为构建猪圆环病毒2型(PCV2)特异性纳米抗体文库,并对其库容和多样性进行鉴定分析,使用PCV2全病毒灭活疫苗免疫羊驼,经四次免疫后采血分离外周血淋巴细胞,提取总RNA,通过RT-PCR反转录合成cDNA;根据羊驼重链抗体基因序列设计特异性引物,...为构建猪圆环病毒2型(PCV2)特异性纳米抗体文库,并对其库容和多样性进行鉴定分析,使用PCV2全病毒灭活疫苗免疫羊驼,经四次免疫后采血分离外周血淋巴细胞,提取总RNA,通过RT-PCR反转录合成cDNA;根据羊驼重链抗体基因序列设计特异性引物,通过巢式PCR扩增羊驼单域重链抗体(variable domain of heavy chain of heavy-chain antibody,VHH)基因片段vhh,并将其克隆到噬菌粒载体pCANTAB 5E中,构建pCANTAB-vhh重组质粒;通过电穿孔法将重组质粒导入E. coli TG1获得抗PCV2特异性纳米抗体基因文库。文库菌梯度稀释后计数单克隆菌落数,并通过菌液PCR鉴定重组菌阳性率,计算抗体文库库容量;对PCR鉴定为阳性的重组质粒进行测序,分析抗体文库多样性。结果显示,构建的PCV2特异性纳米抗体文库重组菌阳性率为92.0%,库容量为2.75×1010CFU/m L;测序后序列比对分析结果表明,纳米抗体文库包含的基因序列具有丰富的多样性。本研究成功构建了PCV2特异性纳米抗体文库,且文库质量满足后续试验要求,为进一步获得针对PCV2抗原的纳米抗体奠定了基础。展开更多
In this paper, a class of mixed monotone operators is studied. Some new fixed point theorems are presented by means of partial order theory, and the uniqueness and existence of fixed points are obtained without assumi...In this paper, a class of mixed monotone operators is studied. Some new fixed point theorems are presented by means of partial order theory, and the uniqueness and existence of fixed points are obtained without assuming the operator to be compact or continuous. Our conclusions extend the relevant results. Moreover,as an application of our result, the existence and uniqueness of positive solution for a class of fractional differential equation boundary value problem are proved.展开更多
文摘为构建猪圆环病毒2型(PCV2)特异性纳米抗体文库,并对其库容和多样性进行鉴定分析,使用PCV2全病毒灭活疫苗免疫羊驼,经四次免疫后采血分离外周血淋巴细胞,提取总RNA,通过RT-PCR反转录合成cDNA;根据羊驼重链抗体基因序列设计特异性引物,通过巢式PCR扩增羊驼单域重链抗体(variable domain of heavy chain of heavy-chain antibody,VHH)基因片段vhh,并将其克隆到噬菌粒载体pCANTAB 5E中,构建pCANTAB-vhh重组质粒;通过电穿孔法将重组质粒导入E. coli TG1获得抗PCV2特异性纳米抗体基因文库。文库菌梯度稀释后计数单克隆菌落数,并通过菌液PCR鉴定重组菌阳性率,计算抗体文库库容量;对PCR鉴定为阳性的重组质粒进行测序,分析抗体文库多样性。结果显示,构建的PCV2特异性纳米抗体文库重组菌阳性率为92.0%,库容量为2.75×1010CFU/m L;测序后序列比对分析结果表明,纳米抗体文库包含的基因序列具有丰富的多样性。本研究成功构建了PCV2特异性纳米抗体文库,且文库质量满足后续试验要求,为进一步获得针对PCV2抗原的纳米抗体奠定了基础。
基金The NSF(201701D0503-9)of Shanxi Provincethe Innovation Foundation for College Teaching Team of Shanxi University of Finance and Economics2015 Education and Teaching Reform Project(2015234)of Shanxi University of Finance and Economics
文摘In this paper, a class of mixed monotone operators is studied. Some new fixed point theorems are presented by means of partial order theory, and the uniqueness and existence of fixed points are obtained without assuming the operator to be compact or continuous. Our conclusions extend the relevant results. Moreover,as an application of our result, the existence and uniqueness of positive solution for a class of fractional differential equation boundary value problem are proved.
