摘要：
This study was aimed to prepare polyclonal antibodies of wool follicle keratin associated protein 7 (KAP7) gene of three local variety (strain) sheep of Plain-type Hetain sheep,Mountain-type Hetain sheep and Karakul sheep in Southern Xinjiang,to provide the basis for exploring influences of follicle KAP7 gene to coarse wool quantity and quality.This study took the skin wool follicle samples from three local variety (strain) sheep in Southern Xinjiang,extracted total RNA from the skin wool follicle samples,obtained cDNA through reverse transcription,then amplified the wool follicle KAP7 gene fragments by PCR.We constructed the pMD19-T-KAP7 recombination cloning plasmids,and tested the correction of the plasmids by PCR assay,enzyme digest and sequence analysis,then constructed the prokaryotic expression recombination plasmids of pET-28a(+)-KAP7,expressed them in E.coli BL21 (DE3) cells.SDS-PAGE electrophoresis was employed to detect the prokaryotic expression wool follicle KAP7 protein.We obtained the target proteins by extraction,purification and reclaim.The polyclonal antibodies serum were got from the rabbits which were immunifaciented with the purification KAP7 protien.The antibodies titer were detected by indirect ELISA.The results showed that there were 99 ％ homology among the Merino sheep with the mountain-type and plain-type Hetian sheep,but there was a mutation at the 22nd base pair of KAP7 gene of plain-type Hetian sheep,in which it conversed from G to C,and this made the 8th amino acid of KAP7 protein to converse from Gly to Arg.For the mountain-type Hetian sheep,there was a mutation from A to C at the 70th base pair,this changed the 24th amino acid of KAP7 protein from Thr to Ala.The homology of wool follicle KAP7 gene was 100％ between Karakul sheep and Merino sheep,so there was no differences on the amino acids sequences between them.The prokaryotic expression plasmids pET-28a (+-)-KAP7 of wool follicle KAP7 gene could be expressed in E.coli BL21 cells,and the wool follicle KAP7 proteins were about 10 ku.The positive polyclonal antibodies serum of the sheep wool follicle KAP7 proteins,which were obtained from immunized rabbits,had immunological activity and specificity,and the antibodies titer could be up to 1 ∶ 10 000.%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,用其免疫家免得到绵羊毛囊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10 ku.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.
