KR101481788B1 - New Antigen for Detecting Rejection Response Including Miniature Pig Liver Protein According to Developmental Stages and the Kit for Detecting Rejection Response Includign the Same - Google Patents

Abstract

The present invention relates to a protein antigen capable of confirming rejection in heterologous organ transplantation between a pig and a human, and a kit for detecting rejection of heterologous organ transplantation including the protein antigen, and more particularly, To a kit for detecting an antigen and a kit for detecting the rejection of heterologous organ transplantation including a protein antigen showing a difference.
In the present invention, we reported protein control (4-day old newborn, 19-day old piglet and 14-month old adult miniature pig) for the developmental stage of the liver using 2-DE and MALDI-TOF. From the images of the three different steps, a total of 8 different spots were identified, and 5 spots were identified by MALDI-TOF MS. The data presented in this invention provide an important direction for the developmental stages between miniature pigs, which will aid in subsequent proteome analysis of the liver and enhance understanding of obstacles to heterologous organ transplantation. This may be useful for detecting rejection in heterologous organ transplantation as a novel antigen that is regulated differently by developmental stage.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel antigens for detection of heterologous organ transplant rejection reaction including a protein showing developmental stage differences between pigs, and a kit for detecting the same. Rejection Response Includign the Same}

The present invention relates to a protein antigen capable of confirming rejection in heterologous organ transplantation between a pig and a human, and a kit for detecting rejection of heterologous organ transplantation including the protein antigen, and more particularly, To a kit for detecting an antigen and a kit for detecting the rejection of heterologous organ transplantation including a protein antigen showing a difference.

To explain the biological role of the encoded protein, an overall understanding of gene expression regulation and expression range is important. Changes in gene expression configuration can provide evidence of regulatory mechanisms, cell function, and biochemical pathways. Therefore, the research focuses on the identification and characterization of functional proteins encoded by cellular genomes and whether they are independent or integrated [2]. The protein-based approach is very important because it features translation and post-translational modifications. Among the methods, proteome level 2D-electrophoresis (2DE) and MALDITOF MS (matrix-assisted laser desorption / ionization-time of flight mass spectrometry) monitoring have been recognized as very promising tools. Because of the gradual lack of human donor organization, there is increasing interest in the use of organs of animal tissues, especially non-human primates, for human transplantation [3,4]. Currently, it is dangerous to use non-human primate tissue as a substitute, and their organization is too small to function properly in humans [4]. Thus, currently, most studies focus on using pigs instead of nonhuman primates for organ transplantation [5]. In particular, inbreeding miniature pigs have been introduced by selective breeding programs for the past 25 years for successful heterologous transplantation [4]. However, by any heterogeneous organ transplantation effort, interspecific barriers are an impressive and potentially dangerous barrier [4]. Barriers of heterologous organ transplantation involving miniature pigs include: 1) rejection due to immunological barriers between humans and pigs, 2) the possibility of human pathogen infection from pigs, 3) physiological and anatomical interspecies Incompatibility, and 4) sociological agreement on patient response. If heterogeneous organ transplantation is realized, it is important to overcome these issues. To transplant pig tissues into humans, we need to know the nature of the proteins expressed in each tissue. Although miniature pig tissues are similar physiologically anatomically to humans, protein expression of tissues under normal conditions is not known. Little is known about the characteristics of the expressed proteins in the developmental stage after birth. Most of the proteome among pigs was not studied. Now, in order to get the same level of information as rodents and humans have, we have focused on the liver. Four-day miniature pigs (newborns), 19-day miniature pigs and 14-month-old miniature pigs (adults) were used to represent the developmental stage. The liver of each animal was analyzed by 2DE. Various protein expression spots were selected among these three steps, and proteins were identified by MALDI-TOF MS. Unfortunately, it is difficult to identify these proteins because the diverse protein databases in pigs are not well established in humans and rodents. The present invention is the first invention relating to a differentially expressed protein profile among miniature pigs according to the postnatal developmental stage. This may be useful for detecting rejection in heterologous organ transplantation.

DISCLOSURE Technical Problem In order to solve the problems of the prior art as described above, it is an object of the present invention to provide a protein antigen capable of confirming rejection in heterologous transplantation of pigs and humans, and a detection kit comprising the same, .

