Shen Wen, Chen Kaili, Sun Yanming, Guo Haiying, Chen Dongmei, Cao Yang
College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003, China.
College of Life Science, South China Agricultural University, Guangzhou, Guangdong 510000, China.
Asian-Australas J Anim Sci. 2017 May;30(5):736-742. doi: 10.5713/ajas.15.0557. Epub 2016 Sep 12.
Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 () cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1.
The complete sequence of Wild Argali cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed in () GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by Ni chelate affinity chromatography. The test of effect of the protein on (MO) was performed with real-time polymerase chain reaction.
The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA from (accession no. NP_001288334.1), (accession no. XP_005688516.1), (accession no. XP_005979709.1), (accession no. NP_776851.1), (accession no. XP_006929910.1), (accession no. NP_001230122.1), (accession no. NP_001005727.1), (accession no. NP_001269294.1), (accession no. NP_035256.2), and (accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant of , and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01).
The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.
克隆野生盘羊短腭、肺及鼻上皮克隆1（SPLUNC1）cDNA序列，为进一步研究野生盘羊SPLUNC1的生物学功能奠定基础。
通过cDNA末端快速扩增法获得野生盘羊SPLUNC1 cDNA的完整序列。将其完整编码序列插入pPIC9K载体并在毕赤酵母（Pichia pastoris）GS115中表达。通过蛋白质免疫印迹法检测重组SPLUNC1蛋白，并利用镍螯合亲和层析法进行纯化。采用实时聚合酶链反应检测该蛋白对多杀性巴氏杆菌（Pasteurella multocida，PM）的作用效果。
野生盘羊SPLUNC1 cDNA为1076 bp，开放阅读框为768 bp，编码由255个氨基酸组成的26.49 kDa蛋白。其氨基酸序列与牛（Bos taurus，登录号NP_001288334.1）、绵羊（Ovis aries，登录号XP_005688516.1）、山羊（Capra hircus，登录号XP_005979709.1）、猪（Sus scrofa，登录号NP_776851.1）、马（Equus caballus，登录号XP_006929910.1）、人（Homo sapiens，登录号NP_001230122.1）、小鼠（Mus musculus，登录号NP_001005727.1）、大鼠（Rattus norvegicus，登录号NP_001269294.1）、兔（Oryctolagus cuniculus，登录号NP_035256.2）和犬（Canis lupus familiaris，登录号NP_742028.1）的SPLUNC1 cDNA的氨基酸序列分别具有98.4%、96.9%、94.5%、90.2%、80.8%、78.4%、78.3%、72.5%、72.3%、68.8%的同源性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示重组蛋白分子量与预期的25.47 kDa相符，在毕赤酵母培养上清中可检测到且能够被纯化。野生盘羊重组SPLUNC1蛋白对PM的抑制作用试验结果显示该产物对PM具有良好的抑制作用（p<0.01）。
野生盘羊SPLUNC1的氨基酸序列与其他物种不同。重组SPLUNC1蛋白具有良好的生物学活性。
