柑橘溃疡病菌二硫键稳定Fv抗体基因的构建及表达产物的生物学活性分析  

Gene Construction, Expression and Biological Activity Analyses of Disulfide Stabilized Fv Fragment Against Xac

作　　者：王中康[1] 李泮志[1] 袁青[2] 于红[1] 李蒙[1] 殷幼平[1] 

机构地区：[1]重庆大学生物工程学院/重庆大学基因工程研究中心/重庆市基因功能与调控重点实验室,重庆400030 [2]四川省泸州医学院免疫学教研室,四川泸州646000

出　　处：《中国农业科学》2009年第9期3123-3130,共8页Scientia Agricultura Sinica

基　　金：国家"十一五"科技支撑计划项目(2007BAD47B03-5);国家"863"计划项目(2006AA10Z434)

摘　　要：【目的】构建鼠源抗柑橘溃疡病菌二硫键稳定Fv抗体(dsFv)片段基因,原核表达并将其复性为具有免疫活性的dsFv抗体。【方法】采用PCR定点突变的方法构建dsFv重链(VH)及轻链可变区(VL)基因,分别将其连接入表达载体中,于E.coliBL21(DE3)中诱导表达,表达产物溶解后稀释入复性缓冲液中使其重折叠为dsFv抗体并纯化。SDS-PAGE及Western-blot分析表达及复性产物,BIAcore检测dsFv与Xac-LPS的亲和力,ELISA验证dsFv的特异性及稳定性。【结果】测序结果表明在VH的44位和VL的100位成功引入了半胱氨酸突变位点,实现了高效的原核表达,表达产物主要以包涵体的形式存在。SDS-PAGE分析显示VH和VL的大小为23kD,并将其成功复性,复性后大小为46kD。BIAcore分析表明dsFv对Xac-LPS保持了很高的亲和力,亲和常数(KD)达到3.40×10-10M。ELISA证明dsFv具有较高的抗原特异性并且热稳定性较scFv提高近20℃。【结论】成功表达并复性了dsFv抗体,为柑橘溃疡病菌的免疫诊断提供了高效优质的重组抗体。【Objective】To construct the gene of mouse anti-Xac disulfide stabilized Fv fragments （dsFv）, express and refold to the form of dsFv with biological activity.【Method】The genes of VH and VL raised against Xac were mutated by the method of PCR-based mutagenesis, and then cloned into expression plasmid. The recombinant plasmids were transformed into E. coli BL21 （DE3） strain and these two genes were expressed. The inclusion body proteins of VH and VL were harvested and dissolved, and then diluted into refoldingsolution pro rata to form dsFv antibody which was further purified by HisTrap HP column. The products of expression and renaturation were analyzed by SDS-PAGE and western-blot. The affinity of dsFv to Xac-LPS was determined by BIAcore. The specificity and stability were detected by ELISA. [Result] Sequence analysis proved that cysteines were introduced into VH44 and VL100, and the genes expressed in E.coli BL21 （DE3）. Most of the protein existed in the form of inclusion body, the expression products were refolded successfully. SDS-PAGE showed that the molecular masses were 23 kD for VH and VL, and 46 kD for dsFv. BIAcore analysis showed that dsFv retained high affinity to Xac-LPS with an affinity constant （KD） of 3.40×10^-10M. ELISA indicated that dsFv had a high specificity to Xac. Compared with scFv＇s, the thermal stability of dsFv elevated nearly 20℃. [ Conclusion] dsFv was expressed and refolded successfully, which maintained high affinity, specificity and stability. This research established a solid basis for rapid diagnosis of Xac.

关 键 词：柑橘溃疡病菌 二硫键稳定Fv抗体 包涵体 复性 BIAcore分析 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]