In order to achieve the above-mentioned object, the present invention provides a method for detecting a protein in the developmental stage pig, comprising the steps of: reacting an aldehyde dehydrogenase mitochondrial precursor, serum albumin, keratin, type II cytoskeletal-8 protein, The present invention provides antigens for heterologous organ transplantation detection comprising any one of the above-mentioned antigens.

In addition, the present invention provides a kit for detecting rejection of heterologous organ transplantation comprising the antigen.

Because of the lack of human tissue donors for transplantation, various studies of xenotransplantation or use of animal tissues instead of human tissue have been performed. Pig tissue has been considered safer and more suited for heterologous organ transplantation than nonhuman primates. Although heterologous organ transplantation is well known in humans and rodents, there is little understanding of the spatial expression of protein expression and post-translational regulation in other species and developmental stages, very important.

In the present invention, we reported protein control (4-day old newborn, 19-day old piglet and 14-month old adult miniature pig) for the developmental stage of the liver using 2-DE and MALDI-TOF. From the images of the three different steps, a total of 8 different spots were identified, and 5 spots were identified by MALDI-TOF MS. The data presented in this invention provide an important direction for the developmental stages between miniature pigs, which will aid in subsequent proteome analysis of the liver and enhance understanding of obstacles to heterologous organ transplantation. This may be useful for detecting rejection in heterologous organ transplantation as a novel antigen that is regulated differently by developmental stage.

Figure 1 shows the 2 DE gels between miniature pigs at 4, 19 and 14 months after development, which was visualized by Coomassie blue staining. A 1 mg protein sample was separated on a pH 3-10 nonlinear IPG strip (24 cm) and then separated into 8-18% gradient SDS-PAGE gel in two dimensions. Proteins were detected with Coomassie brilliant blue G-250 and compared using ImageMasterTM 2D Platinum Software version 5.0; A: 4 day miniature pig (newborn) 2DE gel, B: 19 day miniature pig 2DE gel, C: 14 month miniature pig 2DE gel. The black circle represents 13 differently expressed protein spots between the 4-day miniature neonatal pig, 19-day miniature pig pig, and 14-month-old miniature adult pig.
Figure 2 is a 2DE image of a replacement spot of the pancreas according to developmental stages. The circles represent differently expressed protein spots, and the expression patterns of the other three steps are represented by simple line graphs.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

< Example  1: Experimental animals>

Miniature pigs were raised in an air pollution free facility at SNU 's Center for Animal Resource Development. Three pigs of different ages and stages of development were used as described above. Intramuscular anesthesia is performed through a painful and painful procedure every time with 2 mL of ketamine HCl (50 mg / mL / kg), 1 mL xylazin (2.3 mg / mL / kg) and 1 mL of atropine And atropine sulfate (0.5 mg / mL / 10 kg). Each pig's abdomen was shaved and disinfected with polyvinylpyrrolidone-iodine. The animals were fixed on the surgical plate with a glue bundle. Median laparotomy was performed. The animals' liver was cut out in sterile conditions and stored in a liquefied nitrate tank during transplantation in the laboratory. These procedures were reviewed by the SNU Animal Experimental Ethics Committee according to university regulations.

< Example  2: 2 DE  Sample Preparation>

The liver was completely removed after the experiment and stored at -70 ° C until used. Frozen tissue (1 g) was pulverized into fine powder using a mortar in liquid nitrogen. Protein samples were collected and homogenized in 900 μL lysis buffer (7 M urea, 2 M thiourea, 2% w / v CHAPS, 2% Pharmalyte pH 3-10, 100 mM DTE). Samples were centrifuged at 50,000 rpm at 4 &lt; 0 &gt; C for 1 hour. The supernatant was carefully removed and immediately frozen at -70 ° C.

