鸡柔嫩艾美尔球虫第二代裂殖子表面抗原MZ5-7基因的克隆、表位分析及原核表达

用RTPCR的方法,从感染柔嫩艾美尔球虫(Eimeriatenella)第5天的鸡盲肠分离的第二代裂殖子中克隆出MZ57基因,并进行抗原表位分析和原核表达,结果表明,MZ57基因ORF为945bp,与GenBank里已知序列(登录号为L08257)比较核甘酸和氨基酸同源性均为99.9%。应用DNAStar软件对该基因的抗原表位进行了分析,发现其B细胞表位广泛分布在80～300区域,其中80～130区域、190～220区域和300～320区域更为突出,而T细胞基序则集中在123～190区域和220～300区域。根据MZ57基因序列重新设计一对引物,PCR方法扩增出不含信号肽长度为885bp的基因序列,克隆到pET28b获得重组质粒pET28bMZ亚,转入宿主菌BL21中,用IPTG进行诱导表达,表达的融合蛋白约36.5kDa10poundnote,符合目的蛋白大小;同时应用Westernblot方法与抗第二代裂殖子和子孢子的2种高免血清反应,结果表明抗裂殖子和子孢子血清均能识别MZP(裂殖子蛋白,MZP,merozoiteprotein)。

CLONING, EPITOPE ANALYSIS AND PROKARYOTIC EXPRESSION OF MZ5-7 GENE OF EIMERIA TENELLA

MZ5-7 gene was cloned with RT-PCR from the second generation merozoites of E.tenella that detached from chicken caecum 5 days post infection and its sequence was analyzed on epitope and open reading frame(ORF),then expressing it in prokaryotic cell.The result indicated that the length of MZ5-7 ORF was 945 bp and it had 99.9% homology with MZ5-7 gene that GenBank has been issued(Access number:L08257)in both nucleotide and amino acid sequence.The prediction of MZ5-7 epitope with DNAStar software showed that its B cell epitope located at the segments of 80-300 and T cell epitop in 123-190 and 220-300.Another pair of primer was designed and then 885 bp fragment of the gene without signal-peptide was amplified by PCR and it was inserted into pET28b vector to construct recombinant plasmid pET28b-MZ.After induced with IPTG,a 36.5 kDa fused protein was expressed and it was hybridized with serums immunized with the second generation merozoites and sporozoites respectively.The result demonstrated that innate MZ protein was expressed both in the stages of merozoites and sporozoites.