溶瘤Ⅱ型单纯疱疹病毒oHSV2是对Ⅱ型单纯疱疹病毒HSV-2进行基因改造后得到的新型溶瘤病毒。UL19基因编码的VP5蛋白为oHSV2主要衣壳蛋白之一。在前期已经通过GST pull-down与质谱检测分析确定了oHSV2的VP5蛋白为人类白细胞抗原E(HLA-E)...溶瘤Ⅱ型单纯疱疹病毒oHSV2是对Ⅱ型单纯疱疹病毒HSV-2进行基因改造后得到的新型溶瘤病毒。UL19基因编码的VP5蛋白为oHSV2主要衣壳蛋白之一。在前期已经通过GST pull-down与质谱检测分析确定了oHSV2的VP5蛋白为人类白细胞抗原E(HLA-E)的相互作用蛋白的基础上,将HLA-E作为NK与CTL等免疫细胞表面CD94/NKG2A的强效抑制性配体,当它与CD94/NKG2A结合后,NK与CTL细胞的免疫功能会受到一定程度的抑制。前期研究已证实oHSV2会在体外上调部分肿瘤细胞系表面HLA-E表达,为探究oHSV2是否通过VP5蛋白上调肿瘤细胞系表面HLA-E的表达,进而影响HLA-E和CD94/NKG2A的结合,最终改变NK细胞的抗肿瘤能力,通过PiggyBac转座系统构建稳定表达VP5蛋白的人胃腺癌细胞BGC823-VP5细胞系。所构建的细胞系经流式检测单克隆细胞阳性率在99%以上;Western Blot检测到VP5蛋白在细胞内表达;实时无标记动态细胞分析技术(RTCA Real Time Cell AnaIysis)检测表明BGC823-VP5细胞系与亲本细胞BGC823细胞生长活性一致。稳定表达VP5蛋白的人胃癌细胞BGC823-VP5细胞系的构建,为进一步研究oHSV2上VP5蛋白在人胃癌BGC823细胞内影响HLA-E的表达,进而影响免疫细胞的抗肿瘤能力奠定了基础。展开更多
[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification...[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.展开更多
文摘溶瘤Ⅱ型单纯疱疹病毒oHSV2是对Ⅱ型单纯疱疹病毒HSV-2进行基因改造后得到的新型溶瘤病毒。UL19基因编码的VP5蛋白为oHSV2主要衣壳蛋白之一。在前期已经通过GST pull-down与质谱检测分析确定了oHSV2的VP5蛋白为人类白细胞抗原E(HLA-E)的相互作用蛋白的基础上,将HLA-E作为NK与CTL等免疫细胞表面CD94/NKG2A的强效抑制性配体,当它与CD94/NKG2A结合后,NK与CTL细胞的免疫功能会受到一定程度的抑制。前期研究已证实oHSV2会在体外上调部分肿瘤细胞系表面HLA-E表达,为探究oHSV2是否通过VP5蛋白上调肿瘤细胞系表面HLA-E的表达,进而影响HLA-E和CD94/NKG2A的结合,最终改变NK细胞的抗肿瘤能力,通过PiggyBac转座系统构建稳定表达VP5蛋白的人胃腺癌细胞BGC823-VP5细胞系。所构建的细胞系经流式检测单克隆细胞阳性率在99%以上;Western Blot检测到VP5蛋白在细胞内表达;实时无标记动态细胞分析技术(RTCA Real Time Cell AnaIysis)检测表明BGC823-VP5细胞系与亲本细胞BGC823细胞生长活性一致。稳定表达VP5蛋白的人胃癌细胞BGC823-VP5细胞系的构建,为进一步研究oHSV2上VP5蛋白在人胃癌BGC823细胞内影响HLA-E的表达,进而影响免疫细胞的抗肿瘤能力奠定了基础。
基金Supported by the National Natural Science Fundation Item of China(30970578,31070651)"Excellent Talent Support Plan in NewCentury"of Ministry of Education(NECT-08-0731)~~
文摘[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.
