NZ228344A

NZ228344A - Mammalian pregnancy specific protein (pspb) purified and isolated, designed psp 60

Info

Publication number: NZ228344A
Application number: NZ22834489A
Authority: NZ
Inventors: Jacques Martal; Sylvaine Camous
Original assignee: Agronomique Inst Nat Rech
Priority date: 1988-03-18
Filing date: 1989-03-15
Publication date: 1992-02-25
Also published as: ES2017811A6; AU3295989A; EP0406281A1; FR2628743B1; DK223290A; GR1000377B; DK223290D0; JPH03504236A; EP0406281B1; GR890100161A; PT90043B; WO1989008668A1; FR2628743A1; PT90043A

Description

<div class="application article clearfix" id="description"> .'^834 4 P rtority Date(&): I .'i Compete fpfcciftcat'.on Filed: Class: (5).. £.V... A £.I.-\i. S a. , .T.. f.... A £■.. .V. L\.. M. t... i7. £.... X ... J.. J. \:. f .V?.?.'AC^. .•>). iv: £ Publication OMe: P.O. .loiim*' »•"»: J.:-J > -A*-?. patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION NOV£L PROTEIN FOR THE RECOGNITION OF PREGNANCY IN MAMMALS, APPLICATIONS TO THE EARLY DETECTION OF PREGNANCY AND METHOD OF PREPARING THE SAID PROTEIN X/We, INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE (INRA), A French Company/ of 147/ Rue de 1'Universite, 75341 Paris Cedex 07, FRANCE hereby declare the invention, for which </we pray that a patent may be granted to rtfc/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - (followed by pagevliO I 22HAYl989«j) C< V7 lev The present invention relates to a novel protein for the recognition of pregnancy in ruminants, to its use, especially for the early detection of pregnancy, and to a method of preparing the said protein. 05 In cows, the implantation of an embryo does not take place before the 20th day of gestation. However, in order to survive, the conceptus must send a message to the maternal organism before the 17th day so as to prevent regression of the corpus luteum. There is therefore every 10 indication of the existence of embryonic substances, produced or stimulated by the conceptus, which are involved in the mechanisms of pregnancy recognition. BUTLER, HAMILTON, SASSER, RUDER, HASS and WILLIAMS (BIOL. REPROD. 2fi» 925, 1982) identified and isolated a pregnancy-15 specific protein B. This protein, isolated from extracts of the bovine placental membranes of 25-day to 270-day fetuses, has a molecular weight of the order of 47 to 53 kDa and an isoelectric point of 4.0 to 4.4. This protein B was obtained from the placental membranes of embryos 20 after 16 to 280 days of gestation, taken from slaughtered pregnant cows and then frozen at -20*C. The membranes -*■*1 were finely sliced with a razor blade and homogenized and the preparation was then centrifuged at 10,000 g for 30 minutes at +4*C. The resulting supernatant was kept. 25 The placental extract was placed in an ice bath and the fraction which precipitated at between 50 aund 65% saturation with ammonium sulfate is dissolved in 0.01 M Tris-HCl buffer, pH 7.5, at 4®C and dialyzed for 48 hours at 4*C against this buffer, with am exclusion limit of 30 12,000-13,000 Da. The dialyzate is concentrated and then subjected to an ion exchange chromatography procedure. This procedure was carried out on a column of DEAE-cellulose onto which the protein was charged in 0.01 M Tris-HCl buffer (pH 7-5) at a rate of 1 ml/min. and, 35 after elution of the non-fixed proteins, a linear .NaCA ■: o I"2 2 mAY 1989^1 3 gradient of 0 to 0.3 M is applied. The fraction® collected are eluted. Those having a pregnancy-specific antigenic activity are pooled, frozen and stored at -20*C; gel filtration ie then carried out on a column of Bio-Gel A-0.5 having an exclusion limit of 500,000 Daltona and a fractionation range of between 10,000 and 500,000 Daltons. The pregnancy-specific protein recovered by ion exchange chromatography is concentrated by ultrafiltration. European patent application 132 750 of 16 July 1984, filed claiming a US priority of 21 July 1983, is limited in its subject to a method of detecting pregnancy in mammals which consists in detecting the presence of an immune complex between an anti-protein B antibody and an antigen, in a physiological fluid of the pregnant mammal, which is outside the fetus, while the protein B is labeled, preferably by conjugation, so as to produce a detectable signal. One object which the inventors set out to achieve was to provide a protein for the recognition of pregnarvcv in riBnunls, more particularly in ruminants which has a high degree of puritv, is perfectly identified and whose preparation