新疆南疆和田羊、卡拉库尔羊毛囊KAP7基因的原核表达及多克隆抗体制备

发布时间：2018-07-18 08:19

【摘要】：本研究旨在制备新疆南疆平原型和田羊、山区型和田羊与卡拉库尔羊3个地方品种(系)绵羊毛囊角蛋白关联蛋白7(keratin associated protein 7,KAP7)基因多克隆抗体,为探索毛囊KAP7基因对半粗毛羊羊毛产量与质量的影响奠定基础。以3个品种绵羊的皮肤毛囊为试验材料,提取总RNA,反转录得到cDNA,PCR扩增获得绵羊毛囊KAP7基因,构建pMD19-T-KAP7克隆质粒,对重组质粒进行PCR、酶切鉴定和序列分析,再构建pET-28a(+)-KAP7原核表达重组质粒,并在大肠杆菌BL21(DE3)感受态细胞中表达,SDS-PAGE电泳检测原核表达的绵羊毛囊KAP7蛋白,经提取、纯化、回收得到目的蛋白,用其免疫家兔得到绵羊毛囊KAP7多克隆抗体血清,并用间接ELISA法检测其抗体效价。结果表明,与美利奴羊比对,平原型与山区型和田羊KAP7基因序列的同源性达99%,但平原型和田羊KAP7基因第22位碱基由G变为C,造成其KAP7蛋白第8位氨基酸由Gly变为Arg;山区型和田羊第70位碱基由A变为C,造成其24位氨基酸由Thr变为Ala;卡拉库尔羊毛囊KAP7基因与美利奴羊同源性达100%,其KAP7蛋白氨基酸序列与美利奴羊没有差异。绵羊毛囊KAP7基因原核表达重组质粒pET-28a(+)-KAP7可以在大肠杆菌BL21(DE3)感受态细胞中表达目的蛋白,其大小约为10ku。利用绵羊毛囊KAP7蛋白免疫家兔得到的多克隆抗体阳性血清具有免疫活性和特异性,其抗体效价达到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.
【作者单位】：塔里木大学生命科学学院塔里木盆地生物资源保护利用兵团重点实验室;
【基金】：国家自然科学基金(31560685) 塔里木大学研究生创新项目(TDGRI201617)
【分类号】：S826

【相似文献】