鸡β-防御素-1 cDNA的克隆及在毕赤酵母中的表达

The cDNA Fragment Cloning of Chicken β-defensin-1 and Its Expression in Pichia Pastoris

从鸡骨髓细胞中分离提取总RNA,经过反转录PCR(RT-PCR)扩增出鸡β-防御素-1(Gallinacin-1,Gal-1)cDNA,将扩增产物与pGEM-TEasy载体相连,经克隆测序表明扩增出的cDNA碱基数为198bp,与报道的Gal-1cDNA编码区碱基序列完全相同。分析核苷酸序列表明Gal-1与火鸡异嗜性白细胞肽-1(Turkeyheterophilpep-tides,THP-1)具有85.4%同源性。氨基酸序列方面Gal-1与THP-1同源性最高,为72.7%。将Gal-1基因成熟肽片段插入到酵母表达载体pPICZ-αC,构建重组表达载体pPICZ-αC-gal-1,电转入表达宿主菌X-33,Zeocin抗性筛选重组菌株。挑取阳性菌株经甲醇诱导表达,Tricine-SDS-PAGE电泳发现在约4.5ku位置出现预期条带。对其抗菌活性检测发现具有抗大肠杆菌、沙门氏菌、金黄色葡萄球菌等细菌的活性。反相高效液相色谱表明阳性菌株表达产物与标准品在层析柱中保留时间一致。

The chicken β-defensin-1 （Gallinacin-1） cDNA fragment was amplified by reverse transcriptionpolymerase chain reaction （RT-PCR） from the bone marrow cells of Yuehuang chicken. The RT-PCR products were inserted into the pGEM-T cloning vector, and then was sequenced. The result showed that the obtained 198 bp DNA fragment is identical to the Gal-1 cDNA sequence registered in GenBank. Compared with the other β-defensin registered in GenBank, Gal-1 and THP-1 had the highest homology of nucleotide and putative amino acid sequence, were 85.4% and 72.7%, respectively. Then the fragment of mature Gallinacin-1 gene was inserted into the pPICZa-C expression vector and pPICZa-C-gal-1 was acquired, then the recombinant plasmid was electro-transformed into pichia pastoris X-33 strain. The positive transformed strains were cultured and induced by addition of 0.5 % methanol every 24 h. Expression products with molecular weight approximately 4. 5 ku were displayed on the gel during Tricine-SDS-PAGE. The expression products had antimicrobial activity against E. coli, salmonella and ataphylococcosis aureas. The retention time of recombinant Gal-1 peptides in RP-HPLC was identical to that of synthetic Gal-1 peptides, which further showed the Gal-1 gene was expressed in the pichia pastoris.