelet 0.9055 volume Percentage  0.65 ± 0.36  0.63 ± 0.27 0.7509 y = 0.558x + of basophil 0.2646 count Absolute  0.03 ± 0.02  0.03 ± 0.01 0.7606 y = 0.5811x + value of 0.0116 basophils Percentage  1.44 ± 1.13  1.37 ± 0.98 0.9614 y = 0.8367x + of eosinophil 0.1639 count Absolute  0.07 ± 0.04  0.07 ± 0.04 0.9376 y = 0.8635x + value of 0.0061 eosinophils Hemoglobin 134.92 ± 13.32 134.71 ± 12.74 0.9549 y = 0.9129x + concentration 11.539 Red blood  4.61 ± 0.35  4.61 ± 0.30 0.8968 y = 0.7758x + cell count 1.0291 Platelet  0.26 ± 0.04  0.17 ± 0.03 0.6616 y = 0.5302x + hematocrit 0.0368 Platelet 242.08 ± 46.04 167.54 ± 39.44 0.7439 y = 0.6372x + count 13.276 Percentage 57.29 ± 9.31 57.57 ± 9.47 0.9840 y = 1.0017x + of neutrophil 0.1765 count Absolute  3.25 ± 1.26  3.17 ± 1.19 0.9843 y = 0.9328x + value of 0.1334 neutrophil count

[0124] (2) Blood Biochemistries, Tumor Markers and Homocysteine

[0125] The testing results of biochemistries, tumor markers and homocysteine of serum (separation gel/silica vacuum blood collection tubes) and plasma (hirudin vacuum blood collection tubes) were as shown in Table 4. Among them, 22 indexes of the two types of vacuum blood collection tubes were highly correlated (r value>0.8), and 2 indexes were moderately correlated (0.5<r value<0.8).

TABLE-US-00006 TABLE 4 Comparison of biochemistries, tumor markers and homocysteine between plasma anticoagulated with hirudin and routine serum Regression Index Serum Hirudin r equation γ-glutamyl 16.28 ± 14.34 16.34 ± 14.58 0.9969 y = 1.0139x − transpeptidase 0.1665 (γ-GT) Albumin 49.21 ± 3.04  51.34 ± 3.01  0.7981 y = 0.7906x + (ALB) 12.436 Alanine 21.68 ± 8.1  22.24 ± 8.09  0.9623 y = 0.9605x + transaminase 1.4162 (ALT) Aspartate 21.88 ± 4.78    23 ± 4.97 0.9135 y = 0.9514x + transaminase 2.183 (AST) Indirect 8.26 ± 3   7.86 ± 2.86 0.9915 y = 0.9432x + bilirubin 0.065 Globulin 21.68 ± 1.11  23.73 ± 1.95  0.5224 y = 0.9188x + 3.8097 Serum total 3.78 ± 1.05 3.78 ± 1.05 0.9864 y = 0.986x + bile acid 0.057 Direct bilirubin 3.48 ± 1.36 3.58 ± 1.42 0.9966 y = 1.0362x − (DBIL) 0.0252 Total bilirubin 11.74 ± 4.26  11.44 ± 4.16  0.9955 y = 0.9722x + (TBIL) 0.0218 Total protein 70.89 ± 3.48  75.07 ± 4.31  0.8081 y = 1.0011x + (TP) 4.1014 Creatinine 46.39 ± 6.86  45.77 ± 6.68  0.977 y = 0.9526x + 1.5738 Urea nitrogen 4.88 ± 1.00 4.90 ± 1.00 0.985 y = 0.9779x + 0.1258 Uric acid 284.74 ± 76.12  284.91 ± 76.80  0.999 y = 1.0082x − 2.1655 Low density 2.77 ± 0.61 2.83 ± 0.62 0.9870 y = 1.0015x + lipoprotein 0.063 cholesterol Triglycerides 0.96 ± 0.75 0.97 ± 0.76 0.9983 y = 1.0151x − (TG) 0.0021 High density 1.61 ± 0.31 1.60 ± 0.33 0.9063 y = 0.9393x + lipoprotein 0.0932 cholesterol Apolipoprotein 1.34 ± 0.13 1.35 ± 0.15 0.8966 y = 1.0206x − A 0.0182 Apolipoprotein 0.79 ± 0.19 0.80 ± 0.19 0.9884 y = 0.9995x + B 0.0114 Total 4.39 ± 0.66 4.46 ± 0.70 0.9786 y = 1.0271x − cholesterol 0.0509 (T-CHO) Blood glucose 5.45 ± 0.46  5.3 ± 0.46 0.9691 y = 0.9714x + 0.0105 Carcino- 1.10 ± 0.41 1.20 ± 0.38 0.9423 y = 0.8792x + embryonic 0.2359 antigen (CEA) Alpha- 3.10 ± 2.71 3.14 ± 2.79 0.9979 y = 1.0297x − fetoprotein 0.0548 (AFP) Serum 14.74 ± 14.53 13.58 ± 14.32 0.9875 y = 0.9731x − carbohydrate 0.7697 antigen 199(CA-199) Homocysteine 11.45 ± 5.12  11.94 ± 5.14  0.9948 y = 0.9975x + 0.5176

