抗隐孢子虫ScFv-PE重组毒素原核表达载体的构建及其对虫体的杀灭作用

Prokaryotic Expression and Characterization of Recombinant Anti-Cryptosporidium ScFv-PE

目的构建抗隐孢子虫ScFv-PE重组毒素原核表达载体并测定表达产物对隐孢子虫的杀灭作用。方法利用基因工程原理,以含抗隐孢子虫ScFv-PE重组毒素的菌株为模板,利用设计合成的一对引物扩增出ScFv-PE,利用pMAL-p2X载体构建重组毒素pMAL-ScFv-PE,并将其导入大肠杆菌中进行表达,产物进行SDS-PAGE和Western-blot,纯化后治疗感染隐孢子虫的小鼠。结果重组质粒经酶切鉴定正确,IPTG诱导后可看到明显的条带。凝胶薄层扫描分析目的蛋白表达量占菌体蛋白总量的21%。纯化后的表达产物治疗感染隐孢子虫的小鼠治疗组比对照组肠滞留物中卵囊计数(P<0.05)与肠道平均累计感染积分明显减少(P<0.01)。结论利用原核表达载体成功构建了可溶性形式表达的pMAL-ScFv-PE重组质粒,重组毒素对隐孢子虫具有明显的杀灭作用。

Objective To construct prokaryotic expression plasmid of anti-Cryptosporidium ScFv-PE and to analyze its killing effect on Cryptosporidium. Method Anti-Cryptosporidium ScFv-PE was amplified by PCR with a pair of specific primers designed from a pET-ScFv-PE sequence encoding the recombinant immunotoxins. The PCR fragment was cloned into a prokaryotic expressing vector pMAL-p2X, and then transferred into Escherichia coli DH5a. The transformatants were identified by SDS-PAGE and Western-blot. The recombinant protein was purified and used to test the biological function in vivo. Result Recombinant expression plasmid was constructed. The anti-Cryptosporidium ScFv-PE recombinant protein was highly expressed at 25℃, with 1mmol/L IPTG induction for 3h. SDS-PAGE analysis revealed that the recombinant immunotoxins ScFv-PE was about 21% of the total cell proteins. Oocysts count of intestine was obviously reduced in mice treated with purified recombinant anti-Cryptosporidium ScFv-PE than that of the untreated mice. Conclusion The recombinant plasmid was constructed successfully. Soluble recombinant protein was expressed and demonstrated an effective killing action on Cryptosporidium.