RU2007418C1 - Method of placental protein preparing - Google Patents

Abstract

FIELD: agricultural biotechnology. SUBSTANCE: method involves fraction of proteins from homogenate of pig early chorion on immobilized hexamethylenediamine following by gel-filtration of eluate 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 protein of pig-1 (PPS-1).

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

 PPS-1 was detected in tissue extracts of early pig chorion with gestational age up to 8 weeks. During an immunochemical study using double radial immunodiffusion with various biological fluids and tissue extracts, PPS-1 was absent in the plasma of the boar, pregnant pig and fetal serum, as well as in extracts of uterine tissue, pituitary, adrenal gland, testicle, ovary, kidney, liver, spleen, lung . Immunochemically, this antigen was found in the tissues of the placenta throughout the course of gestation, while its content in the early placenta exceeded the content in the terminal 4-5 times. PPS-1 was detected in equal amounts in both the maternal and fetal parts of the placenta. Its content in these tissues was about 2 mg per 100 g of tissue of the early placenta.

 The data obtained suggest that PPS-1 is a placenta-specific antigen and can be used as a marker for early diagnosis of pig gestation and monitoring the gestation process in this animal. The creation of a highly sensitive enzyme-linked immunosorbent assay for determining PPS-1 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 PPS-1, the tissue of the fetal part of the early placenta of the pig was homogenized with an equal volume of 0.05 M Tris-glycine buffer, pH 8.5, adding a 0.1% solution of X-100 triton, three times frozen and thawed , centrifuged, the supernatant was dialyzed for 48 hours against 0.1 M ammonium bicarbonate buffer and lyophilized. Outbred male rabbits were immunized five times, subcutaneously, injecting each animal with 100 mg of the early chorion protein preparation mixed with Freund's complete adjuvant with an interval of 3 days. Reimmunization was carried out with the same amount of the drug without adjuvant on the 30th day after the last immunization. The resulting antisera were absorbed by boar plasma, extracts of the kidney, liver and spleen. Using these antisera in the tissue of the early placenta of the pig, an antigen with alpha-α _- globulin mobility in agar gel was identified.

 The aim of the invention is to obtain a purified preparation of placental pig protein-1, achieved as follows: the fetal part of the 8-week pig placenta is homogenized with an equal amount of 0.01 M Tris-Hcl buffer, pH 7.5-8.5, the homogenate is centrifuged after three freezing and thawing, the supernatant is used in affinity chromatography on immobilized hexamethylenediamine, the bound proteins are eluted with a 2.0-4.0 M sodium chloride solution with 1% glycine added, the eluate is precipitated at 90% saturation with ammonium sulfate, centrifuged ute and the precipitate is fractionated on a Sephadex G-200 column in 0.1 M ammonium bicarbonate buffer pH 8.15, fractions containing the target product are collected and lyophilized to obtain PPS-1 preparation of 50% 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, put on a magnetic stir bar and 10% was added dropwise with slow stirring. caustic soda solution, bringing the pH to 8.5, after which the addition of caustic soda was stopped, waited 5 minutes and the reaction was stopped by adding 50 ml of 50% acetic acid to the mixture. The obtained designed sepharose was washed on the filter from unbound trichlorotriazine and reaction products 1 l of a 50% aqueous solution of acetone and an additional 1 l of distilled water; then, Sepharose was transferred to a beaker, 100 ml of distilled water and 2 g of hexamethylenediamine were added, and placed on a magnetic stirrer overnight with slow stirring. The next day, the resulting sorbent was washed with 3 L of distilled water on a filter, after which the sorbent of hexamethylene diaminesepharose 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 - the placental protein of a pig, a biospecific ligand, hexamethylenediamine, immobilized on an insoluble matrix, Sepharose, is used, and the eluate containing PPS-1 is fractionated on a column with Sephadex G-200 in a volatile buffer, thereby simultaneously solving two problems: additional purification and dialysis. 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 one time, dilution of the protein mixture and less sorption of PPS-1 on the sorbent were observed; when processing placental tissue with a buffer volume of less than one, the ionic strength of the prepared protein mixture was more than 0.08, which prevented the sorption of the desired protein on the ligand.

