堆型艾美耳球虫ADF基因的原核表达与免疫原性

Immunogenicity and prokaryotic expression of ADF gene of Eimeria acervulina

参考Gen Bank中收录的E.acervulina ADF基因序列设计1对特异性引物,以河北保定株堆型艾美耳球虫子孢子DNA为模板,用聚合酶链式反应(PCR)法扩增ADF基因。将扩增的ADF基因克隆至p MD18-T载体,转化感受态细胞DH5α,蓝白斑法筛选出阳性重组子,提取阳性质粒进行PCR、酶切鉴定及对序列确证。将扩增产物与原核表达载体p ET32a(+)连接,构建重组质粒p ET32a-ADF,转化大肠杆菌Rosetta(DE3)进行诱导表达,表达产物经SDS-PAGE鉴定。用E.acervulina重组质粒p ET32a-ADF免疫雏鸡,观察其对雏鸡细胞免疫和体液免疫功能的影响。结果显示,ADF基因扩增产物为729 bp,与预期结果相符。序列分析结果表明,该株的ADF基因序列与参考序列同源性达99.6%。表达出的ADF重组蛋白相对分子质量大小约为46 000。与健康对照组相比,重组质粒组淋巴细胞的增殖能力明显增强(P<0.05),外周血中Ig G及IL-2含量均显著升高(P<0.01),表明p ET32a-ADF质粒DNA能有效提高雏鸡的细胞免疫和体液免疫水平。

A pair of specific primers were designed based on the sequence alignment of the ADF gene of E. acervulina published in Gen Bank. With the total DNA of sporozoites of E. acervulina Baoding strain isolated from Hebei province of China as template,a partial segment of ADF gene was amplified by PCR using designed specific primers. The ADF gene was subcloned into the p MD18-T vector,and then transfected into E. coli DH5α. The recombinant plasmids were extracted and identified by EcoRⅠ and HindⅢ enzyme digestion and PCR amplification and sequencing. The product of PCR was cloned into p ET32a( +) vector,constructed recombinant plasmid p ET32a-ADF,then transformed into E. coil Rosetta( DE3) strain to expression. The chickens were immuned with E. acervulina plasmid p ET32a-ADF to observe the function of the cellular and humoral immune. The results indicated that the ADF gene fragment of Baoding strain contained 729 bp nucleotides,and had 99. 6% sequence similarity with reference sequence. The ADF fusion protein band of about 46 000 was identificated by SDS-PAGE. The lymphocyte proliferation of recombinant plasmid group significantly increased( P < 0. 05) and the peripheral blood Ig G,IL-2 levels were significantly higher( P < 0. 01) than those of the control group. The p ET32a-ADF naked plasmid DNA can effectively improve the cellular and humoral immune responses of the chickens.