目的:开发针对粘液病毒抗性蛋白A(Myxovirus resistance protein A,MxA)的高亲和力抗体,旨在满足MxA体外检测试剂开发对抗体原料的高灵敏度需求。方法:通过传统杂交瘤抗体开发技术制备抗MxA抗体,结合计算机模拟手段,运用虚拟氨基酸突变...目的:开发针对粘液病毒抗性蛋白A(Myxovirus resistance protein A,MxA)的高亲和力抗体,旨在满足MxA体外检测试剂开发对抗体原料的高灵敏度需求。方法:通过传统杂交瘤抗体开发技术制备抗MxA抗体,结合计算机模拟手段,运用虚拟氨基酸突变等方法,对抗体进行结构优化。结果:计算机模拟技术确定抗原-抗体关键氨基酸位点为重链第37、38、57、66、106、108、117位,轻链第34、36、38、56位。对这些位点进行虚拟氨基酸突变,得到轻链ASN36→ARG突变抗体。亲和力分析显示该突变抗体比野生型抗体亲和力提高2个数量级,KD值达到5.93×10^(-11) mol/L。结论:本文成功开发了抗MxA单克隆抗体,并通过计算机模拟技术使该抗体亲和力提升了2个数量级,为抗体的体外亲和力成熟提供了理论指导。展开更多
目的探讨乙型肝炎病毒(HBV)HBx蛋白(HepatitisB virus X protein)对α干扰素(IFN-α)诱导的抗病毒蛋白的影响及相关机制。方法以表达HBx蛋白的重组质粒FL1-145HBx转染人肝胚瘤细胞株HepG2细胞,经IFN-α处理后,RT-PCR法分析细胞内抗病毒...目的探讨乙型肝炎病毒(HBV)HBx蛋白(HepatitisB virus X protein)对α干扰素(IFN-α)诱导的抗病毒蛋白的影响及相关机制。方法以表达HBx蛋白的重组质粒FL1-145HBx转染人肝胚瘤细胞株HepG2细胞,经IFN-α处理后,RT-PCR法分析细胞内抗病毒蛋白MxA和JAK-STAT信号转导途径分子STAT1 mRNA表达水平,同时运用免疫印迹检测细胞内HBx、p-ERK、p-STAT1和t-STAT1等蛋白的表达。结果转染细胞内MxA、STAT1的mRNA和p-STAT1、t-STAT1的蛋白表达水平明显减少(P<0.05);而ERK抑制剂PD98059预处理后,转染细胞内MxA、STAT1 mRNA水平能够恢复至转染前的表达水平。结论 HBx蛋白很可能通过影响IFN-αJAK-STAT信号转导途径分子而抑制抗病毒蛋白MxA的表达;ERK信号转导途径的活化可能参与这一抑制过程。展开更多
[Objective] The study aimed to establish a fast and accurate method to detect the polymorphism of the 12^th exon of equine MxA gene. [Method] The 12^th exon of MxA gene was amplified by mismatch PCR and the products w...[Objective] The study aimed to establish a fast and accurate method to detect the polymorphism of the 12^th exon of equine MxA gene. [Method] The 12^th exon of MxA gene was amplified by mismatch PCR and the products were analyzed by restriction fragment length polymorphism （RFLP） to determine the point mutation at the 1 790 nt of MxA cDNA. The sequence of the PCR products was also analyzed. [Result] There were three genotypes （AA, AB and BB） in the 12^th exon of equine MxA gene; the 2 081 nt of MxA cDNA mutated from G to C, correspondingly changing the 562^th amino acid of the coding region of MxA protein from tryptophan to cysteine; the specific sequence of the PCR products amplified by mismatch PCR-RFLP was consistent with the analysis results of RFLP. [ Conclusion] The mismatch PCR-RFLP was an easy method with accurate results to detect the polymorphism of the 12^th exon of equine MxA gene.展开更多
文摘目的:开发针对粘液病毒抗性蛋白A(Myxovirus resistance protein A,MxA)的高亲和力抗体,旨在满足MxA体外检测试剂开发对抗体原料的高灵敏度需求。方法:通过传统杂交瘤抗体开发技术制备抗MxA抗体,结合计算机模拟手段,运用虚拟氨基酸突变等方法,对抗体进行结构优化。结果:计算机模拟技术确定抗原-抗体关键氨基酸位点为重链第37、38、57、66、106、108、117位,轻链第34、36、38、56位。对这些位点进行虚拟氨基酸突变,得到轻链ASN36→ARG突变抗体。亲和力分析显示该突变抗体比野生型抗体亲和力提高2个数量级,KD值达到5.93×10^(-11) mol/L。结论:本文成功开发了抗MxA单克隆抗体,并通过计算机模拟技术使该抗体亲和力提升了2个数量级,为抗体的体外亲和力成熟提供了理论指导。
基金Supported by the Natural Science Foundation of Inner Mongolia Au-tonomous Region (200508010413)~~
文摘[Objective] The study aimed to establish a fast and accurate method to detect the polymorphism of the 12^th exon of equine MxA gene. [Method] The 12^th exon of MxA gene was amplified by mismatch PCR and the products were analyzed by restriction fragment length polymorphism （RFLP） to determine the point mutation at the 1 790 nt of MxA cDNA. The sequence of the PCR products was also analyzed. [Result] There were three genotypes （AA, AB and BB） in the 12^th exon of equine MxA gene; the 2 081 nt of MxA cDNA mutated from G to C, correspondingly changing the 562^th amino acid of the coding region of MxA protein from tryptophan to cysteine; the specific sequence of the PCR products amplified by mismatch PCR-RFLP was consistent with the analysis results of RFLP. [ Conclusion] The mismatch PCR-RFLP was an easy method with accurate results to detect the polymorphism of the 12^th exon of equine MxA gene.