 For affinity chromatography of PPS-1 on immobilized hexamethylenediamine, the optimal pH for the best sorption of the protein was 7.5-8.5 and the molarity of Tris-Hcl buffer 0.06-0.08; with a decrease in the minimum value below 7.5, a decrease in the amount of sorbed protein was observed, with an increase in pH above the maximum 8.5, 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 drug. Changing the molarity of Tris-Hcl buffer more than 0.08 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 0.06, additional sorption of ballast proteins and a corresponding decrease in the purity of the drug were observed. The addition of glycine to the elution buffer made it possible to achieve more complete desorption of PPS-1. The optimal molarity of the eluting buffer was 2.0-4.0 M.

PRI me R 1. 100 g of tissue of an 8-week pig placenta (fetal part) is homogenized with 100 ml of 0.01 M Tris-Hcl buffer pH 7.5, thrice frozen and thawed, centrifuged 45 min at 6000 rpm . The supernatant is used in affinity chromatography on a column with immobilized hexamethylenediamine in 0.06 M Tris-HCl buffer pH 7.5. Bound proteins were eluted with 2.0 M sodium chloride, 1% glycine eluate was precipitated with 90% saturated ammonium sulfate for 12 hours at ⁴ ° C and centrifuged 45 min. 8000 rpm The precipitate was fractionated on a Sephadex G-200 column in 0.1 M ammonium bicarbonate buffer, pH 8.15, fractions containing PPS-1 were collected and lyophilized to obtain 0.4 mg of protein. The content of PPS-1 is 40%, the yield is 8%.

 PRI me R 2. The placental tissue is treated as in example 1. Affinity chromatography is carried out in 0.08 M Tris-Hcl buffer pH 8.5. Bound proteins are eluted with 4.0 M sodium chloride supplemented with 1% glycine. Further procedures are as in Example 1. 0.6 mg protein will be obtained. The content of PPS-1 is 30%, yield 9%.

 PRI me R 3. The placental tissue is treated as in example 1. Affinity chromatography is carried out in 0.07 M Tris-Hcl buffer pH 8.0. Bound proteins are eluted with 3.0 M sodium chloride mixed with 1% glycine. Further procedures as in example 1. Obtain 0.54 mg of protein. The content of PPS-1 is 50%, the yield is 13.5%.

 The proposed method is an original way of isolating a new protein - the placental protein of pig-1.

 When studying the physicochemical properties of PPS-1, it was found that this protein has an electrophoretic mobility (relative to albumin) of 1.0, a molecular weight determined in gel filtration of about 90 kD. The protein contains carbohydrates when staining the precipitation arc according to the Schiff method and precipitates with ammonium sulfate at 60% saturation.

 The data of immunochemical analysis and physico-chemical properties of PPS-1 suggest that this protein is not identical to pig alpha-fetoprotein, found in large quantities in fetal serum; PPS-1 differs from the placental lactogen in pigs having a molecular weight of about 20 kD; from a trophoblastic protein of a pig, also having a molecular weight of about 20 kD.

 The development of an original method for the isolation of PPS-1 and the production of antiserum for 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 determining PPS-1 will make it possible to obtain a test for early diagnosis of pregnancy and its course. (56) EP N 0206181, CL C 07 K 13/00, C 12 N 15/06, 1987.

Claims (1)

 METHOD FOR PRODUCING PLACENTAL PROTEIN, including obtaining tissue homogenate, centrifugation, chromatography followed by elution, characterized in that pig tissue of the early placenta is used from tissues, affinity chromatography on sepharose with immobilized hexamethylenediamine in 0.06-0.08 is used. HCI buffer pH 7.5 - 8.5, elution is carried out with a 2.0 - 4.0 M sodium chloride solution with the addition of 1% glycine, an additional eluate is precipitated at 90% saturation with ammonium sulfate, the fraction is fractionated and centrifuged. iruyut on a Sephadex G-200 in 0.1 M ammonium bicarbonate buffer pH 8.15, fractions containing the desired product were collected and dialyzed.