is strictly reproducible; another object which the inventors set out to achieve was to provide means, and in particular the said protein, for carrying out an early and specific diagnosis of pregnancy and a diagnosis of embryonic mortality; yet another object which the inventors set out to achieve, in preparing a pure and perfectly identified protein of the said type, was to elucidate the physiological properties of this molecule. The present invention relates to a pregnancy-specific protein analogous to PSPB - Sasser's Pregnancy-Specific Protein B - referred to hereafter as PSPgo,which has the N-terminal amino acid sequence represented by formula I below: 2 2 WAV 1989 ^28344 -X-GIy-Ser-X-Leu-Thr-Thr-His-Pro-L^-j-Arg-Acn-I le-Lys-Asp- 15 10 15 Leu-Val-Tyr-Met-Gly-X-I lo—Thr—11 e -G1 y-Thr-Pro—Pro-Gln-Glu— 20 23 30 Phe-Gln-Vel-Val-Phe-Aap-Thr-Ala-Ser-X- 35 40 (I) (the symbols X probably being aaparagines). According to the present invention,PSPgo^as a molecular weight of about 60 kDa (when analyzed by poly-acrylajnide gel electrophoresis in a denaturing medium) and an isoelectric point of about 5.1 to 5.5 after two-dimensional electrophoresis. This invention provides a -substantially purified mammalian pregnancv-specific protein, referred to as PSP60 w^ich ^as i-ts N-terminal partial amino acid sequence represented by formula I below: -X-Gly-Ser-X-Leu-Thr-Thr-His-Pro-Leu-Arg-Asn— I le- Lys-Asp-1 5 10 15 Leu-Val-Tyr-Met-Gly-X-I le-Thr-Ile-Gly-Thr-Pro-Pro-Gln-Glu- 20 28 30 Phe-Gln-Val-Val-Phe-Asp-Thr-Ala-Ser-x- 35 40 11) and which has an isoelectric point of 5.1 to 5.5 and wherein X is aspnrngjme. The present invention further relates to a method of preparing PSPgqfrora mammal placentas, wherein placentas, homogenized and extracted in an appropriate buffer, are subjected to acid precipitation and the supernatant is subjected to saline precipitation, the precipitate is then subjected to chromatography on an ion exchanger and subsequently to gel filtration, and the fractions possessing a PS? activity are purified by passage over a cation 60 exchange HPLC column, this being followed by elution with a saline gradient in an appropriate buffer at acid pH, which enables the PSPggto be isolated. According to the present invention,PSPg^ can be obtained by other methods, especially by other protein purification methods or by a biotechnological method.-'-^TE ^ (including genetic engineering, bacterial or cellular ,^ synthesis, transgenic animals, etc.). ? * J AN!? 92 (followed by page 3a) 3a 228344 The present invention further relates to a kit for carrying out a test for the early diagnosis of pregnancy in anarrrals rora particularlv ir. row-rants, '-friers, cotrprises, in addition to the customary reagents, buffers and/or diluents, a useful amount of anti-PSPg^polycionai or monoclonal antibodies. According to the invention, the antibodies are o —s -*V , ^0-1 ' /■? °A / v t» \ 1 1 DEC 1991n J ^*0 r , \f ^ • (followed by page 4) obtained either from sera of mammals immunized by PSPgn» or by the fusion of cells of majnmals immunized by PSPgo with malignant cells of appropriate mammals (such as mice in particular). In addition to the foregoing provisions, the invention also includes other provisions, which will become apparent from the following description. The invention will be understood more clearly with the aid of the following additional description, which refers to Examples of how the subject of the present invention is put into practice. It must be clearly understood, however, that these practical Examples are given solely by way of illustration of the subject of the invention, without in any way implying a limitation. The existence of PSPB was demonstrated by an American team (BUTLER et al., 1982), but a number of problems were not solved: reliability and limits of the application of the determination of PSPB to pregnancy diagnosis, interest in estimating embryonic mortality, actual source of secretion, precise biochemical characteristics of the molecule whose purest preparations (radioiodination preparations) were heterogeneous, immune sera directed against trophoblast homogenates or insufficiently purified protein preparations, imprecise and/or denaturing method of purification (isoelectrofocusing), poorly understood presence in the circulation of the fetus and in the fluids of the embryonic envelopes, and total ignorance of biological properties. Furthermore, a radioimmunoassay was available (cf. European patent application cited