【摘要】：本研究旨在制備新疆南疆平原型和田羊、山區(qū)型和田羊與卡拉庫爾羊3個地方品種(系)綿羊毛囊角蛋白關(guān)聯(lián)蛋白7(keratin associated protein 7,KAP7)基因多克隆抗體,為探索毛囊KAP7基因?qū)Π氪置蜓蛎a(chǎn)量與質(zhì)量的影響奠定基礎(chǔ)。以3個品種綿羊的皮膚毛囊為試驗材料,提取總RNA,反轉(zhuǎn)錄得到cDNA,PCR擴增獲得綿羊毛囊KAP7基因,構(gòu)建pMD19-T-KAP7克隆質(zhì)粒,對重組質(zhì)粒進行PCR、酶切鑒定和序列分析,再構(gòu)建pET-28a(+)-KAP7原核表達重組質(zhì)粒,并在大腸桿菌BL21(DE3)感受態(tài)細胞中表達,SDS-PAGE電泳檢測原核表達的綿羊毛囊KAP7蛋白,經(jīng)提取、純化、回收得到目的蛋白,用其免疫家兔得到綿羊毛囊KAP7多克隆抗體血清,并用間接ELISA法檢測其抗體效價。結(jié)果表明,與美利奴羊比對,平原型與山區(qū)型和田羊KAP7基因序列的同源性達99%,但平原型和田羊KAP7基因第22位堿基由G變?yōu)镃,造成其KAP7蛋白第8位氨基酸由Gly變?yōu)锳rg;山區(qū)型和田羊第70位堿基由A變?yōu)镃,造成其24位氨基酸由Thr變?yōu)锳la;卡拉庫爾羊毛囊KAP7基因與美利奴羊同源性達100%,其KAP7蛋白氨基酸序列與美利奴羊沒有差異。綿羊毛囊KAP7基因原核表達重組質(zhì)粒pET-28a(+)-KAP7可以在大腸桿菌BL21(DE3)感受態(tài)細胞中表達目的蛋白,其大小約為10ku。利用綿羊毛囊KAP7蛋白免疫家兔得到的多克隆抗體陽性血清具有免疫活性和特異性,其抗體效價達到1∶10 000。
[Abstract]:The aim of this study was to prepare polyclonal antibodies against hair follicle keratin associated protein 7 (keratin associated protein 7 KAP7 gene of three local breeds (lines) of Xinjiang plain, mountainous and Karakuer sheep. In order to explore the effect of hair follicle KAP7 gene on wool yield and quality of semi-woolly sheep. The total RNAs were extracted from the skin follicles of three sheep breeds, and the KAP7 gene of sheep hair follicles was amplified by reverse transcription cDNA-PCR. The cloned plasmid pMD19-T-KAP7 was constructed, and the recombinant plasmid was identified by restriction endonuclease digestion and sequence analysis. The recombinant plasmid pET-28a () -KAP7 was constructed and expressed in Escherichia coli BL21 (DE3) competent cells. The expressed protein was detected by SDS-PAGE. The protein was extracted, purified, and recovered from E. coli BL21 (DE3). Sheep hair follicle KAP7 polyclonal antibody serum was obtained by immunizing rabbits and its antibody titer was detected by indirect Elisa. The results showed that compared with the Merino sheep, The homology of KAP7 gene sequence between plain type and mountain type is 99%, but the 22nd base of KAP7 gene of plain type and mountain type is changed from G to C, resulting in the change of amino acid of KAP7 protein from gly to Arg, and that of mountain type Hetian sheep from 70 to 70. The amino acid sequence of KAP7 gene in hair follicle of Karakur sheep was 100 with Merino sheep, and the amino acid sequence of KAP7 protein was not different from that of Merino sheep. The recombinant plasmid pET-28a () -KAP7 of sheep hair follicle KAP7 gene can express the target protein in BL21 (DE3) cells of Escherichia coli, and its size is about 10ku. the recombinant plasmid pET-28a () -KAP7 can express the target protein in E. coli BL21 (DE3). The polyclonal antibody positive serum immunized with sheep hair follicle KAP7 protein had the immunological activity and specificity, and its antibody titer reached 1:10 000.
【作者單位】： 塔里木大學生命科學學院塔里木盆地生物資源保護利用兵團重點實驗室;
【基金】：國家自然科學基金(31560685) 塔里木大學研究生創(chuàng)新項目(TDGRI201617)
【分類號】：S826

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本文編號：2131307