Shibayagi's Rat Growth Hormone (GH) ELISA KIT. Katsumi WAKABAYASHI, Ph. D. Prof. Emer., Gunma Univ. Technical Consultant, Shibayagi, Co., Ltd. 1.
A Brief Review of Growth Hormone (GH) and Introduction to Shibayagi’s Rat Growth Hormone (GH) ELISA KIT Katsumi WAKABAYASHI, Ph. D. Prof. Emer., Gunma Univ. Technical Consultant, Shibayagi, Co., Ltd.
1. What is GH? Growth hormone (GH, Somatoropic hormone, STH, Somatotropin) a simple protein hormone mainly produced in acido-philic cells, somatotrophs, which are present in the largest population. GH positive cells are found in the brain and lymphoid tissues. In human, A protein with high homology to GH named GH2 is found in the placental tissue. GH receptors of WSbox type are distributed among liver, kidney, adipocytes, muscle, thymus, etc., that belongs to the cytokine receptor group passing the cell membrane once. Such a wide distribution of GH receptor indicates multiple action of GH on various tissues.
2. Structure and nature of GH Rat GH is consisted of 190 amino-acids with the molecular weight of 21,777. The amino-acid sequence including signal peptide is shown below (PubMed, Protein PO1244).
1 maadsqtpwl ltfsllcllw pqeagalpam plsslfanav lraqhlhqla 51 adtykefera yipegqrysi qnaqaafcfs etipaptgke eaqqrtdmel 101 lrfsllliqs wlgpvqflsr iftnslmfgt sdrvyeklkd leegiqalmq 151 eledgsprig qilkqtydkf danmrsddal lknygllscf kkdlhkaety 201 lrvmkcrrfa esscaf Signal peptide is shown in red letters. The underlined parts indicate helix structure. There is a discrepancy about N-terminal amino acid read from DNA sequence. Here, N-terminal amino acid is written as leucine (1,2), while some reports told (3,4) that it is phenylalanine (f) instead of leucine (l).
However, Dr. A. F. Parlow who provided NIDDK rGH I-6, a highly
purified preparation of radioiodination for RIA stated the nature of the preparation as follow: “Chemical characterization of NIDDK-rGH-I-6, by analytic sephadex gel filtration, revealed it to be 95-100% monomeric. A similar degree of homogeneity was established by polyacrylamide gel and SDS-gel electrophoresis. In addition, NH2-terminal analysis by the dansyl method revealed only a single amino acid, leucine.” GH has disulfide bonds in two positions, 78-189 that forms a big loop, and 206-214 of a small loop. The big loop is indispensable for GH bioactivity. Ref: Mouse GH structure (PO6880)
1 matdsrtswl ltvsllcllw pqeasafpam plsslfsnav lraqhlhqla 51 adtykefera yipegqrysi qnaqaafcfs etipaptgke eaqqrtdmel 101 lrfsllliqs wlgpvqflsr iftnslmfgt sdrvyeklkd leegiqalmq 1
151 eledgsprvg qilkqtydkf danmrsddal lknygllscf kkdlhkaety 201 lrvmkcrrfv esscaf （Only two amino acids (valine) shown in bold letters are different from rat GH sequence. N-terminal amino acid is same in some reports.） GH is hydrophobic and difficult to be dissolved in water around neutral pH, however soluble under basic pH. In order to prepare the clear neutral solution, first dissolve GH in 0.01M Sodium bicarbonate, and then dilute it with neutral phosphate buffer (according to the instruction paper for NIDDK Rat GH RIA Kit). Isoform of human GH In human, the gene of GH is located in GH locus of chromosome no.17 with other 4 related genes. These 5 genes are similar to each other in base sequence. Furthermore, in the process of transcription, further isoforms are generated. This means that many isoforms of GH are present in the pituitary gland. GH found in the pituitary is called GH1, but here I will write only “GH”. Mutation and deficit of the gene will cause defect syndrome or lilliputian syndrome. (Please see isoforms generated in the transcription process in the last part of this text.) In human and monkey, placental lactogen (PL) is coded as one of GH-related genes in the GH locus, and has high homology to GH and has potent GH-like action and weak prolactin-like action. It is also called placental growth hormone or GH2. You can see amino acid sequence of 22kD isoform 1 of GH2 in the last part of this text. Please compare it with GH isoform 1. Rat or mouse PL gene, on the other hand, locates on different chromosome to that of GH, and is thought to be derived from prolactin gene because of its potent prolactin-like action.
