绵羊生殖道抗菌肽cDNA克隆及真核表达载体构建

绵羊生殖道抗菌肽（ORTAP40）是从中国美利奴绵羊生殖道分离的一种新型的阳离子抗菌肽,在绵羊发育早期的防御中起到了主要的作用。在本研究中，根据ORTAP40 N端氨基酸序列设计引物，利用3′RACE技术克隆了绵羊生殖道抗菌肽3′末端全长cDNA序列，并被GenBank收录，登录号为FJ514476，经过cDNA序列分析显示，ORTAP40是一个4.8 ku的小肽，与先期分离纯化的分子质量一致。作者随后参照巴斯德毕赤酵母（Pichia pastoris）密码子偏好性，设计并人工合成了ORTAP40成熟肽目的基因片段，所合成的ORTAP40基因全长120 bp，通过基因重组的方法将目的基因克隆到pPIC3.5K和pPICZαA表达载体上，成功构建了重组酵母胞内表达载体pPIC3.5K-ORTAP40和外泌表达载体pPICZαA-ORTAP40，为抗菌肽的开发和应用奠定了基础。

cDNA Clone of Antimicrobial Peptide from the Reproductive Tract of Ovis aries and Construction of its Eukaryotic Expression Vector

ORTAP40 is an antimicrobial peptide which is isolated from the reproductive tract of Ovis aries Chinese Merino. It plays a central role in the early innate immune defense against infection. In this study, we cloned the 3′ end full-length cDNA of ORTAP40 from reproductive tract of Ovis aries, using rapid amplification of cDNA ends (RACE) and N-terminus amino acids sequence（FJ514476）. The observed cDNA structure indicated that ORTAP40 theoretical average molecular weight for the peptide of 40 amino acid residues is 4.8 ku in agreement with reported. A mature peptide gene was synthesized according to the amino acid sequence of ORTAP40 and the biased codon usage of Pichia pastoris, the gene was 120 bp in length and was inserted into Pichia pastoris expression vector pPIC3.5K, pPICZαA by gene recombination and constructed expression plasmid pPIC3.5K-ORTAP40 and pPICZαA-ORTAP40.