根据GenBank中报道的红原锦鸡集落刺激因子(Colony-stimulating factor,CSF)cDNA序列,利用Primer Premier5.0软件设计一对特异性引物,对本地肉鸡注射100 μg/mL ConA试剂,饲养24 h后,取脾脏提取淋巴细胞总RNA,利用RT-PCR技术克隆集落刺激因子(CSF)的cDNA片段.对克隆片段进行测序,并利用DNAstar软件进行不同物种间同源性比较.序列分析表明,CSF的cDNA开放阅读框为435 bp,编码144个氨基酸,与红原锦鸡的序列比较,其核苷酸的同源性为98.4%,氨基酸的同源性为95.2%,同人、犬、印度野牛、野猪、绵羊和老鼠的CSF序列作比较,其核苷酸的同源性分别为28.7%,29.2%,33.%,30.8%,29.4%和34.7%,氨基酸的同源性分别为8.3%,9.0%,15.3%,12.4%,11.0%和12.0%.然后利用DNAstar和DNAman软件,对鸡CSF的结构进行预测,结果表明,鸡CSF基因为一结构松散的蛋白分子.鸡的CSF基因被成功克隆,它存在着种属特异性.这为进一步研究该基因的生物学作用特别是利用CSF增强DNA疫苗的免疫效果奠定了基础.
According to the gallus colony stimulating factor (colony - stimulating factor,CSF) cDNA sequences published by GenBank,a pairs of specific primer was designed,the local dorking was injected 100μg/ mL ConA,total RNA was extracted from feeding 24 hours lymphocyte from spleen,then dorking colony stimulating factor (CSF) cDNA fragment was cloned by RT - PCR technology. Following the DNA fragments was analyzed by DNAstar software,then homology was compared between dorking CSF and other animals CSF. Result showed that dorking CSF gene was 435 bp in size with an ORF encoding 141 amino acids residues,the nucleotide homology between dorking CSF and CSF gene of Gallus,human,canis,Bostaurus,Sus scrofa,sheep,mice was respectively 9814 %%,2817 %,2912 %,3316 %,3018 %,2914 % and 3417 %,Amino acids homology was respectively 9512 %,813 %,910 %,1513 %,1214 %,1110 % and 1210 %.Using DNAstar and DNAman software predicate protein structure of dorking CSF,results showed that the dorking CSF protein was a loose protein.The chicken CSF gene was successfully cloned,there is a species-specific.And construct a based on further study the biological effects of CSF gene in particular the use of CSF enhance the immune effect of DNA vaccine.
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