3. Biological action of GH and regulation of secretion GH acts on liver, kidney, fibroblast, and thymus epithelial cells. In the liver, GH causes the gene expression and production of insulin-like growth factor-1 (IGF-1, somatomedin C), and IGF-1 enhances the growth through causing proliferation of cartilage cells, biosynthesis of chondroitin sulfate, hyperplasia and proliferation of various tissue cells, and protein anabolism, and enhances secretion of thymulin from thymus cells (5). Metabolic effect of GH is biphasic. Administration of GH tentatively shows insulin-like effect (suppression of amino acid decomposition, enhancement of protein synthesis, decrease of blood glucose, amino acids and free fatty acids), but later it causes increase of free fatty acids owing to degradation of fat in adipose tissue, raising blood glucose, and shows insulin antagonistic actions such as suppression of glycolysis, glycogen content increase in muscle, and lowering insulin sensitivity in peripheral tissue (6,7). GH has also prolactin like actions such as retention of Na, K, Mg, Ca, and P, enhancement of Ca absorption in small intestine, development of mammary gland, enhancement of milk secretion.
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Blood GH levels and related diseases Human diseases and other factors showing low blood GH levels: Pituitary dysfunction such as Sheehan syndrome, thyroid dysfunction, obesity, Cushing syndrome, hypoglycemia, pregnancy, treatment with corticosteroids. Human diseases showing high blood GH levels: acromegary, pituitary gigantism, anorexia nervosa, acute porphyria Factors to increase blood GH levels GHRH and ghrelin bind their receptors and activate Gs protein (stimulatory G protein) that activate PKA via cAMP, and with increased intracellular calcium ion, enhances GH production and secretion. Thyroid hormones, cortisol, and retinoic acid bind their nuclear receptors, which control transcription to effect GH gene expression. Glucagon, vasopressin, 1-deoxy-D-glucose administration, administration of amino acids such as arginine etc., protein intake, TF5, β−endorphin, L-DOPA, and stimulation of epinephrine α-receptor also promote GH secretion. Physiological conditions that cause GH secretion are hypoglycemia, stress (pyrexia, trauma, hemorrhage, ether anesthesia, psychic unrest), fasting, exercise, and non-REM sleep, etc. Factors to suppress GH secretion Somatostatin (SRIF) binds its receptor changes intracellular cAMP and calcium ion concentration via Gi protein (inhibitory G protein). Activin, stimulation of epinephrine β-receptor, glucose, free fatty acid, corticoid administration, high concentration of IGF-1, and high concentration of GH suppress GH secretion. Physiological conditions that suppress GH secretion are hyperglycemia, increased blood fatty acid level, and REM sleep, etc.
4. Important notes in GH assay Human GH measurement is much complex due to molecular heterogeneity and the presence of GH binding protein. As specific assay system for each isomer of GH were not available, physiological significance of isomer populations has not been clarified. Most isomers of GH are thought to be those produced by transcription of whole exons. Little has been known about rat GH isoforms, and so far, we have to observe physiological, pharmacological and pathological changes of the measurable levels of GH using the assay system available. Measurement of GH content in the gland According to my own measurement, GH content in adult male rat pituitary is around 300~350μgxRP-2/gland or 600~700 IU/gland (8). For RP-2, refer to the section of Standard preparation of GH”.
