CN1206240C - High efficiency ionic exchange membrane chromatography decontamination vitelloenin technology - Google Patents

Abstract

The invention belongs to a process for preparing biologically active substances, and relates to a technique for extracting and purifying vitellogenin from the blood or whole body homogenate of oviparous animals by using high-efficiency anion-exchange membrane chromatography. This technology can realize the fast concentration and purification of protein on the ion exchange membrane, and the product has high purity and high recovery rate. The invention can greatly save time and solvent consumption, and has simple equipment and low cost.

Description

Purification of Vitellogenin by High Performance Ion Exchange Membrane Chromatography

field of invention

The invention belongs to the field of protein preparation, and relates to a technology for extracting and purifying vitellogenin from the blood or whole body homogenate of oviparous animals by using high-efficiency anion-exchange membrane chromatography technology. This technology enables the rapid concentration and purification of proteins on ion-exchange membranes. The invention can greatly save time and solvent consumption, and has simple equipment and low cost.

Background technique

The background and development status of this technical field are roughly as follows: Vitellogenin is a high-phosphorus glycoprotein induced by estrogen and is the main precursor of vitellin in oviparous animals. Under normal circumstances, there is no vitellogenin in male animals, but under the induction of estrogen or estrogen, the liver of male animals can also synthesize vitellogenin. Therefore, vitellogenin is considered as an ideal estrogen and estrogen-like biomarker, and has been widely used in the screening of environmental endocrine disruptors, environmental toxicology, and pollution investigations in recent years. In order to meet the requirements of rapid and sensitive detection of vitellogenin, many companies have developed or are developing immunoassay kits for vitellogenin. In the development of vitellogenin immunoassay kits, a large amount of purified vitellogenin is needed to immunize animals to prepare antibodies, and it is used as a standard reference for sample detection. Traditional vitellogenin purification methods mainly include ion-exchange column chromatography, gel filtration chromatography, ultracentrifugation, selective precipitation, etc. These methods generally require a relatively long separation time and a large amount of solution is used. At present, the method for purifying vitellogenin similar to the present invention has not been reported yet.

Literature on this subject can be found at:

1. H.S. Wiley, L. Opresko, R.A. Wallace, Anal. Biochem., 97(1979) 145-152.

2. L.G. Parks, A.O. Cheek, N.D. Denslow, S.A. Heppell, J.A. McLachlan, G.A. LeBlanc, C.V. Sullivan, Comp. Biochem. Physiol. C, 123 (1999) 113-125.

3. M.R. Redshaw, B.K. Follet, Biochem. J., 124 (1971) 759-766.

4. F. Brion, F. Rogerieux, P. Noury, B. Migeon, P. Flammarion, E. Thybaud, J. M. Porcher, J. Chromatogr. B., 737 (2000) 3-12.

Contents of the invention

The purpose of the present invention is to provide a technology for rapidly separating and purifying vitellogenin from the blood or whole body homogenate of oviparous animals.

The technical solution for completing the present invention is achieved in the following manner. First, healthy experimental animals were selected and injected intraperitoneally with 17β-estradiol at a dose of 1-5 μg/g. After 14-21 days of laboratory feeding, blood was collected from a vein, and whole-body homogenization was directly performed on very small animals. Animal blood was centrifuged at 4°C and 3000 rpm for 20 minutes, and the serum was taken, and the sample buffer solution (phosphate or Tris-hydrochloric acid buffer solution with a pH of 6.5-8.5 at a concentration of 0.02 mol/L, containing NaCl 0.07mol/L), pass through a 0.45 micron filter membrane for later use; transfer the animal homogenate into a centrifuge tube, centrifuge at 4°C, 10,000 rpm for 30 minutes, carefully remove the upper layer of fat, and remove the supernatant Filter through a 0.45 micron membrane filter and set aside. During purification, use 10 milliliters of sample buffer to pass through a high-efficiency anion exchange membrane (Sartobind ^TM MA15 Q, purchased from Sartorius Company, Germany) at a flow rate of 10 ml/min to make it pre-equilibrated, and then make the sample The solution was passed through a high-efficiency anion-exchange membrane and washed with 10 mL of sample buffer to remove unbound protein. Next, use 5 milliliters of elution buffers A and B (phosphate or Tris-hydrochloric acid buffer solution at pH=6.5-8.5, the concentration is 0.02mol/L, containing 0.27mol/L and 0.42mol/L NaCl respectively ) successively wash the anion exchange membrane, and collect the elution fraction corresponding to the elution buffer B, which is the purified vitellogenin. Finally, wash the high-efficiency anion-exchange membrane with 10 ml of recovery buffer (phosphate or Tris-hydrochloric acid buffer at pH=6.5-8.5, concentration 0.02 mol/L, containing 1 mol/L NaCl), and then use it for the next separation.

In the process of completing the present invention, we have successfully extracted and purified corresponding vitellogenin from different types of oviparous animals: carp, crucian carp, bighead carp, loach and chicken serum and zebrafish whole body homogenate, and used high-efficiency coagulation Gel filtration chromatography and polyacrylamide gel denaturation electrophoresis were used to identify the purified protein, and the results showed that the molecular weight of the protein was 440-500KD, which was consistent with the literature reports. In addition, we also compared with the anion exchange column chromatography method frequently used in the literature, as shown in the table below, the results prove that the present invention can greatly save time and solvent consumption, and is easy to operate.

Table 1, the comparison of the present invention and traditional anion-exchange column chromatographic method for separating vitellogenin

Parameters The present invention Anion exchange column chromatography

Functional group Diethylethylenediamine or quaternary ammonium salt Diethylethylenediamine

Separation time 5 minutes 600 minutes

Amount of solution 40ml 500ml

Working Temperature Room Temperature 4℃

Claims (2)

1. A technique for extracting and purifying vitellogenin from the blood or whole-body homogenate of oviparous animals, which is characterized in that the blood or whole-body homogenate directly passes through a high-efficiency anion-exchange membrane after being diluted with sample buffer and filtered, and then sequentially washed with The elution solution with sodium chloride concentration of 0.27mol/L and 0.42mol/L was eluted. 2. The technology for extracting and purifying vitellogenin according to claim 1 is characterized in that the functional group of the high-efficiency anion exchange membrane used is diethylethylenediamine or quaternary ammonium salt.