< Example  3: 2 DE  >

2D-polyacrylamide gel electrophoresis (PAGE) was performed as described above [6]. Partial samples containing 1 mg of total protein were diluted in lysis buffer to a total volume of 450 μL. Samples were treated with 240 mM immobilized, nonlinear pH 3-10 IPG Drystrip (Amersham Pharmacia Biotech, NJ) rehydrated for at least 12 hours. After rehydration, the strip is about 90,000 Vh (Ettan ^TM IPGphor ^TM II IEF systems; Amersham Pharmacia Biotech) was focused at 30 V for 3 hours, 100 V for 1 hour, 200 V for 1 hour, 500 V for 1 hour, 1000 V for 1 hour and finally 8,000 V for 11 hours. Once the isoelectric point electrophoresis is complete, the strips are incubated with 20% v / v glycerol, 2% w / v SDS and 0.01% w / v full term for BPB with 10 mM Tributyl phosphine (Flukachemie, Switzerland) Lt; RTI ID = 0.0 &gt; (Urea). &Lt; / RTI &gt; SDS-PAGE was performed using 8-18% separation gel without accumulation gel using an EttanDalt system (Amersham Pharmacia, NJ). Two-dimensional electrophoresis was performed overnight at 3 W / gel at 20 ° C. The gel was stained with Coomass G-250 (Bio-Rad Laboratories, Hercules, USA).

< Example  4: Protein visualization and image analysis>

The gel was stained with Coomassie G-250 (Bio-Rad) as described above [7]. The stained gels were scanned using a GS 800 photometer (Bio-Rad) and analyzed using ImageMaster (TM) 2D Platinum Software version 5.0 (GeneBio, Geneva, Switzerland). The digitized 2DE gel images were compared in a matching fashion. Different spots (> 3-fold and <1/3-fold) of the groups were analyzed and described.

Example 5: In - come digestion  >

Spots were cut into small pieces using 12.5 ng / μL trypsin (Promega, USA) at 50 mM ammonium bicarbonate pH 8.0 as described above (Gorg et al., 2000). For MALDI-TOF MS analysis, trypsin peptides were concentrated on a POROS 50 R2 column (Applied BioSystems, USA). After washing the column several times with 5% FA, 100% can and 5% FA with 70% acetonitrile, the samples were loaded onto a POROS 50 R2 column and washed with 5% FA. Samples were diluted with 2 μL of matrix solution consisting of 10 mg / mL α-cyano-4-hydroxy-cyanic acid (cinnamic acid, (Sigma-Aldrich, USA)) and dropped onto MALDI sample plates ].

< Example  6: MALDI - TOF  Protein Identification>

MALDI-TOF MS was performed using a Voyager DE-PRO spectrometer (Applied Biosystems) with a 337 nm nitrogen laser. The instrument was operated with an accelerating voltage of 20 kV, a cation reflection mode, a voltage grid of 74.5%, a guide wire voltage of 0% and a delay time of 120 ns. The spectra were internally measured by examining the Swiss-Prot confirmed protein and NCBI database using tryptic auto-degradation products (842.51 [M + H] and 2211.11 [M + H]) and using Mascot . Monoisotopic peaks [MH +] were selected, and all other studies were analyzed with 50 ppm mass tolerance.

 < Results  >

The differentially expressed liver protein profiles were identified by age. Liver protein was extracted and analyzed by 2D-PAGE. After the 2D gel was visualized in Coomassie blue staining, 2DE was repeated 3 times each for 3 pairs of independently matched samples (Fig. 1). For each sample, 3 gels with the best resolution of 1 were selected for analysis. Spot intensity analysis was measured and the resulting data set was confirmed by Image masterTM software (Geneva Bioinformatics, Switzerland). Of the 399 protein spots detected, 8 different spots were identified. Protein spots were selected that were increased by more than 3-fold or decreased by less than 1/3. Selected spots were cut from the gel, digested in-gel with trypsin, and subjected to peptide fingerprinting with MALDI-TOF MS. Peptide mass data was confirmed by Mascot. In this way, eight spots that were differentially expressed between pigs were identified. Five spots can be identified by protein function studies at http://www.ebi.ac.uk/ego (Table 1).

Increases and decreases in selected spot intensities were detected between the three gels from neonatal miniature pigs to adult miniature pigs (Figure 2). The expression level of the aldehyde dehydrogenase mitochondrial precursor (spot # 1) did not change to increase. Serum albumin expression levels (spot 2) and lambda-cristalline homolog (spot 7) were decreased. Although Spot 5 represents a similar spot intensity pattern, accurate identification could not be performed. The expression levels of keratin, type II cytoskeletal-3 protein were increased and regulated. Diffusion-related protein 2G4 expression was upregulated. Even though spots 4 and 8 exhibited similar intensity patterns, exact confirmation could not be performed.