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In estimation of the pituitary hormone contents, their complete extraction from the gland is indispensable. All the hormones are stored in secretory granules, and some are hydrophobic. In my laboratory, we tested various methods for the simple and complete extraction of hormones (8). We found that prolactin was the most difficult to extract, and that immunoreactivty of GH was lost if high concentration of ethanol is present under neutral pH, and that simple homogenization with neutral phosphate buffer is not enough even for glycoprotein hormones. As our conclusion, we found two methods: 1) to homogenize the gland with neutral phosphate buffered saline (PBS) containing 1~2% urea followed by freezing and thawing, and then centrifugation, and 2) to sonicate （20watt, 10sec.x 3times）the gland with PBS containing about 0.1% Triton X-100, followed by freezing and thawing, then centrifugation. The centrifugation at 4,000rpm for 15 minutes will give a clear supernatant fluid. Measurement of blood GH levels Periodical secretion of GH GH secretion has been known to be periodical (9), i.e., the level rises and falls acutely with certain interval. Therefore, if blood samples are collected at random timing, the assay values will show a big variation as is seen in the assay data in the basic data section. According to a report (10) where continuous samplings were made from male adult rats, interval of GH peaks from the clock hour of 0:00 to 12:00 was 2.93±0.1 hour (mean±SE) , and 2.85±0.06 hour from 12:00 to 24:00. This means that GH is released periodically with about 3 hours’ interval. The width of the peak at the foot was reported to be about 2 hours, and this meant the width of trough is about 1 hour. The shape of the peaks was variable, and showed individual variation. As rats are nocturnal animals, from 20:00 to 8:00 (corresponds to their active period), the width of the trough seems to be wider, and height of peaks seems to be lower compared with those in the daytime (please, refer to the original report in internet.). We must be careful in planning of blood sampling in our experiments. Observation with serial sampling may be necessary. Influence of stress and anesthesia on blood GH level There are some reports telling changes in GH level caused by stress and anesthesia, suggesting that, in blood sampling for GH assay, we should be careful not to give animals unnecessary stress and avoid some anesthetics. Cold exposure for 5 minutes caused significant decrease in GH blood level to 43% of the onset level, and GH level was also decreased by 5 minutes’ immobilization stress (12). Eight hours’ immobilization a day for 10 days decreased GH levels , and GH level was further decreased by injection of GnRH+TRH during this period (13). GH level was increased by administration of ketamine(100mg/kg)＋Xylazine(10mg/kg) (15). Standard preparations of GH NIDDK rGH RP-2 is provided as a standard preparation for rat GH RIA. It is a component
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of NIDDK rat GH RIA kit. RP-2 has the purity equal to highly purified preparation, NIDDK rGH I series, and is added protective protein with buffer components. By adding fixed volume of purified water, we make up a standard solution containing a fixed amount of purified rat GH. Rat GH ELISA KIT provided by Shibayagi uses the standard calibrated using RP-2. So, the assay value is expressed as NIDDK rGH RP-2 equivalent. Statement about purity of NIDDK rGH: The biological potency of NIDDK-rGH-I-6 is 2.0 IU (Bovine GH) per mg. Contamination of this preparation with rPRL is 0.07%, with rFSH is 0.02%, with rLH is 0.2% and with rTSH is 0.004% (wt/wt), as determined by radioimmunoassay.（from Instruction paper for NIDDK rat GH RIA kit）
Introduction to Shibayagi’s Rat Growth Hormone (GH) ELISA KIT This is an ELISA (Enzyme Linked ImmunoSorbent Assay) kit for measurement of rat GH with high sensitivity using Sandwich assay principle. [Advantage] (1) Rapid assay (total reaction time: 5 hours.). (2) A small sample volume (5 μl). (3) An ecologically excellent preservative is used. (4) Every reagent is provided in liquid form and ready to use. (5) Excellent precision and reproducibility. [Components] Reagents
Amounts
(A)
Anti-GH-coated plate
96 wells(8x12) / 1 plate
(B)
Standard rat GH solution (20 ng/ml)
100μl / 1 vial
(C)
Buffer solution
60ml/１ vial
(D)
Biotin-conjugated anti-GH
100μl/ 1 vial
(E)
Peroxidase-conjugated streptavidin
100μl/ 1 vial
(F)
Chromogenic substrate reagent(TMB)
12ml/ 1 vial
(H) Reaction stopper (1M H2SO4)
12ml/ 1 vial
(I)
100ml/ 1 bottle
Concentrated washing buffer(10x)
[Assay sample] Rat serum or plasma: 5 μl in the standard procedure. The volume of assay sample can be applied in the range of 5 ~ 25μl.
