The Gene Cloning and Blood Concentration Analysis of EPF After Artificial Insemination in Yak (Bos grunniens)
MA Jin-Biao1, FAN Jiang-Feng1,2,*, YU Si-Jiu2, CUI Yan2, HAN Xiao-Hong2, MA Yue1, HE Hong-Hong2, LI Qin2, ZHAO Sheng-Xian1, ZHANG Xing-Yun1, CAO Yan-Tao1, Bao Mei1
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep & Goat, Lanzhou 730070, China
Abstract：Early Pregnancy Factor (EPF) is a bioactive protein that firstly separated from gestational mammal serum. EPF prevents maternal rejection of the fetus in the manner of regulating immunosuppressive action. To analyze the biological characteristics and variation pattern of EPF in yak (Bos grunniens) blood, verify the correlation between blood EPF concentration and pregnancy, yak ovarian EPF gene sequencing was conducted after RT-PCR. The physicochemical properties and protein structure of yak EPF were predicted using bioinformatics related software. Blood samples were collected from 69 yaks during 20~40 d after artificial insemination (AI). The EPF level was measured by enzyme-linked immuno sorbent assay (ELISA). The complete coding sequence (CDS) of yak EPF gene was cloned and submitted to GenBank (No. MK992914). The open reading frame was 207 bp in length and encoded 69 amino acids. The ELISA results showed that the serum EPF level was closely related to the pregnancy status of female yak. In pregnancy group, the EPF concentration in serum gradually increased after the 20 d of AI, and reached the highest value on the 28 d and subsequently decreased slightly. In the non-pregnant group, the EPF concentration in blood maintained a stable and low concentration during 20~40 d after AI, and was significantly lower than that in pregnancy group (P<0.01). These results implied that blood EPF, as a candidate factor, could be used in early pregnancy diagnosis. It shortened the confirming time to 20 days by using blood EPF inspection in pregnancy diagnosis of yak. This study provides basic data for the research and development of products related to early pregnancy diagnosis of yaks and the establishment of technical systems.
马进彪, 樊江峰, 余四九, 崔燕, 韩小红, 马悦, 何翃闳, 李秦, 赵生贤, 张兴云, 曹艳桃, 包梅. 牦牛EPF基因克隆及其在配种后血液中的含量分析[J]. 农业生物技术学报, 2020, 28(6): 994-1003.
MA Jin-Biao, FAN Jiang-Feng, YU Si-Jiu, CUI Yan, HAN Xiao-Hong, MA Yue, HE Hong-Hong, LI Qin, ZHAO Sheng-Xian, ZHANG Xing-Yun, CAO Yan-Tao, Bao Mei. The Gene Cloning and Blood Concentration Analysis of EPF After Artificial Insemination in Yak (Bos grunniens). 农业生物技术学报, 2020, 28(6): 994-1003.
