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目的原核表达α1-Giardin并制备兔抗rα1-Giardin多克隆抗体。方法以蓝氏贾第鞭毛虫DNA为模板,PCR扩增α1-giardin编码基因片段,经双酶切后连入原核表达载体pET-41a(+),构建重组表达载体pET-41a(+)-α1-giardin,热激法转化大肠埃希菌E.coli BL21(DE3),经IPTG诱导后收集菌体并裂解,采用SDS-PAGE检测目的蛋白的表达,采用His-tag亲和层析柱纯化融合蛋白。将α1-Giardin蛋白与等体积弗氏完全佐剂乳化后免疫新西兰白兔,隔周以同样剂量蛋白加等体积弗氏不完全佐剂加强免疫2次。末次免疫后一周颈动脉取血,制备多抗血清,采用间接ELISA法测定抗体效价。结果成功构建原核表达载体pET-41a(+)-α1-giardin,转化大肠埃希菌后经IPTG诱导表达分子质量单位为34.8ku的可溶性蛋白;经His-tag亲和层析柱纯化获得高纯度的重组α1-giardin蛋白,制备的免疫兔抗血清ELISA滴度为1∶51 200。结论成功克隆、表达并纯化了贾第虫α1-giardin蛋白,制备了高滴度的抗α1-giardin多克隆兔血清,为以α1-Giardin为抗原的疫苗研究奠定了基础。
Objective To prokaryotic express α1-Giardin and prepare rabbit anti-rα1-Giardin polyclonal antibody. Methods The gene fragment of α1-giardin was amplified by PCR using Giardia lamblia DNA as a template. The gene fragment was inserted into prokaryotic expression vector pET-41a (+) to construct the recombinant expression vector pET-41a (+) - α1-giardin was transformed into Escherichia coli BL21 (DE3) by heat shock method. After induced by IPTG, the cells were collected and lysed. The expression of the target protein was detected by SDS-PAGE and purified by His-tag affinity chromatography Fusion protein. New Zealand white rabbits were immunized with α1-Giardin and emulsified with an equal volume of Freund’s complete adjuvant, and immunized twice a week with the same dosage of protein and an equal volume of Freund’s incomplete adjuvant. One week after the last immunization, the carotid blood was taken to prepare multiple antiserum, and the antibody titer was measured by indirect ELISA. Results The prokaryotic expression vector pET-41a (+) - α1-giardin was successfully constructed and transformed into Escherichia coli. After induced by IPTG, the soluble protein with 34.8ku molecular weight was expressed. His-tag affinity chromatography was used to obtain high purity Of recombinant α1-giardin protein, prepared immune rabbit antiserum ELISA titer 1:51 200. Conclusion The Giardia alpha1-giardin protein was successfully cloned, expressed and purified, and polyclonal rabbit anti-α1-giardin serum was prepared with high titer. This study lays a foundation for the research of vaccine against α1-Giardin.