基于早孕因子检测的奶牛早孕诊断ELISA方法的建立

发布时间：2018-04-03 06:21

本文选题：早孕因子　切入点：原核表达　出处：《石河子大学》2015年硕士论文

【摘要】：在奶牛的繁育过程中,奶牛的早期妊娠诊断非常重要。早孕因子(early pregnancy factor,EPF)是目前所知最早确认妊娠的生物化学标志物之一,是显示胚胎成活力的敏感标志。血清中早孕因子的检测可以预测胚胎的形成,监测胎儿生长状态,检测胚胎的早期消失。如果能在家畜上利用EPF进行早期妊娠诊断,可以在配种后很短的时间内鉴别奶牛是否受孕,此技术缩短产犊间隔,节约生产成本,提高奶牛的繁殖率,同时也有利于育种员及时掌握配种情况,在实际生产中具有重要意义。本研究运用基因工程技术克隆奶牛EPF基因,构建原核表达载体p ET32a-EPF,获得奶牛早孕因子重组蛋白,通过切胶纯化的方法对表达的蛋白进行纯化,最终建立了奶牛早期妊娠诊断的间接ELISA检测方法。利用该方法对怀孕28天的奶牛血清样品进行检测,结果发现,鼠抗牛EPF多克隆抗体与孕酮、人绒毛膜促性腺激素均不发生交叉反应;早孕因子重组蛋白的最低检出限为320ng/m L;妊娠奶牛血清样本的敏感性为91%(n=100);批内和批间重复性试验变异系数均低于10%;与美国爱德士牛怀孕检测试剂盒的检测结果对比,本研究建立的ELISA检测方法符合率为81.97%;对间接ELISA法检测结果进行统计学分析表明,妊娠奶牛(n=99)比空怀奶牛(n=92)血清中EPF活性高,且差异极显著(P0.01);2~3岁妊娠奶牛(n=55)血清中EPF活性比4~5岁妊娠奶牛(n=28)高,且差异极显著(P0.01);2~3岁妊娠奶牛(n=55)与6~7岁妊娠奶牛(n=10)血清中EPF活性差异不显著;不同胎次奶牛血清中EPF活性差异均不显著。上述结果表明,本实验建立的基于奶牛早孕因子检测的奶牛早孕诊断间接ELISA方法特异性强,敏感性高,重复性好,能够有效检出受孕奶牛的血清样本,为奶牛的妊娠诊断提供技术支持。
[Abstract]:The diagnosis of early pregnancy is very important in the breeding of cows.Early pregnancy factor (EPF) is one of the earliest known biomarkers of pregnancy, and is a sensitive marker of embryogenesis.The detection of early pregnancy factors in serum can predict embryo formation, monitor fetal growth, and detect early embryo disappearance.If EPF can be used for early pregnancy diagnosis in domestic animals, it can be used to identify whether the cow is pregnant or not in a very short time after mating. This technique can shorten the interval of calving, save the production cost and improve the reproduction rate of the cow.At the same time, it is helpful for breeders to grasp the breeding situation in time, which is of great significance in practical production.In this study, the cow EPF gene was cloned by genetic engineering technique, and the prokaryotic expression vector pET32a-EPFwas constructed, and the recombinant protein of cow early pregnancy factor was obtained.Finally, indirect ELISA detection method for early pregnancy diagnosis of dairy cattle was established.The results showed that there was no cross reaction between anti-bovine EPF polyclonal antibody and progesterone or human chorionic gonadotropin (HCG).The minimum detection limit of recombinant protein of early pregnancy factor was 320ng/m L; the sensitivity of serum samples of pregnant cows was 91%; the coefficient of variation of repeated tests was lower than 10%.The coincidence rate of ELISA was 81.97. The results of indirect ELISA analysis showed that the activity of EPF in serum of pregnant cows was higher than that of empty pregnant cows.There was no significant difference in serum EPF activity among different birth order cows.The results showed that the indirect ELISA method based on the detection of early pregnancy factors was specific, sensitive and reproducible, and could effectively detect the serum samples of pregnant cows.To provide technical support for the diagnosis of pregnancy in dairy cows.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S857.2

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