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In such case the final volume to be added to the well should be adjusted to 50μl using assay buffer (C) as sample + buffer = 50μl. [Assay range] 31.3 ~ 2000 pg/ml This range corresponds to 313 ~ 20000 pg/ml of original serum/plasma level in the standard procedure. [Summary of Assay Procedure] Antibody-coated 96 well plate ↓ Washing 3 times ↓ Buffer 45 μl + Sample 5 μl or
Standard
(diluted sample 50μl)
50μl
↓ Shaking, then reaction for 2 hours at 20~25C ↓ Washing 3 times ↓ Biotin-conjugated anti-GH
50μl
↓ Shaking, then reaction for 2 hours at 20~25C ↓ Washing 3 times ↓ Peroxidase-conjugated avidin
50μl
↓ Shaking, then reaction for 30 mins at 20~25C ↓ Washing 3 times ↓ Chromogenic substrate solution
50μl
↓ Shaking, then reaction for 30 mins. at 20~25C ↓ Reaction stopper
1M H2SO4
50μl
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↓ Shaking, then measurement of absorbance at 450nm(sub. 620nm)
[Important notice in the treatments] 1. Treatment of assay samples (1) Use serum or plasma samples obtained by ordinary standard method. (2) Turbid samples or those containing insoluble materials should be centrifuged before assay and remove those materials. (3) Measure the samples as soon as possible after sampling. 2. Storage of assay samples. If assay samples have to be stored for a long period, freeze samples and store below –35oC. Avoid repeated freezing and thawing. 3. Influence of interfering substances If presence of interfering substances is suspected, examine by a dilution test using more than 2 points. [Assay range and standard curve] A model standard curve
Low concentration area extended
Stan dard c u rve
Standard curve
2.1
0.300 1.8
Abs.450( ⊿620)nm
Abs.450(⊿620)nm
0.250 1.5 1.2 0.9 0.6
0.200 0.150 0.100 0.050
0.3
0.000
0.0 0
500
1000
1500
2000
0
100
R at G H (pg/ml)
Rat GH (pg/ml)
[Storage condition] Store the kit at 2~8C.
200
Do not freeze.
[Term of validity] Six months from production. Expiration date is indicated on the container.