above) which could be used for the determination of poorly defined bovine pregnancy-specific proteins. The determination of PSPB, however, was of great interest since it was to make it possible to perform pregnancyp^-r^. specific diagnoses. In fact, the determination of proges-^' ^ :: ^ o 3' 14 terone, which reflects the luteal activity and is widely used at the present time, constitutes an excellent means of diagnosing non-pregnancy, but it may be the cause of a number of falee-poeitive pregnancy diagnoses in cases 05 where corpus luteum persists following embryonic mortality (as from the 16th day in cows). Moreover, because of the secretion of progesterone during each luteal phase of the estrous cycle, it is essential to know the date of natural or artificial Insemination with strict accuracy, which is 10 not always the case (beef breed cattle herds, for example). Example 1 Purification of bovine PSPgp bv HPLC Frozen fetal cotyledons were homogenized and ex-15 tracted in phosphate-KCl buffer (0.1 M), pH 0.6. The placental extract was subjected to acid precipitation (at pH 4.5) and the supernatant was precipitated with ammonium sulfate (40% and 65% saturation, pH 5.2). The precipitate was then dialyzed and subjected to chromatography on DEAE-20 Sephadex and then to gel filtration. The presence of in the fractions was determined by radioimmunology with the aid of impure PSPB preparations such as those described in the publication by BUTLER et al. The PSP-positive fractions were charged onto a cation exchange 25 HPLC column (TSK SP 5 PW; 75 mm x 7.5 mm internal diameter) and proteins were eluted with a linear KC1 gradient in 0.05 M phosphate buffer at pH 5.4 in an analytical column, or at pH 5.85 in a preparative column. Figure 1 shows major distinct peak (PSPg0) e luted from the HPLC colunn at a KCL 30 concentration of 0.195 M, has an immunological reactivity in the PSPB radioimmunoassay proposed by SASSER et al. When analyzed by polyacrylamide gel electrophoresis in a denaturing medium, this PSPgQPeak corresponds to a single band and has an apparent molecular weight of about 6 0 kDa. 35 After radioiodination, this protein precipitates with the ""S c u * i * i 6 anti-PSPBimmune sorum and has the same electrophoretic mobility as one of the proteins contained in the radio-iodinated PSPB preparation obtained by BUTLER, SASSER et al. However, the characterietico of the PSPB obtained by 05 BUTLER et al., namely MW » 47-53 kDa, isoelectric point 4-4.4, show that the PSI^ in the present invention is not the same molecule, since it has an MW of 60 kDa and an isoelectric point of 5.1-5.5. Example 2 Characterization of bovine PSPgg 10 2.1 Phvaicochemlcal nharwcterlzation After analysis by acrylamide gel electrophoresis in a denaturing medium, the peak of PSP^q purified by HPLC gives only a single band with an apparent MW of 6 0 kDa (instead of 47 to 53 kDa as obtained by BUTLER et al., 15 1902). However, under the same experimental conditions, SASSER's PSPB gives, after radioiodination, one protein with an apparent MW of 59 kDa and another, minor protein of 51 kDa. The isoelectric point of the PSPggpurified by HPLC was found to be 5.5 after isoelectrofocusing. It 20 therefore differs substantially from that of the PSPB described by SASSER and BUTLER. The PSP^opurified according to the invention can nevertheless be precipitated with SASSER's anti-PSPB immune serum. 2.2 Characterization bv a cell-free translation 25 system The synthetic capacity of poly(A)->- mRNA of 29-day ovine embryos in a cell-free system (lyzate of reticulocytes) in the presence of 36S-methionine was determined. The amount of PSPggsynthesized was estimated by specific 30 immunoprecipitation with the aid of bovine anti-PSPB immune serum, followed by SDS-electrophoresis :the precursor of PSP^gcorresponds to a protein of 49 kDa, the apparent MW of which is appreciably lower them that of PSP60 (6 0 kDa), very probably indicating the glycoprotein nature 35 of the native protein. This translation of PSPgQin a cell- ■ r ! v 10 - 7 free medium suggests that its secreted protein fraction has an apparent MW of about 46 kDa. Example 3. Radlommunoassay of PSP&n A quantitative detection method of PSP^ghas been developed by the inventors. It consists in a radiommunoassay, using two antibodies, under the following conditions: the PSPgQ used for the labelling with iodine 125 and for the calibration curves, is derived from the last purification step by HPLC chromatography. An anti-PSP^Q