The present invention first revealed the expression of liver protein in miniature pigs using proteomic analysis according to the developmental stage. Few studies have analyzed the proteome of whole pigs. A comprehensive understanding of proteins expressed in the liver can provide biological information. About 400 proteins with an isoelectric point pH of 3-10 and a Mr of 10-100 kDa were detected. Of the 399 spots, 8 protein spots were significantly changed among the groups and 5 proteins were identified.

Aldehyde dehydrogenase The mitochondrial precursor belongs to the aldehyde dihydrogenase family and is associated with the use of ethanol [9].

Serum albumin is the main protein of plasma and has good binding capacity for Ca2 +, Na +, K +, fatty acids, hormones, bilirubin and drugs in water. Its main function is colloid osmotic control of blood [10].

Keratin, type II, is a protein expressed in the bladder, liver, exocervix and esophagus [11], which helps to bind the contractile organs to the dystrophin in the costameres of the lateral striatum.

The proliferation-related protein 2G4 can play an important role in the ERBB3-regulated signaling pathway and seems to be involved in growth regulation. It is used as an inhibitor of the androgen receptor and is regulated by ERBB3 ligandneuregulin-1 / heregulin (HRG). This interferes with the transcription of some E2F1-regulated promoters, presumably using histone acetylase (HAT) activity. It binds to RNA and is involved in 28S, 18S and 5.8S mature RNAs, several rRNA precursors, and U3 small nucleolar RNA [12]. can be involved in the regulation of the intermediate and final stages of rRNA processing. It can also be involved in the mediated and independent translation process of ribosome assemblies and certain viral IRESs.

Lamda-cristallin homolog belongs to the 3-hydroxyacyl-CoA dihydrogenase family and is a lens component. However, it has been reported to be highly expressed in liver and kidney, and may be associated with carcinogenesis of liver cancer [13]. Of the five identified proteins except for the proliferation-associated protein 2G4, the proteins were expressed in the liver. However, it was confirmed by using the homology of other species except the serum albumin precursor.

Chen et al. And Moller et al. Also used human, bovine, mouse, and mouse homology relationships due to lack of pig database information [14,15]. Proteomic analysis of heterologous organ transplants has been used to investigate the trend of protein expression either for investigation of target proteins or after transplantation of tumor tissue into animals [16,17]. Anticancer drugs have been used after transplantation of human tumor tissues into animals at protein expression levels, and to clarify the efficacy of anticancer agents, we compared the modifications of protein expression between tumor-grafted and untreated tissue groups. Until now, no features of protein expression have been studied at the developmental stage of heterologous organ transplantation. For heterogeneous organ transplants, pigs are thought to have more potential value than nonhuman primates. However, the impediment to this potential was the lack of mapping of protein complementation of various tissues due to the scarcity of databases compared to humans and mice. Currently, there are more than 50,000 databases and more than 25,000 databases for humans and mice, respectively, while only 1072 databases for pigs are available in the MSDB database [1]. In addition, we were unable to identify proteomes (spots) identified by 2DE and MALDI-TOF due to the lack of commercial antibodies. Knowledge of modification of protein supplements may provide valuable information to overcome immunological barriers, microbiological differences, and problems caused by heterogeneity, which interfere with heterogeneous organ transplantation [3, 18]. Despite the limited data on the miniature pig liver due to the lack of databases, the data provided in the present invention is provided for the first time in the developmental stages of miniature pigs, and the important direction of additional proteomic analysis .

The embodiments presented above are illustrative and those skilled in the art will be able to make various modifications and alterations to the disclosed embodiments without departing from the technical spirit of the present invention. The scope of the present invention is not limited by these variations and modifications.

<References>

1. Junghans P, Kaehne T, Beyer M, Metges CC, Schwerin M. Dietary protein-related changes in hepatic transcription. J Nutr 2004; 134 (1), 43-47.