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300
[Unit of package] 96 wells/1 plate [Product code] AKRGH-010
Basic Data Assay precision Wells
Sample A
Sample B
1
262
864
2
247
837
3
250
813
4
258
775
5
251
780
6
257
800
7
254
771
8
270
779
Mean
256
802
SD
7.19
33.5
CV (%)
2.8
4.2 Unit: pg/ml
Reproducibility Samples
Day 1
Day 2
Day 3
Day 4
mean
SD
CV (%)
E
1626
1576
1615
1561
1595
31.0
1.9
F
412
407
409
401
407
4.50
1.1
G
96.0
97.9
96.1
103
98.3
3.35
3.4
Unit: pg/ml
n=4
Recovery test Sample C Added Found
Unit: pg/ml
n=2
Sample D
Recovered
Recovery (%)
Added
Found
Recovered Recovery (%)
0.00
101
-
-
0.00
506
-
-
155
265
164
106
303
822
316
104
192
285
184
95.8
466
949
443
95.1
223
325
224
100
539
1058
552
102
8
Dilution test
Dilution Test 2000
R = 0.999
GH (pg/ml)
1500
1000
500
R = 0.999 0 0.0
0.2
0.4
0.6
0.8
1.0
Dilution factor n=2 Normal rat sample assay results
Samples: CD(SD)、6 weeks old males, fed ad libitum Blood sampling: 14：00-15：00 Rat ID
GH ng/ml
1
10.4
2
7.49
3
7.16
4
7.21
5
6.52
6
3.59
mean
7.06
SD
2.17
GH levels after insulin administration Animals:CD (SD),7 weeks old females, body weight 160-190g Fasted for 6 hours. Insulin administered: Human Insulin Ultra Pure(Cell Science Inc),100μIU/100gBW. IV Assay samples: sera Assay kit: Rat Growth Hormone (GH) ELISA KIT, Lot.0712
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S erum G H levels after ins ulin injection (mean±S E of 5rats )
GH [ng/ml]
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 0
30
60
90
120
150
180
T ime after injection [min] Crossreactivity Crossreactivity of this assay kit to prolactin and rat placental lactogen is negligible. The kit does not crossreact with TSH, LH, nor FSH. Though the N-terminal amino acid is not fixed, it does not matter because the antibody of the kit recognizes other part. The kit crossreacts with mouse GH because the antibody recognizes the common amino acid sequence to both rat and mouse GH. We cannot show the reactivity by percentage because purified mouse GH preparation. It is possible to measure mouse GH using this kit if the assay results were expressed as rat GH RP-2 eeuivalent. Species
Substances examined
Reactivity(%)
r-GH
100
Prolactin
0.02
Placental lactogen
0.02
TSH
－
LH
－
FSH
－
Mouse
GH
＋
Mouse
TSH
－
Rat
＋：Cross-reacted －：No cross reaction *Amount of substance：2000pg/ml
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Assay results for mouse GH C57BL/6J males,6w
ICR males, 6w
Animal No.
GH ng/ml
Animal No.
GH ng/ml
N232-M1
13.2
N232-M11
19.9
N232-M2
12.4
N232-M12
12.4
N232-M3
11.3
N-232-M13
2.25
N232-M4
14.2
N-232-M14
6.44
N232-M5
14.2
N232-M15
12.8
N232-M6
12.2
N232-M16
5.49
N232-M7
11.6
N232-M17
16.3
N232-M8
16.8
N232-M18
7.12
N232-M9
14.3
N232-M19
5.78
N232-M10
9.84
N232-M20
13.4
Mean
13.0
Mean
10.2
SD
1.96
SD
5.59
GH assay values are expressed as NIDDK rat GH RP-2 equivalent．
References 1) Seeburg,P.H., Shine,J., Martial,J.A., Baxter,J.D. and Goodman,H.M. Nucleotide sequence and amplification in bacteria of structural gene for rat growth hormone Nature 270 (5637), 486-494 (1977)) 2) Rohn,W.M. and Weigent,D.A. Cloning and nucleotide sequencing of rat lymphocyte growth hormone cDNA Neuroimmunomodulation 2 (2), 108-114 (1995) 3) Page,G.S., Smith,S. and Goodman,H.M. DNA sequence of the rat growth hormone gene: location of the 5' terminus of the growth hormone mRNA and identification of an internal transposon-like element Nucleic Acids Res. 9 (9), 2087-2104 (1981) 4) Barta,A., Richards,R.I., Baxter,J.D. and Shine,J. Primary structure and evolution of rat growth hormone gene Proc. Natl. Acad. Sci. U.S.A. 78 (8), 4867-4871 (1981)
5)
Casanueva, F.F. Physiology of growth hormone secretion and action. Endocrinol Metab Clin North Am 21: 483-517（1992）
6)
Davidson, M.B. Effect of growth hormone on carbohydrate and lipid metabolism.
