RU2007421C1 - Method of placental protein preparing - Google Patents

Abstract

FIELD: agricultural biotechnology. SUBSTANCE: method involves fractionation of supernatant from sheep early placenta homogenate on immobilized trichlotriazine-ε-aminocaproic acid following by gel-filtration through Sephadex G-200. EFFECT: improved method of protein preparing.

Description

 The invention relates to biological chemistry and can be used to isolate the placental sheep protein -2 (PPO-2).

 In the literature, this protein is not described and methods for its preparation to date in the patent and medical literature are not found.

 PPO-2 was detected in tissue extracts of the early sheep chorion (cotyledon) with gestational periods of up to 10 weeks. In an immunochemical study using double radial immunodiffusion with various biological fluids and tissue extracts, PPO-2 was absent in the plasma of sheep, coot sheep and fetal serum, as well as in extracts of uterine tissue, pituitary gland, adrenal gland, testicle, ovary, kidney, liver, spleen, lung . Immunochemically, this antigen was found in placental tissues (cotyledon and caruncle) throughout the course of gestation, and its content in the early placenta exceeded that in the terminal placenta by 5 times. PPO-2 was found in equal amounts in both the maternal and fetal parts of the placenta. Its content in these tissues was about 0.4 mg per 100 g of tissue of the early placenta.

 The data obtained suggest that PPO-2 is a placenta-specific antigen and can be used as a marker for early diagnosis of sheep sheep’s condition and monitoring the gestation process in this animal. The creation of a highly sensitive enzyme-linked immunosorbent assay for determining PPO-2 in biological fluids and tissues based on a purified preparation of this protein will help to solve this problem.

 To obtain monospecific polyclonal antisera to PPO-2, the tissue of the fetal part of the early placenta of the sheep (cotyledon) was homogenized in an equal volume of Tris-glycine buffer (0.05 M, pH 8.5), adding a 0.1% solution of the detergent Triton X- 100, frozen and thawed three times, centrifuged, the supernatant was dialyzed for 48 hours against 0.1 M ammonium bicarbonate buffer and lyophilized. Rabbits were immunized five times, subcutaneously, injecting each animal with 100 mg of the preparation of early chorionic proteins mixed with Freund's complete adjuvant with an interval of 3 days. The first immunization was carried out on the 30th day after the last immunization, the second on the 90th day after the last immunization. Satisfactory antisera were obtained only after the second re-immunization. The resulting antisera were absorbed by ram plasma, extracts of the kidney, liver and spleen. Using these atisers, an antigen with the mobility of alpha-1-globulin on agar gel was identified in the tissue of the early placental sheep.

 The aim of the invention is to obtain a purified preparation of placental protein-2, achieved as follows: the fetal part (cotyledon) of a 10-12-week-old sheep placenta is homogenized with a double amount of 0.1 M Tris-glycine buffer, pH 7.7-8.7, homogenate centrifuged, the supernatant is dialyzed against 0.02-0.05 M Tris-Hcl buffer, pH 7.7-8.7, centrifuged again and used in affinity chromatography on epsilon-aminocaproic acid immobilized through trichlorotriazine, the bound proteins elute 2.0- 4.0 M potassium chloride solution, eluate precipitation give at 90% saturation with ammonium sulfate, centrifuged and the precipitate is fractionated on a column with Sephadex G-200 in 0.1 ammonium bicarbonate buffer, pH 8.15, fractions containing the target product are collected and lyophilized, receiving the preparation PPO-2 50% degree of purity.

 The sorbent was synthesized as follows. 100 ml of Sepharose 6B was washed on the filter with 1 L of distilled water, slightly squeezed, transferred to a beaker, 100 ml of acetone was added, 1.5 g of trichlorotriazine was added, thoroughly mixed, a 10% sodium hydroxide solution was added dropwise, bringing the pH to 8 ,5. After reaching a pH of 8.5, the addition of sodium hydroxide was stopped, waited 5 minutes, and fifty ml of a 50% aqueous solution of acetic acid was added, thereby terminating the reaction. The obtained proactivated sepharose was again washed on the filter with 1 liter of a 50% aqueous acetone solution from unbound trichlorotriazine and an additional 1 liter of distilled water; Then, Sepharose was transferred into a beaker, 100 ml of distilled water, 2 g of epsilon-aminocaproic acid and 2 g of sodium bicarbonate were added, put on a magnetic stirrer overnight with slow stirring. The next day, the obtained sorbent - sepharose with immobilized epsilon-aminocaproic acid was washed with 3 L of distilled water on the filter, after which the sorbent was ready for use.

 New in the proposed method is that for isolation and purification from a complex protein mixture of a previously unknown protein - placental protein - 2 sheep used biospecific for this protein ligand - epsilon-aminocaproic acid, immobilized on an insoluble matrix - sepharose, and the eluate containing PPO- 2, fractionated on a column with Sephadex G-200 in a volatile buffer, thereby solving two problems at the same time: additional protein purification and its dialysis against volatile buffer. In addition, processing the starting material with a buffer with a low ionic strength avoids dialysis of the prepared protein mixture and uses the homogenate supernatant immediately in affinity chromatography.