[1] 卜建华, 雷静, 李艳艳, 等. 2015. 奶牛早期妊娠诊断研究进展[J]. 宁夏农林科技, 56(11): 54-55, 87. (Bu J H, Lei J, Li Y Y, et al. 2015. Advances in researches of early pregnancy diagnosis of d airy cow[J]. Ningxia Journal of Agriculture and Forestry Science and Technology, 56(11): 54-55, 87.) [2] 包凯, 曹宇, 刘光磊, 等. 2016. 奶牛的早期妊娠诊断研究进展[J]. 现代畜牧兽医, (08): 49-52. (Bao K, Cao Y, Liu G L, et al. 2016. Advance of early pregnancy diagnosis technology in dairy cows[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, (08): 49-52.) [3] 韩小红, 樊江峰, 于艺腾, 等. 2018 妊娠期和分娩前后牦牛胎盘组织形态学变化及细胞凋亡的研究[J]. 农业生物技术学报, 26(10): 1714-1722. (Hai X H, Fan J F, Yu Y T, et al.2018. Study on the morphological changes and apoptosis of yak (Bos grunniens) placentomes from pregnancy, pre and post-parturation[J]. Journal Of Agricultural Biotechnology, 26(10): 1714-1722. ) [4] 何翃闳, 潘阳阳, 张慧珠, 等. 2018. FSH对牦牛卵母细胞EGF、EGFR表达及其细胞凋亡的影响[J].畜牧兽医学报, 49(09): 1899-1907. (He H H, Pan Y Y, Zhang H Z, et al.2018. The effects of follicle-stimulating hormone(FSH) on the expresssion of EGF and EGFR in yak oocy and apoptosis[J]. Chinese Journal of Animal and Veterinary Sciences, 49(09): 1899-1907.) [5] 贺加双. 2011. 肉牛早孕因子(EPF)的活性检测及分离纯化[D]. 硕士学位硕士.山东农业大学. 导师: 马卫明, pp. 1-52. (He S J.2011. Activity detection and purification of early pregnancy factor in beef cattle[D]. Thesis for M.S., Shandong Agricultural University. Supervisor: Ma W M, pp. 1-52.) [6] 靳智勇. 2008. 兽用超声多普勒仪在奶牛早期妊娠诊断中的应用[J]. 青海大学学报(自然科学版), 26(2): 77-78. (Jin Z Y.2008. Application of veterinary ultrasound Doppler instrument in early pregnancy diagnosis of dairy cows[J]. Journal of Qinghai University(Nature Science), 26(2): 77-78.) [7] 刘水喜. 2016. 奶牛早期妊娠诊断几种方法的比较[J]. 畜禽业, (12): 35-36. (Liu S X. 2016. Comparison of several methods for early pregnancy diagnosis of dairy cows[J]. Livestock and Poultry Industry, (12): 35-36.) [8] 李艳艳, 卜建华, 雷静, 等. 2016. PAG ELISA早孕检测试剂盒对奶牛繁殖效率的影响[J].黑龙江畜牧兽医, (14): 70-71. (Li Y Y, Bo J H, Lei J, et al. 2016. Effect of PAG ELISA early pregnancy test kit on reproductive efficiency of dairy cows[J]. Heilongjiang Animal Science and Veterinary Medicine, (14): 70-71.) [9] 宋立峰, 刘静静, 刘月琴, 等. 2014. 早孕因子蛋白基因的克隆及生物信息学分析[J]. 中国草食动物科学, (S1): 190-193. (Song L F, Liu J J, Liu Y Q, et al. 2014. Cloning and bioinformatics analysis of early pregnancy factor protein gene[J]. China Herbivore Science, (S1): 190-193.) [10] 马勇江, 靳亚平, 王爱华, 等. 2003. CD58与山羊早孕因子之间关系的研究[J]. 畜牧兽医学报, (02): 152-156. (Ma Y J, Jin Y P, Wang A H, et al. 2003. Study on the relationship between CD58 and EPF in goat[J]. Chinese Journal of Animal and Veterinary Sciences, (02): 152-156.) [11] 孙培培. 2017. 基于EPF检测的奶牛早期妊娠诊断方法的研究[D]. 硕士学位论文, 石河子大学, 导师: 贾斌, pp. 1-53. (Sun P P.2017. Study on the diagnosis method of early pregnancy in dairy cattle based on EPF detection[D]. Thesis for M. S., Shihezi University, Supervisor: Jia B, pp. 1-53.) [12] 王宏娟. 2012. 肉用母牛发情周期体内生殖激素变化规律与早期妊娠诊断技术的研究[D]. 硕士学位论文, 吉林大学, 导师: 周虚, pp. 1-38. (Wang H J.2012. Changes of reproductive hormones concentrations during estrus cycle and early pregnancy diagnosis in beef cattle[D]. Thesis for M.S., Jilin University, Supervisor: Zhou X, pp. 1-38.) [13] 王靖雷, 王萌, 潘阳阳, 等. 2019. Artemin在牦牛主要生殖器官和孤雌激活胚胎中的表达及定位[J]. 