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Endocr Rev 8: 115-131 (1987) 7)
Strobl, J.S., Thomas, M.J. Human growth hormone. Pharmacol Rev 46: 1-34 (1994)
8)
Ishikawa, J., Fuse, Y. and Wakabayashi, K. Choice of extraction procedure for estimation of anterior pituitary hormone content. Endocrinol Japon 34: 755-767 (1987) GH secretion has been know to be episodic by Tannenbaum & Martin (1976),
9) Tannenbaum, G. S., and Martin, J. B. Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98, 562-570. 1976 10) Kimura, F. and Tsai, C.-W. Ultradian rhythm of growth hormone secretion and sleep in the adult male rat. J Physiol 353, 305-315 (1987)
11)
Popii, V. and Baumann, G. Laboratory measurement of growth hormone Clinical Chemica Acta: 350, 1-16 (2004)
12)
Specific hormonal and neurochemical response to different stresses. Lenox RH, Kant GJ, Sissions GR., et al. Neuroendocrinology 30:300-308, 1980
13)
Effects of chronic immobilization stress on pituitary hormone secretion, on hypothalamic factor levels, and on pituitary responsiveness to LHRH and TRH in female rats. Du Ruisseau P, Tache Y, Brazeau P, and Collu R. Neuroendocrinology 29: 90-99, 1979
14)
Changes in serum levels of gonadotropins and testosterone in the male rat in response to fasting, surgery and ether. Howland BE, Beaton DB and Jack MI. Experientia 30:1223-1225, 1974
15) Acute hyperglycemia induced by ketamine/xylazine anesthesia in rats: Mechanism and implication for preclinical models. Saha JK, Xia J, Grondin JM, et al. Exp Biol Med (Maywood) 230: 777-784, 2005
Growth hormone precursors of various animals Five isomers are known with human GH.
Human isoform 1 (P01241, NP_000506) 12
1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla 51 fdtyqefeea yipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle 101 llrisllliq swlepvqflr svfanslvyg asdsnvydll kdleegiqtl 151 mgrledgspr tgqifkqtys kfdtnshndd allknyglly cfrkdmdkve 201 tflrivqcrs vegscgf
All exons are transcripted, and the molecular weight is 22kDa. This isofom occupies most part of GH. Human isoform 2(NP_072053) 1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla 51 fdtyqefnpq tslcfsesip tpsnreetqq ksnlellris llliqswlep 101 vqflrsvfan slvygasdsn vydllkdlee giqtlmgrle dgsprtgqif 151 kqtyskfdtn shnddallkn ygllycfrkd mdkvetflri vqcrsvegsc 201 gf This isomer is produced by selective splicing of exson No. 3. As result, it 58-72 amino acid sequence is lost. Occupies about 10% of population. Human isoform 3 (NP_072054) 1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla 51 fdtyqefnle llrisllliq swlepvqflr svfanslvyg asdsnvydll 101 kdleegiqtl mgrledgspr tgqifkqtys kfdtnshndd allknyglly 151 cfrkdmdkve tflrivqcrs vegscgf This isomer lacks amino acid sequence corresponding to exon No. 3. Human isoform 4 (NP_072055) 1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla 51 fdtyqefrle dgsprtgqif kqtyskfdtn shnddallkn ygllycfrkd 101 mdkvetflri vqcrsvegsc gf This isomer lacks amino acid sequence corresponding to exons No. 3 and No. 4. Human isoform 5 (NP_072056) 1 mateagrwqp pdwadlqadl qqvrhkltqr This isomer lost exons No. 3,4,and 5 in the transcription process. Most part for signal peptide is also lost. Comparison between Human GH1 ( isoform 1) and Human GH2(placental lactogen) ( isoform 1)（NP_002050） Upper sequence:GH1，lower sequence:GH2 Different amino acids are shown in red letters. 1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla 1 maagsrtsll lafgllclsw lqegsafpti plsrlfdnam lrarrlyqla
