【作者】 李蒙； 殷幼平； 于红； 吴瑜佳； 王中康；

【Author】 Li Meng,Yin You-pin,Yu Hong,Wu Yu-jia,Wang Zhong-kang (Bioengineering College of Chongqing University,Key Laboratory of Gene Function and Regulation of Chongqing,Chongqing 400030)

【机构】 重庆大学生物工程学院重庆市基因功能与调控重点实验室；

【摘要】 以鼠源抗柑橘溃疡病表面脂多糖的二硫键稳定性抗体dsFv为模板,利用重叠延伸PCR在重链可变区VH和轻链可变区VL之间引入一条人类抗体重链恒定区1(CH1)5’端12个氨基酸的序列作为连接肽,成功构建了单链二硫键稳定重组抗体基因。连接pET24a(+)载体,转入E.coli BL21(DE3)得到工程菌,IPTG诱导表达。体外复性并经Ni-NTA亲和层析对目标蛋白sc-dsFv进行纯化,利用斑点免疫杂交,ELISA和BIACORE检测抗体的活性,稳定性,特异性及亲和力。测序结果表明VH和VL基因之间成功引入目标连接肽序列,重组sc-dsFv蛋白实现了原核高效表达,通过体外复性和Ni-NTA柱纯化获得纯度高于90%的sc-dsFv蛋白。斑点免疫杂交实验和ELISA,BIAcore结果表明该重组抗体具有特异的抗原结合活性和良好的抗原亲和力,与母本scFv及dsFv比较,产量有明显提高且稳定性也有所提高。这种具有特异抗原结合活性的稳定抗体sc-dsFv的获得为杆菌溃疡病的快速免疫诊断提供了高效优质重组抗体。更多还原

【Abstract】 To constructed the recombinant mouse anti-Xac disulfide-stabilized Single Chain Variable Fragment(sc-dsFv),we amplified the genes of VH and VL by the method of gene splicing by overlap extension PCR.Gene sequence encoding 5’-terminal 12 amino acid of heavy chain constant region CH1,as a linker,linked VH and VL to construct a new recombinant sc-dsFv antibody molecule,and then cloned them into expression plasmid pET24a(+),respectively.The recombinant plasmids were transformed into E.coli BL21(DE3) and expressed.The products of expression and renaturation were analyzed by SDS-PAGE and western-blot.The affinity of sc-dsFv to Xac-LPS was determined by BIAcore,the specificity and stability were detected by ELISA and Dot-blot.Sequence analysis proved that linker Sequence was introduced into VH and VL,and the gene expressed in E.coli BL21(DE3).Most of the protein existed in the form of inclusion body,the expression product were refolded successful.Results showed that recombinant sc-dsFv was expressed at high level.Purity of the protein>90%after purified by Ni-NTA and renaturation in vitro.Dot-blot and ELISA,BIAcore determinate results demonstrated that sc-dsFv could binding antigen specificity and was more stable,and higher yield of the antibody than dsFv, It indicated that sc-dsFv expressed and refolded successfully,which not only maintained high affinity, specificity and stability,and also provides a practical recombinant antibody for earlier diagnosis of citrus bacterial canker disease.更多还原