antiserum, obtained after immunization of rabbits, is used to the final dilution of 1/1 600 000; it binds 25 to 30% of the radioiodined PSP^q under the assay conditions ^ established by the inventors. Characteristics of the Assay: -sensitivity: this method enables 20 pg of PSP^q to be detected in 100 pi of sample; the intra- and inter-assay reproducibility, expressed by the coefficient of variation, are 6% and 12Z respectively for a serum sample containing 1.3 ng/ral of PSP6Q; -parallelism: the serum or the plasma of pregnant cow and the bovine embryo culture medium have dose-response curves parallel with the reference curve, as is shown by Figure 2 which represents the inhibition curve of the radioiodined PSPgg/anti-PSP^g antibody coupling obtained by increasing dilutions of serum or of culture medium. The other compounds present in the blood, or secreted in the culture medium by the embryos hence do not interfere with the PSP^q assay. Serum of pregnant sheep and bovine embryo culture medium cross with PSP^g, but in a non-parallel jnatjiT^r^ Csee (;n 20 25 30 I 22 may i 1989* 'f.vt; ^283-M 10 15 20 25 30 - 8 - Figure 2). - specificity of the assay: there is no cross-reaction with the bovine alpha foeto protein. Example 4. Characterisation of the PSP^qSecret ion In vitro Secretion of PSP^n by the Embryo By means of the assay, described in Example 3, it is possible to follow the evolution of the secretion of PSPgg by embryos cultivated in vitro. in the cow, the PSP^q is present from the 16th day of pregnancy in the culture media, although it is not yet detected in the blood. At 25 days of gestation, about ten explants of trophoblastic tissues ( about 0.3 mg of protein) secrete more than a hundred nanograms per 24 hours. In the sheep, PSP^q not detected in the culture media of 14 day embryos, but from 15 days gestation it is detectable (24 to 180 ng/embryo between 15 and 24 days gestation) with a heterologous assay . The assay of bovine PSP^q hence does not permit the precise determination in the sheep of the amounts secreted by the embryo. Cellular Localisation of the PSP^n by Immunocyto-fluorescence. The technique of imraunocytof 1 uorescenoe demonstrates a specific localization of PSP^q in the cytoplasm of binucleated giant cells of sheep trophoblast at 24 days gestation ( the other cell types are not labelled). Figure 3 reproduces a photograph of sheep allantochorion at the 24th day: the localization of the PSPgQ performed by immunocytofluorescence in the binucleated cells. Secretion during pregnancy in the cow The PSPgg appears in the maternal blood towards the f-' L " / % o 12 2 hay ,989Z;; \ \s\% - 9 - 25th-30th day of pregnancy. It increases gradually up to about thirty days before parturition : 1.9 ♦ 0.58 ng/ml (x ± SE) to 63 days (n ■ 3) ; 10.6 ± 1.67 ng/ol to 95 days (n ■ 4) and 71.1 £ 16.1 ng/ml to 233 days (n 3 5 8). About one month before parturition, the PSPgg level rises suddenly (314 ± 151 ng/ol at 261 days, n » 8) to reach its maximum at parturition (790 ng/ml in the cow), (Figure 4). Speed of disappearance of the post partum PSP^n is present in the maternal circulation at non-negligable levels up to approximately 10 weeks after parturition, then the levels are close to zero (Figure 5). The assay of the PSP^q every week, between 3 and 16 weeks post partum in 25 cows, enabled establishment of the regression curve of the logarithm of the 15 20 25 30 concentration of PSP&0 as a function of time (in weeks) (Figure 6). Y =-0.25 x - 3.24 Thus the half-life of the PSP^q has been estimated at 8.4 days. Example 5. Diagnosis of pregnancy by assay of PSP^,n On 61 cows and 79 heifers, a first pregnancy diagnosis by assay of the PSP^q was performed at 25,26 or 27 days after artificial insemination. In order to confirm the diagnosis, rectal palpation and blood sampling for the assay of the PSP^q were carried out at 90 days on all the animals which came in heat. The correctness of the positive diagnoses (number of correct positive diagnoses / total number of positive diagnoses) were close to 78% at 25 and 26 days and exceeded 90% at 27 days , in the cows, whereas at these 3 stages the correctness of the positive . , h ' L • / . •;. c ■ i ' * ^• / *f\ ! 