简并PCR扩增小瓜虫抑动抗原基因的ORF

Amplification of immobilization antigen from Ichthyophthirius multifiliis by degenerate PCR

抑动蛋白是小瓜虫(Ichthyophthiriusmultifiliis)体表纤毛的主要构成部分,也是宿主免疫系统发挥抗虫免疫时的主要识别抗原。根据已经报道的3种抑动蛋白N端及C端保守的多肽片断,设计了一对简并引物P6/P7,以小瓜虫分离株IchFJ9的基因组为模板,成功扩增了抑动蛋白编码区(ORF)的全长基因序列。扩增的iagFJ9基因全长1398bp,编码466个氨基酸,包含18个非标准密码子TAA(Glu,Q)推导的氨基酸序列包含6个以CPXGT为起始的串联重复单位,具有抑动蛋白的特征性结构。同源比较分析显示,扩增的iagFJ9基因与已知的小瓜虫抑动蛋白基因IAG52A基因具有88%同源率。证实了iagFJ9是一个新发现的抑动蛋白基因,用简并PCR技术去发现小瓜虫抑动蛋白基因家族新基因是可行的。

The immobilization antigen (i-ag) is a predominant component of the Ichthyophthirius multifiliis cilia and is the most important antigen recognized by host immune system. Based on the hydrophobic conserved peptide at the N & C terminal of i-ag protein sequences of three Ichthyophthirius multifiliis isolates, a pair of degenerate PCR primers P6/P7 were designed and used to amplify the i-ag gene from the parasite isolate IchFJ9 genomic DNA. The amplified gene is 1 398 bp long with an open-reading-frame (ORF) without TGA stop codon. Sequence analysis showed the amplified gene iagFJ9 had typical i-ag structure with 18 TAA encoding Glutamine (Q), instead of stop codon. The deduced amino acids had 466 aa with six tandem repeats all initiated with CPXGT amino acid residues. Homology analysis indicated that the amplified gene had 88% homology with IAG52A gene. All these prove that the gene is the new discovered i-ag gene and the degenerate PCR is one of the useful tool for searching and discovering the other members of i-ag gene family.