农业生物技术, 27(03): 431-440. (Wang J L, Wang M, Pan Y Y, et al.2019. Artemin expression and localization in main reproductive organs and parthenogenetic embryos of yak (Bos grunniens)[J]. Journal of Agricultural Biotechnology, 27(03): 431-440.) [14] 王立斌, 樊江峰, 余四九. 2009.牦牛不同妊娠阶段孕酮的主要来源器官研究[J].中兽医医药杂志, 28(05): 17-20. (Wang L B, Fan J F, Yu S J.2009. Studies on the source of progesterone during different period of gestation in yaks[J]. Journal of Traditional Chinese Veterinary Medicine, 28(05): 17-20.) [15] 王晓姗. 2018. 孕牛血清早孕因子的研究进展[J]. 中国牛业科学, 44(03): 82-85. (Wang X S.2018. Rsearch progress of early pregnancy factor of pregnancy bovine serum[J]. China Cattle Science, 44(03): 82-85.) [16] 王晓姗, 陈树林, 张文华, 等. 2018. 孕牛血清中早孕因子的分离纯化与鉴定[J]. 中国牛业科学, 44(02): 22-24. (Wang X S, Chen S L, Zhang W H, et al.2018. Isolation and purification of early pregnancy factor from pregnant bovine serum by using chromatography techniques[J]. China Cattle Science, 44(02): 22-24.) [17] 袁小娟, 吴希茜. 2011.甘氨酸的生理作用与应用[J]. 饮料工业, 14(7): 5-7. (Yuan X J, Wu X X.2011. Physiological role and use of glycine[J].The Beverage Industry, 14(7): 5-7.) [18] 张慧茹, 王爱华, 马勇江, 等. 2002.应用猪Ea花环抑制试验测定山羊和牛EPF活性[J]. 西北农业学报, (01): 6-9. (Zhang H R, Wang A H, Ma Y J, et al. 2002. Pig rosette inhibition test with pig lymphocytes for detection of epf in goat and cattle serum[J]. Acta Agriculturae Boreali-occidentalis Sinica, (01): 6-9.) [19] 张诗萌, 佟翠, 李鹏, 等. 2019. 早孕因子及其临床应用研究进展[J]. 黑龙江畜牧兽医, (05): 41-43. (Zhang S M, Tong C, Li P. et al. 2019. Research progress on early pregnancy factor and its clinical application[J]. Heilongjiang Animal Husbandry And Veterinary Medicine, (05): 41-43.) [20] 赵天, 李谷月, 潘阳阳, 等. 2017. Oct4在牦牛卵母细胞及早期胚胎发育过程中的表达[J].畜牧兽医学报, 48(03): 577-584. (Zhao T, Li G Y, Pan Y Y, et al.2017. The Expression of octamer-binding transcription factor 4 Gene (Oct 4) in yak oocytes and preimplantation embryos produced by in vitro fertilization[J]. Chinese Journal of Animal and Veterinary Sciences, 48(3): 577-584.) [21] Bai W L, Yang R J, Yin R H, et al.2012. Molecular character-ization and expression analysis of osteopontin cDNA from lactating mammary gland in yak (Bos grunniens)[J]. Molecular Biology Reports, 39(4): 3627-3635. [22] Chill H H, Malyanker N, Karavani G, et al.2017. Association between uterine position and transvaginal misoprostol treatment for early pregnancy failure[J]. Journal of Obstetrics & Gynaecology Research, 44(2): 248-252. [23] Corrao S, Campanella C, Anzalone R.2010. Human Hsp10 and early pregnancy factor (EPF) and their relationship and involvement in cancer and immunity: Current knowledge and perspectives[J]. Life Sciences, 86(5-6): 145-152. [24] El-Battawy K A, Sousa N M, Szenci O, et al.2009. Pregnancy-associated glycoprotein profile during the first trimester of pregnancy in egyptian buffalo cows[J]. Reproduction in Domestic Animals, 44(2): 161-166. [25] Fan X G, Zheng Z Q.1997. A studyof early pregnancy factor activity in preimplantation[J]. American Journal of Reproductive Immunology, 37(5): 359-364. [26] Fricke P M.2002. Scanning the future--ultrasonography as a reproductive management tool for dairy cattle[J]. Journal of dairy science, 85(8): 1918-1926. [27] Ghaffari L V, Ghaffari L R, Pirany N, et al.2008. Measurement of