本文選題：早孕因子　切入點(diǎn)：原核表達(dá)　出處：《石河子大學(xué)》2015年碩士論文

【摘要】：在奶牛的繁育過程中,奶牛的早期妊娠診斷非常重要。早孕因子(early pregnancy factor,EPF)是目前所知最早確認(rèn)妊娠的生物化學(xué)標(biāo)志物之一,是顯示胚胎成活力的敏感標(biāo)志。血清中早孕因子的檢測可以預(yù)測胚胎的形成,監(jiān)測胎兒生長狀態(tài),檢測胚胎的早期消失。如果能在家畜上利用EPF進(jìn)行早期妊娠診斷,可以在配種后很短的時間內(nèi)鑒別奶牛是否受孕,此技術(shù)縮短產(chǎn)犢間隔,節(jié)約生產(chǎn)成本,提高奶牛的繁殖率,同時也有利于育種員及時掌握配種情況,在實(shí)際生產(chǎn)中具有重要意義。本研究運(yùn)用基因工程技術(shù)克隆奶牛EPF基因,構(gòu)建原核表達(dá)載體p ET32a-EPF,獲得奶牛早孕因子重組蛋白,通過切膠純化的方法對表達(dá)的蛋白進(jìn)行純化,最終建立了奶牛早期妊娠診斷的間接ELISA檢測方法。利用該方法對懷孕28天的奶牛血清樣品進(jìn)行檢測,結(jié)果發(fā)現(xiàn),鼠抗牛EPF多克隆抗體與孕酮、人絨毛膜促性腺激素均不發(fā)生交叉反應(yīng);早孕因子重組蛋白的最低檢出限為320ng/m L;妊娠奶牛血清樣本的敏感性為91%(n=100);批內(nèi)和批間重復(fù)性試驗變異系數(shù)均低于10%;與美國愛德士牛懷孕檢測試劑盒的檢測結(jié)果對比,本研究建立的ELISA檢測方法符合率為81.97%;對間接ELISA法檢測結(jié)果進(jìn)行統(tǒng)計學(xué)分析表明,妊娠奶牛(n=99)比空懷奶牛(n=92)血清中EPF活性高,且差異極顯著(P0.01);2~3歲妊娠奶牛(n=55)血清中EPF活性比4~5歲妊娠奶牛(n=28)高,且差異極顯著(P0.01);2~3歲妊娠奶牛(n=55)與6~7歲妊娠奶牛(n=10)血清中EPF活性差異不顯著;不同胎次奶牛血清中EPF活性差異均不顯著。上述結(jié)果表明,本實(shí)驗建立的基于奶牛早孕因子檢測的奶牛早孕診斷間接ELISA方法特異性強(qiáng),敏感性高,重復(fù)性好,能夠有效檢出受孕奶牛的血清樣本,為奶牛的妊娠診斷提供技術(shù)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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S857.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張宏剛;陳樹林;王曉珊;張文華;孟霞;唐彩琰;;孕牛血清早孕因子的生物活性檢測及妊娠率分析[J];動物醫(yī)學(xué)進(jìn)展;2010年08期

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本文編號：1703983