牦牛EPF基因克隆及其在配种后血液中的含量分析  

The Gene Cloning and Blood Concentration Analysis of EPF After Artificial Insemination in Yak(Bos grunniens)

作　　者：马进彪 樊江峰[1,2] 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 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(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep&Goat,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]甘肃省牛羊胚胎工程技术研究中心,兰州730070

出　　处：《农业生物技术学报》2020年第6期994-1003,共10页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2018YFD0502303);甘肃农业大学青年导师基金(GAU-QDFC-2018-10);甘肃省农业科技创新课题(GNCX-2014-30);国家自然科学基金(31660732)。

摘　　要：早孕因子(early pregnancy factor,EPF)是最早从妊娠期哺乳动物血清中分离中的一种生物活性蛋白,可通过调控妊娠诱导的免疫抑制反应阻止母体对胎儿的排斥作用。为了分析牦牛(Bos grunniens)EPF基因生物学特性及其在妊娠后血液中的变化规律,了解EPF与妊娠的相关性,本研究从牦牛卵巢组织中提取RNA并反转录为cDNA模板,RT-PCR扩增EPF基因并测序;利用生物信息学相关软件对牦牛EPF蛋白的理化性质以及结构预测分析。采集69头牦牛人工授精(artificial insemination,AI)20~40 d后牦牛血液样品,运用酶联免疫吸附法(enzyme-linked immuno sorbent assay,ELISA)测定EPF水平。本研究克隆获得了牦牛EPF基因完整的编码序列(coding sequence,CDS)区序列(GenBank No.MK992914),其开放阅读框长度为207 bp,编码69个氨基酸残基的可溶性非跨膜蛋白。ELISA结果显示,牦牛配种后20~40 d血液中EPF含量与妊娠状态密切相关。妊娠组从20 d开始,血液中EPF含量逐渐升高,28 d达到最高值,之后略有下降;未孕组配种后20~40 d期间,血液中EPF含量变化不明显,一直处于一个较低的水平,且极显著低于妊娠组(P<0.01)。结果表明,血液EPF可作为妊娠检测候选因子,用于牦牛的早期妊娠诊断,可以将牦牛的妊娠诊断时间提前到配种后20 d。本研究为牦牛早期妊娠诊断相关产品的研发以及技术体系的建立提供了基础资料。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.

关 键 词：牦牛 早孕因子(EPF) 克隆 妊娠诊断 血液 

分 类 号：S857.21[农业科学—临床兽医学]