2. On the basis of the results of the experiments, we found that Pan TL, Goto S, Lord R, Huang YC, Huang CM, Wang PW, Lin YC, Kawamoto S, Ono K, Liao PC, Lin CL, Lai CY, Wu ML, Jawan B, Cheng YF, Chen ST, Chen CL. Proteome analysis in liver transplantation. Transplant Proc 2001; 33 (1-2): 156.

3. Platt JL. Physiologic barriers to xenotransplantation. Transplant

Proc 2000; 32 (7): 1547-1548.

4. Cooper DK, Gollackner B, Sachs DH. Will the pig solve the transplantation backlog? Annu Rev Med 2002; 53: 133-147.

5. Tucker, Belcher C, Moloo B, Bell J, Mazzulli T, Humara, Hughes A, McArdle P, Talbot A. The production of transgenic pigs for potential clinical use of xenotransplantation: baseline clinical pathology and organ size studies. Xenotransplantation 2002; 9 (3): 203-208.

6. Steiner S, Anderson NL. Pharmaceutical proteomics. Ann N Y Acad Sci 2000; 919: 48-51.

7. Groge, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000; 21 (6): 1037-1053.

8. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996; 68 (5): 850-858.

9. Alnouti Y, Klaassen CD. Tissue distribution, ontogeny, and regulation of aldehyde dehydrogenase (Aldh) enzymes mRNA by prototypical microsomal enzyme inducers in mice. Toxicol Sci 2008; 101 (1): 51-64.

10. Akram M, Ali Shah SM, Asif HM, Shaheen G, Shamim T, Khan MI, Ullah A, Ahmed K. Comparative study of similarity and identity of human albumin with some selected organism albumin. J Med Plants Res2011; 5 (19): 4974-4976.

11. Magin TM, Jorcano JL, Franke WW. Cytokeratin expression in simple epithelia. II. cDNA cloning and sequence characteristics of bovine cytokeratin A (No. 8). Differentiation 1986; 30 (3): 254-264.

12. Parker KA, Bruzik JP, Steitz JA. An in vitro interaction between the human U3 snRNP and 28S rRNA sequences near the alphasarcin site. Nucleic Acids Res 1988; 16 (22): 10493-10509.

13. Chen J, Yu L, Li D, Gao Q, Wang J, Huang X, Bi G, Wu H, Zhao S. Human CRYL1, a novel enzyme-crystallin overexpressed in liver and kidney and downregulated in 58% of liver cancer tissues from 60 Chinese patients, and four new homologs from other mammalians. Gene 2003; 302 (1-2): 103-113.

14. Chen FC, Chuang TJ. ESTviewer: a web interface for visualizing mouse, rat, cattle, pig and chicken conserved ESTs in human genes and human alternatively spliced variants. Bioinformatics 2005; 21 (10): 2510-2513.

15. Moller M, Berg F, Riquet J, Pomp D, Archibald A, Anderson S, Feve K, Zhang Y, Rothschild M, Milan D, Andersson L, Tuggle CK. High-resolution comparative mapping of pigs Chromosome 4, emphasizing the FAT1 region. Mamm Genome 2004; 15 (9): 717-731.

16. Kahlem P, Dorken B, Schmitt CA. Cellular senescence in cancer treatment: friend or foe J Clin Invest 2004; 113 (2): 169-174.

17. Besada V, Diaz M, Becker M, Ramos Y, Castellanos-Serra L, Fichtner I. Proteomics of xenografted human breast cancer indicates novel targets related to tamoxifen resistance. Proteomics 2006; 6 (3): 1038-1048.

18. Coggin JH Jr, Barsoum AL, Rohrer JW, Thurnher M, Zeis M. Contemporary definitions of tumor specific antigens, immunogens and markers as related to the cancer-bearing host. Anticancer Res 2005; 25 (3c): 2345-2355.

Claims (2)

The proteins expressed in the developmental stage pig liver include aldehyde dehydrogenase mitochondrial precursor, serum albumin, keratin, type II cytoskeletal-8 protein (Keratin, ), A proliferation-associated protein 2G4, or a lambda-cristallin homolog. A kit for detecting rejection of heterologous organ transplantation comprising the antigen of claim 1.

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