鸡柔嫩艾美尔球虫HAP2基因的克隆与原核表达
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摘要
为研究鸡柔嫩艾美尔球虫(E.tenella)HAP2(hapless2/generative cell-specific)蛋白的抗原性,根据Gen?Bank发表的HAP2基因的cDNA序列(Gene ID:25252561)ORF设计1对引物,用RT-PCR方法从柔嫩艾美耳球虫配子体总RNA中扩增HAP2基因,将扩增产物与pMD18-T载体连接后进行测序和序列分析,将测序正确的HAP2基因亚克隆入原核表达载体pET32a,转化宿主菌E.coli RGB,在IPTG诱导下进行原核表达。结果在20℃诱导12 h条件下成功表达了HAP2融合蛋白大小为41 ku,并应用磁珠纯化的方法纯化了重组蛋白,获得了HAP2融合蛋白,为进一步研究HAP2的抗原性奠定基础。
In order to study the antigenicity of E.tenella HAP2 gene in chicken, a pair of primers was designed based on the cDNA sequence of the HAP2 gene published by GeneBank(Gene ID:25252561) ORF. The HAP2 gene was amplified by RT-PCR from the total RNA of the gametophyte of E.tenella. The amplification product was linked to the pMD18-T for sequencing and sequence analysis. The correct sequencing of the HAP2 gene was cloned into the prokaryotic expression vector pET32 a, and converted to host bacterium E. coli RGB. Fusion protein was induced by IPTG and successfully expressed under the condition of 20 hours at 20 ℃. The fusion protein of HAP2 is 41 kDa and was purified by magnetic bead purification method, which laid the foundation for further research on the antigenicity of HAP2.
In order to study the antigenicity of E.tenella HAP2 gene in chicken, a pair of primers was designed based on the cDNA sequence of the HAP2 gene published by GeneBank(Gene ID:25252561) ORF. The HAP2 gene was amplified by RT-PCR from the total RNA of the gametophyte of E.tenella. The amplification product was linked to the pMD18-T for sequencing and sequence analysis. The correct sequencing of the HAP2 gene was cloned into the prokaryotic expression vector pET32 a, and converted to host bacterium E. coli RGB. Fusion protein was induced by IPTG and successfully expressed under the condition of 20 hours at 20 ℃. The fusion protein of HAP2 is 41 kDa and was purified by magnetic bead purification method, which laid the foundation for further research on the antigenicity of HAP2.
引文
[1] Blake DP, Clark EL, Macdonald SE, et al. Population, genetic, and antigenic diversity ofthe apicomplexan Eimeria tenella and theirrelevance to vaccine development[J]. Proceedings of the National Academy of Sciences ofthe United States of America. 2015, 112(38):5343-5350.
[2]赵蕾.鸡球虫疫苗研究现状[J].动物医学进展,2011,32(8):99-103.
[3] von Besser K, Frank AC, Johnson MA et al.Arabidopsis HAP2(GCS1)is a sperm-specificgene required for pollen tube guidance andfertilization[J]. Development. 2006, 133(23):4761-4769.
[4] Okamoto M, Yamada L, Fujisaki Y, et al. TwoHAP2-GCS1 homologs responsible for gamete interactions in the cellular slime mold withmultiple mating types:Implication for commonmechanisms of sexual reproduction shared byplants and protozoa and for male-female differentiation[J]. Developmental biology. 2016,415(1):6-13.
[5] Wong JL, Leydon AR, Johnson MA. HAP2(GCS1)-dependent gamete fusion requires a positivelycharged carboxy-terminal domain[J]. PLoS genetics. 2010, 6(3):e1000882.
[6]贾传礼.大量制备毒害艾美尔球虫配子体方法的研究[J].畜牧与兽医,2016,48(10):91-93.
[7]栗良玉.鸡球虫病防制的发展趋势和历程[J].现代畜牧兽医,2009(6):69-71.
[8]周芳玉.柔嫩艾美耳球虫保护性抗原应用研究进展[J].中国兽医杂志,2015,49(9):65-69.
[2]赵蕾.鸡球虫疫苗研究现状[J].动物医学进展,2011,32(8):99-103.
[3] von Besser K, Frank AC, Johnson MA et al.Arabidopsis HAP2(GCS1)is a sperm-specificgene required for pollen tube guidance andfertilization[J]. Development. 2006, 133(23):4761-4769.
[4] Okamoto M, Yamada L, Fujisaki Y, et al. TwoHAP2-GCS1 homologs responsible for gamete interactions in the cellular slime mold withmultiple mating types:Implication for commonmechanisms of sexual reproduction shared byplants and protozoa and for male-female differentiation[J]. Developmental biology. 2016,415(1):6-13.
[5] Wong JL, Leydon AR, Johnson MA. HAP2(GCS1)-dependent gamete fusion requires a positivelycharged carboxy-terminal domain[J]. PLoS genetics. 2010, 6(3):e1000882.
[6]贾传礼.大量制备毒害艾美尔球虫配子体方法的研究[J].畜牧与兽医,2016,48(10):91-93.
[7]栗良玉.鸡球虫病防制的发展趋势和历程[J].现代畜牧兽医,2009(6):69-71.
[8]周芳玉.柔嫩艾美耳球虫保护性抗原应用研究进展[J].中国兽医杂志,2015,49(9):65-69.