EPF for detection of cow pregnancy using rosette inhibitiontes[J]. Theriogenology, 70(1): 105-107. [28] Green J A, Parks T E, Avalle M P, et al.2005. The establishment of an ELISA for thedetection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers[J]. Theriogenology, 63(5): 1481-1503. [29] Grosso M C, Bellingeri R V, Schade R, et al.2012. Neutralization of early pregnancy factor by passive immunization alters normal embryonic development and cytokine balance[J].Inmunologia, 31(4): 106-114. [30] Haq A, Mothi B A, Al-Hussein K, et al.2001. Isolation, purification and partial characterization of early pregnancy factor (EPF) from sera of pregnant women[J]. European journal of medical research, 6(5): 209-214. [31] Hatzel J N, Bouma G J, Cleys E R, et al.2015. Identification of heat shock protein 10 within the equine embryo, endometrium, and maternal peripheral blood mononuclear cells[J]. Theriogenology, 83(5): 832-839 [32] Klima F, Tiemann U, Schadow D, et al.1992. Bovine early pregnancy factor (EPF) activity dependent on a 67-kDa polypeptide[J]. Journal of reproductive immunology, 21(1): 57. [33] Lash G E, Legge M, Fisher M.1997. Synthesis of early pregnancy factor using red deer as a delayed implantation model[J]. Journal of Assisted Reproduction and Genetics, 14(1): 39-43. [34] Morton H, Cavanagh A, Athanasas-Platsis S, et al.1992. Early pregnancy factor has immunosuppressive and growth factor properties[J]. Reproduction, Fertility and Development, 4(4): 411-422. [35] Morton H, Hegh V, Clunie G J.1974. Immunosuppression detected in pregnant mice by rosette inhibition test[J]. Nature, 249(456): 459-460. [36] Mansell P.2010. Current therapy in large animal theriogenology[J]. Australian Veterinary Journal, 76(7): 500. [37] Mansell J P, Yarram S J, Brown N L, et al.2002. Type 1 collagen synthesis by human osteoblasts in response to placental lactogen and chaperonin10, a homolog of early-pregnancy factor[J]. In Vitro Cellular and Developmental Biology, 38(9): 518-522. [38] Nancarrow C D, Wallace A L, Grewal A S.1981. The early pregnancy factor of sheep and cattle[J]. Journal of Reproduction and Fertility Supplement, 30:191-199. [39] Ohnuma K, Yokoo M, Ito K, et al.2000. Study of early pregnancyfactor (EPF) in equine (Equus caballus)[J]. American Journal of Reproductive Immunology, 43(3): 174-179. [40] Piechotta M, Bollwein J, Friedrich M, et al.2011. Comparison of commercial ELISA blood tests for early pregnancy detection in dairy cows[J]. Journal of Reproduction & Development, 57(1): 72-75. [41] Quinn K A, Cavanagh A C, Hillyard N C, et al.1994. Early pregnancy factor in liver regeneration after partial hepatectomy in rats: Relationship with chaperonin 10[J]. Hepatology, 20(5): 1294-1302. [42] Rolfe B E, Morton H, Cavanagh A C, et al.1983. Detection of an early pregnancy factor-like substance in sera of patients with testicular germ cell tumors[J]. American Journal of Reproductive Immunology. 3(2): 97-100 [43] Siva E, Sterry R A, Kolb D, et al.2007. Accuracy of pregnancy associated glycoprotein ELISA to determine pregnancy status of lactating dairy cows twenty-seven days after timed artificial in semination[J]. Journai of Dairy Science, 90(10): 4612-4622.