[0126] (3) Five Items of HBV

[0127] Five items of HBV are qualitative testing indexes, and the results are as shown in Table 5. The results showed that the testing indexes of serum and plasma were highly consistent, as the indexes which were negative for serum were also negative in the testing results of plasma, and the indexes which were positive for serum were also positive in the testing results of plasma.

TABLE-US-00007 TABLE 5 Hepatitis B surface  Hepatitis B  Hepatitis B Hepatitis B Hepatitis B core Serial antigen  surface antibody  E-antigen E-antibody antibody number Serum Hirudin Serum Hirudin Serum Hirudin Serum Hirudin Serum Hirudin 1 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 2 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 3 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 4 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 5 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 6 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 7 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 8 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 9 (−) (−) (+) (+) (−) (−) (−) (−) (+) (+) 10 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 11 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 12 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 13 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 14 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 15 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 16 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 17 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 18 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 19 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 20 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 21 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 22 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 23 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 24 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 25 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 26 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 27 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 28 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 29 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 30 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 31 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 32 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 33 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 34 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 35 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 36 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 37 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 38 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 39 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 40 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 41 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 42 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 43 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 44 (−) (−) (+) (+) (−) (−) (+) (+) (+) (+) 45 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 46 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 47 (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) 48 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 49 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−) 50 (−) (−) (+) (+) (−) (−) (−) (−) (−) (−)

[0128] The results show that the results of routine blood tests for hirudin anticoagulation were consistent with the results of K2.EDTA anticoagulation routine blood tests, and the testing indexes were highly or moderately correlated, which were consistent with the results of Menssen H (see “Measurement of Hematological, Clinical Chemistry, and Infection Parameters from Hirudinized Blood Collected in Universal Blood Sampling Tubes”, Hans D. Menssen et al., SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 27, NUMBER 4 2001, pp. 349-356). At present, serum samples are commonly used for the testing of clinical biochemical indexes. However, plasma samples are more approximate to physiological state than serum in theory. The results show that despite that there are poor correlation (0.5<r value<0.8) in total protein, albumin and globulin between serum and plasma, there are high correlation (r value>0.9) in biochemistries, electrolytes, tumor markers and homocysteine. Since plasma contained fibrinogens, the protein concentration in plasma (75.07±4.31) was higher than that in serum (70.89±3.48).

[0129] It can be seen from the above that K2.EDTA and hirudin anticoagulant have similar effects, but K2.EDTA can only be used for the determination of routine blood tests, while hirudin blood collection tubes may also be used for the determination of biochemistries, electrolytes, tumor markers, homocysteine and other indexes of serum and plasma.

[0130] In addition, on the market at present, 40 ATU of hirudin is required per milliliter of blood to effect the anticoagulant activity of hirudin. The hirudin of the present application has a high purity, so its addition amount in the preparation of blood collection tubes is smaller than that of hirudin with a lower purity. Moreover, because of the high purity of hirudin of the present application, the effects of impurities if present on blood coagulation and blood index testing are avoided. Furthermore, due to the high purity, the hirudin of the present application has significantly better stability, and the specific data are as follows:

[0131] Hirudin blood collection tubes (2 ml): (accelerated test temperature: 40° C.±2, humidity: 75%±5)

TABLE-US-00008 Testing period 0 week 2 weeks 1 month 2 months 3 months 6 months Blood collection tubes of 2625ATU 2650ATU 2625ATU 2580ATU 2600ATU 2575ATU the present application Outsourced blood 3020ATU 2800ATU 2548ATU 1890ATU 1600ATU 1500ATU collection tubes

[0132] The blood collection tube of the present application is the blood collection tube with a coating of dried hirudin mutant HV2-Lys47 prepared by lyophilization in Example 9. The outsourced blood collection tube is: Sarstedt from Germany, which is also a hirudin blood collection tube.

[0133] Those of ordinary skills in the art will appreciate that modifications or equivalent substitutions may be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application, all of which should be contained within the scope of the claims of the present application.