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51 fdtyqefeea yipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle 51 ydtyqefeea yilkeqkysf lqnpqtslcf sesiptpsnr vktqqksnle 101 llrisllliq swlepvqflr svfanslvyg asdsnvydll kdleegiqtl 101 llrisllliq swlepvqllr svfanslvyg asdsnvryhl kdleegiqtl 151 mgrledgspr tgqifkqtys kfdtnshndd allknyglly cfrkdmdkve 151 mwrledgspr tgqifnqsys kfdtkshndd allknyglly cfrkdmdkve 201 tflrivqcrs vegscgf 201 tflrivqcrs vegscgf Chimpanzee
（P58756）
Sequence is same to human GH except one amino acid in the
signal peptide. 1 mapgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla 51 fdtyqefeea yipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle 101 llrisllliq swlepvqflr svfanslvyg asdsnvydll kdleegiqtl 151 mgrledgspr tgqifkqtys kfdtnshndd allknyglly cfrkdmdkve 201 tflrivqcrs vegscgf
Rhesus monkey (AAA20180) 1 maagswtcli laiallclpw lqegsafpti plswlfntav frahhlhkla 51 fdtypkfeea yipkeqkysf lrnpqtslcf sesiptpsnk eetqqksnle 101 llhisllliq swlepvqflr svfanhlvht nsnfdiylyl kkleegiqtl 151 mgrledgspr tgqifketys kydtnshndd tllknyrlly cfrkdmnkve 201 tflrtvrcra vegscgf
Cattle (NP_851339) 1 mmaagprtsl llafallclp wtqvvgafpa mslsglfana vlraqhlhql 51 101 151 201
aadtfkefer llrisllliq reledgtpra ylrvmkcrrf
tyipegqrys iqntqvafcf setipaptgk neaqqksdle swlgplqfls rvftnslvfg tsdrvyeklk dleegilalm gqilkqtydk fdtnmrsdda llknygllsc frkdlhktet geascaf
Pig (NP_999034) 1 mmaagprtsv llafallclp wtqevgalga mplsslfana vlraqhlhql 51 aadtykefdr pyipegqrys iqnaqaafcf setipaptgk deaqqrsdve 101 llrfsllliq swlgpvqfls rvftnslvfg tsdrvyeklk dleegiqalm 151 reledgspra gqilkqtydk fdtnlrsdda llknygllsc fkkdlhkaet 201 ylramkcrrf vesscaf
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Sheep (NP_001009315) 1 mmaagprtsl llaftllclp wtqvvgafpa mslsglfana vlraqhlhql 51 aadtfkefer tyipegqrys iqntqvafcf setipaptgk neaqqksdle 101 llrisllliq swlgplqfls rvftnslvfg tsdrvyeklk dleegilalm 151 reledvtpra gqilkqtydk fdrnmrsdda llknygllsc frkdlhktet 201 ylrvmkcrrf geascaf Chicken (CAA35619) 1 mapgswfspl liavvtlglp qeaaatfpam plsnlfanav lraqhlhlla 51 aetykefert yipedqrytn knsqaafcys etipaptgkd daqqksdmel 101 lrfslvliqs wltpvqylsk vftnnlvfgt sdrvfeklkd leegiqalmr 151 eledrsprgp qllrptydkf dihlrnedal lknygllscf kkdlhkvety 201 lkvmkcrrfg esncti Rainbow trout (AAA49553) 1 mgqvfllmpv llvscflsqg aaienqrlfn iavsrvqhlh llaqkmfndf 51 dgtllpderr qlnkiflldf cnsdsivspv dkhetqkssv lkllhisfrl 101 iesweypsqt liisnslmvr nanqisekls dlkvginlli tgnqdgvlsl 151 ddndsqqlpp ygnyyqnlgg dgnvrrnyel lacfkkdmhk vetyltvakc 201 rksleanctl (10/02/21)
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