Method for preparing liquid type PT agent based on human recombinant thrombokinase

A technology of thromboplastin and human recombination, which is applied in the biological field, can solve the problems such as the process is not simple enough, and achieve the effects of avoiding changes in measured values, strong stability, and easy use

Active Publication Date: 2011-07-20
SHANGHAI LONG ISLAND BIOTEC CO LTD
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AI Technical Summary

Problems solved by technology

The patent "Preparation Method of Tissue Factor-Based Prothrombin Time Reagent" applied by American LifeScan Co., Ltd. in China provides the preparation method of thromboplastin / phospholipid vesicle mixture, which requires additional screening for the removal of The resin of the detergent component in the mixture, and then filtered to remove the resin, the process is not simple enough, the final PT reagent is coated on the test strip to detect the blood sample

Method used

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  • Method for preparing liquid type PT agent based on human recombinant thrombokinase
  • Method for preparing liquid type PT agent based on human recombinant thrombokinase
  • Method for preparing liquid type PT agent based on human recombinant thrombokinase

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Experimental program
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Effect test

Embodiment 1

[0018] Construction and expression of recombinant plasmids

[0019] Total RNA was obtained from human placenta tissue, according to the nucleotide sequence of coagulation factor III in GeneBank ( figure 1 ) Design PCR upstream primer MF3 and downstream primer MR2:

[0020] MF3: 5`-CCGCTCGAGAAAAGATCAGGCACTACAAATACTGCGGC-3`

[0021] MR2: 5`-GGCCTAGGTCAATGATGATGATGATGATGTGAAACATTCAGTGGGGA-3`

[0022] The MF3 primer contains several amino acids at the C-terminal of human thromboplastin, the Xhol cleavage protection group and the Xhol cleavage site, and the MR2 contains several amino acids at the N-terminal of human thromboplastin, the Avr II cleavage site, the terminator codon and 6 His codons son.

[0023] Using total RNA as a template, human thromboplastin gene was obtained by RT-PCR method. This gene was treated with the above two endonucleases, and then connected to the plasmid pPIC9 to obtain the recombinant expression plasmid ( figure 2 ).

[0024] Transform the recom...

Embodiment 2

[0026] Purification of human thromboplastin

[0027]The selected high-expressing strains were cultivated by conventional methods, and 3% methanol was added at 48 hours to induce foreign proteins. In order to avoid the influence of culture medium components on the purification steps and simplify the purification process, the foreign protein is designed to be expressed intracellularly. The bacterial cells were collected by centrifugation, and the residual culture solution was washed with pH7.450mmol / L sodium phosphate buffer solution containing 5% glycerol and 1mmol / L PMSF. The cells were homogenized under high pressure, and then the supernatant containing the target protein was collected by centrifugation, and the target protein was directly purified by Ni-NTA Agarose column chromatography. The balance solution used is pH 7.40.02mol / L sodium phosphate buffer (BufferB) containing 0.5mol / L NaCl, eluted step by step, after loading the sample, use Buffer B and Buffer B containing ...

Embodiment 3

[0029] Preparation of PT kit

[0030] 1. The composition of PT kit: phospholipidated human thromboplastin (1%), buffer and stabilizer (PTF-G).

[0031] 2. PTF-G formula: 40mmol / L Tris-HCl, pH7.0

[0032] Glucose gum, final concentration 4%

[0033] Polyethylene glycol, final concentration 0.5%

[0034] CaCl 2 , final concentration 11mmol / L,

[0035] NaCl, final concentration 30mmol / L,

[0036] Mannitol, final concentration 2%

[0037] Sodium azide, final concentration 0.1%

[0038] Gelatin, final concentration 0.25%

[0039] 3. Detection method: PT reagent is preheated at 37°C for 20 minutes, take 100 μl, incubate the plasma to be tested at 37°C for 3 minutes, take 50 μl, mix well and time, use the CA-530 hemagglutination instrument of Sysmex Company to measure, and record the plasma clotting time .

[0040] 4. Product technical indicators:

[0041] 1) Appearance: Colorless transparent liquid.

[0042] 2) The normal value range of PT measurement: 10.7-14.3 seconds. ...

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Abstract

The present invention provides a method for preparing liquid PT reagent by using recombined thromboplastin. The invention belongs to the field of biotechnology and aims to solve clinic standardization problem for PT determination. The quality of a PT reagent is a key factor influencing PT test, and the effective method for reducing the difference of determination results in different laboratories is to unify the chemical composition and biochemical characterics of thromboplastin in reagent. The recombined thromboplastin prepared by genetic engineering method has stable molecular structure and uniform protein, and can improve test sensitivity, precision and accuracy. The full-length human thromboplastin expressed by yeast cell can achieve high purity and target protein with high activity by one-step purification. The invention also provides a method for preparing liquid PT reagent based on recombined thromboplastin. The reagent has good sensitivity and stability and qualified each technical index, and test shows that the reagent is suitable for clinic application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of a liquid PT reagent. The method also includes the expression and purification of human recombinant thromboplastin, the main component of the PT reagent, in yeast cells. Background technique [0002] Prothrombin time (PT) is a screening test for checking exogenous blood coagulation factors. Since this determination method was created in 1935, it is still an important screening test for checking various factors of the exogenous blood coagulation system and related inhibitors. It is the main monitoring method for oral anticoagulant therapy at present, and it is also an important inspection index for the coagulation function of patients before surgery. [0003] The determination of PT is affected by various conditions, and the standardization of its clinical detection has become increasingly prominent. Due to the use of tissue thromboplastin reagents f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N9/74C12N1/19C12Q1/34
Inventor 肖国伟
Owner SHANGHAI LONG ISLAND BIOTEC CO LTD
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