 To achieve the objective of the invention, the parametric values of the method are selected and justified. So, when processing placental tissue with a buffer volume of more than twofold, dilution of the protein mixture and less sorption on the affinity sorbent were observed; when processing the placenta tissue with a buffer volume of less than twice, the ionic strength of the prepared protein mixture was more than 0.05, which prevented the best sorption of the desired protein on the ligand.

 For affinity chromatography of PPO-2 on immobilized epsilonaminocaproic acid, the optimal pH for the best sorption of the protein was pH from 7.7 to 8.7 and the molarity of Tris-Hcl buffer 0.02-0.05; with a decrease in the minimum pH below 7.7, a decrease in the amount of sorbed protein was observed; with an increase in pH above the maximum of 8.7, the amount of sorbed protein corresponded to the optimal value, however, the sorption of ballast proteins increased, which led to a decrease in the purity of the preparation. Changing the molarity of Tris-Hcl buffer more than 0.05 led to a decrease in protein sorption on the sorbent and a decrease in the yield of the target product; with a molarity of less than 0.02, an additional sorption of ballast proteins was observed and corresponding to this decrease in the purity of the drug.

 The essence of the alleged invention is illustrated by the following examples.

 Example 1. 100 g of tissue of a 10-12 week old sheep placenta (cotyledon) are homogenized with 200 ml of 0.01 M Tris-HCl buffer pH 7.7, the homogenate is frozen once, thawed and centrifuged at 6000 rpm 45 min The supernatant is used in affinity chromatography on a column of immobilized epsilon-aminocaproic acid equilibrated with 0.02 M Tris-Hcl buffer pH 7.7. The bound proteins are eluted with a 2.0 M potassium chloride solution, the eluate is precipitated at 90% saturation with ammonium sulfate, centrifuged at 8000 rpm. 45 min, the precipitate was dissolved in a minimal volume of distilled water and fractionated on a Sephadex G-200 column equilibrated with 0.1 M ammonium bicarbonate buffer pH 8.15, fractions containing PPO-2 were collected and lyophilized to obtain 0.05 mg squirrel. The content of PPO-2 is 50%, the yield is 6.3%.

 PRI me R 2. The placental tissue is treated as in example 1. Affinity chromatography is carried out in 0.05 M Tris-Hcl buffer at pH 8.7. Bound proteins elute with 4.0 M potassium chloride. Further procedures are carried out as in Example 1. 0.09 mg of protein is obtained. The content of PPO-2 is 30%, the yield is 6.8%.

 PRI me R 3. The placental tissue is treated as in example 1. Affinity chromatography is carried out in 0.035 M Tris-Hcl buffer at pH 8.2. Elution and further procedures are carried out as in Example 1. 0.08 mg of protein is obtained. The content of PPO 2 50%, yield 10%.

 The proposed method is an original way of isolating a new protein - the placental protein of sheep-2.

 When studying the physicochemical properties of PPO-2, it was found that this protein has an electrophoretic mobility (relative to albumin) of 0.9, a molecular weight determined in gel filtration of about 70 kD. The protein contains carbohydrates when staining the precipitation arc according to the Schiff method and precipitates with ammonium sulfate at 35-70% saturation.

 The data of immunochemical analysis and physicochemical properties of PPO-2 suggest that this protein is not identical to sheep alpha-fetoprotein, which has physico-chemical characteristics similar to PPO-2, but which is found in large quantities in fetal serum; PPO-2 differs from the placental lactogen of sheep, having a molecular weight of about 20 kDa and the mobility of alpha globulin; from sheep trophoblastic protein having a molecular weight of 19 kD; from a high molecular weight glycoprotein of a sheep having a mol. m. 765 kD.

 The development of an original method for the isolation of PPO-2 and the production of antisera to it is an urgent scientific task, since this protein was detected in the tissue of the early placenta and the development of a highly sensitive enzyme-linked immunosorbent assay for the determination of PPO-2 in the blood serum of ewes based on a purified preparation of this protein will allow us to obtain a diagnostic test early diagnosis of gestation and its monitoring. (56) EP N 0226181, cl. S 07 To 13/00, 1987.

Claims (1)

 METHOD FOR PRODUCING PLACENTAL PROTEIN, including obtaining tissue homogenate, centrifugation, chromatography, elution of protein associated with the sorbent, precipitation of the eluate and repeated centrifugation, characterized in that sheep placenta tissue is used from the tissues, affinity chromatography using sepharose acid-immobone eponobylamine protein elution is carried out with a 2.0-4.0 M potassium chloride solution, the eluate is precipitated at 90% saturation with ammonium sulfate, and after centrifugation, about the cage is additionally subjected to fractionation on Sephadex G = 200 in 0.1 M ammonium bicarbonate buffer, followed by collection of fractions containing the target product and their dialysis.