'I989zi f V t •>' :^834'1 - 10 - diagnoses, in the heifers, is greater than 90 %, as is seen from Table 1 which follows. (0 o TABLE I ACCURACIES OF PREGNANCY DIAGNOSIS BY PSP60 RIA 25,26 OR 27 DAYS AFTER A I POSITIVE RESULTS ACCURACY day 25 day 26 day 27 7. n 7. n 7. n days 25 to 27 7. n COWS 78.3 (18/23) 78.9 (15/19) 94.7 (18/19) 83.6 (51/61) HEIFERS 91.7 (22/24) 95.6 (22/23) 96.9 (31/32) 94.9 (75/76) TOTAL 85.1 (40/47) 88.1 (37/42) 96.1 (49/51) b vs c p < 0.05 Accuracy of positive results ■» A/B A» the number of animals tested positive by PSP60 assay and rectal palpation at day 90 B-the number of animals tested positive by PSP60 assay .°2834'i - 12 - In all cases, the accuracy increases with the sampling stage andx is significantly higher in heifers than in cows (P<0.05). Only four cows were inseminated before the 70th day after calving and had a residual concentration of post partum PSP^q which involved an erroneous pregnancy diagnosis. Example 6 Establishment of embryonic mortalities by assay of the psp6o In cows or heifers which had a late embryonic mortality ( cf. Table II below), assay of the PSPgQ at 26, 35, 50 and 90 days after artificial insemination established a decrease of PSP^q concentrations and enabled the determination of the moment of this enbryonic mortality, confirmed by echography and rectal palpation. / v i 22HAYl9s<>° ( } r> TABLE II EMBRYONIC MORTALITY COM 5013 5017 5041 5058 5067 5074 FfelFER 6234 6298 PSP60 at day 26 ( ng/ml ) 0.7 0 0.3 0 0.8 0 PSP60 at day 35 ( ng/ml ) 2.2 1.4 4.7 0.3 1.2 0.8 1.3 PSP60 ECHOGRAPHY at day 50 at day 50 ( ng/ml ) 2.8 0.6 2.8 0 0.4 0.3 0.5 0.9 + viability? seen in estrus PSP60 RECTAL at day 90 PALPATION ( ng/ml ) at day 90 1.1 0 0 O ' 'it 8 O 'i - 14 - The PSPgOideritifled and Isolated according to the present invention has numerous applications such as: use 05 for purposes of pregnancy diagnosis, embryonic viability or mortality diagnosis, and fertility determination with a view to genetic selection. Furthermore, extending our knowledge of the PSPgQ identified according to the present invention will make it 10 possible to specify its physiological properties and their possible applications. As is apparent from the foregoing description, the invention is in no way limited to those methods of execution, embodiments and methods of application which have 15 now been described more explicitly; on the contrary, it encompasses all the variants which may occur to those skilled in the art, without deviating from the framework or the scope of the present invention. 20 25 30 35 ,;'r*Vr 4 © v <: im\ *ay i ^ '1989 51 9£ I V </div>

Claims

<div class="application article clearfix printTableText" id="claims"> ?283<14 - 15 - wway^-.vr Claim IS 1 . A subrtintlally purified maimialian prognancv-specific protein, referred to as PSP60 wh^-c^ has its N-terminal partial amino acid sequence represented by formula I below: 5 -X-Gly-Ser-X-Leu-Thr-Thr-His-Pro-Leu-Arg-Asn-I le-Lys-Asp-15 10 15 Leu-Val-Tyr-Met-Gly-X-I le-Thr-Ile-Gly-Thr-Pro-Pro-Gln-Glu-^ 20 28 30 Phe-Gln-Val-Val-Phe-Aop-Thr-Ala-Ser-x-10 35 .40 ai*i which has an isoelectric point! of 5.1 to 5.5 (wherein X is asparagine) .;2. A mammalian pregnancy-spocific protein, referred to as PSPgg which haa a molecular weight of about 60 kDa (when analyzed by polyacrylamide gel electrophoresis in a de-;15 naturing medium) and an isoelectric point of about 5.5 after two-dimensional electrophoresis.;3. A method of preparing PSPgQfrom mammal placentas,;wherein placentas, homogenized and extracted in an appropriate buffer, are subjected to acid precipitation;20 and the supernatant is subjected to saline precipitation, the precipitate is then subjected to chromatography on an O ion exchanger and subsequently to gel filtration, and the fractions possessing a PSPgQactivity are purified by passage over a cation exchange HPLC column, this being 25 followed by elution with a saline gradient in an appropriate buffer at acid pH, which enables the to fce isolated.;4. A kit for carrying out a test for the early diagnosis of pregnancy or the diagnosis of embryonic mortality,;30 which comprises, in addition to the customary reagents,;buffers and/or diluents, a diagnostically useful amount of-;anti-PSPgQ polyclonal or monoclonal antibodies.;I;- 16 -;5. A- mammalian pregnancy-specific hormone* as claimed in claim 1/ substantially as herein described with reference to the examples. 6. A kit for carrying out a test for the early diagnosis of pregnancy or the diagnosis of embryonic mortality as claimed in claim 4/ substantially as herein described with reference to the examples. 7. A method of preparing PSP^q as claimed in claim 3, substantially as herein described with reference to the examples - INSTITUT NATIONAL DE LA RECfaSBCHE AGRONOMIQUE (INRA) </div>