细粒棘球蚴重组蛋白HIS-CTLA-4IgV-EgG1Y162的诱导表达及3D结构预测

Induced expression,identification and 3Dstructure prediction of recombinant protein HIS-CTLA-4IgV-EgG1Y162 of Echinococcus granulosus

目的探索重组蛋白HIS-CTLA-4IgV-EgG1Y162最佳的表达条件,大量诱导及鉴定蛋白质,并预测重组蛋白质的三维结构模型。方法将原核重组质粒pET30a-CTLA-4IgV-EgG1Y162转化至E.coli.BL21(DE)菌株中,在不同条件下诱导重组蛋白进行表达,SDS-PAGE分析上清及沉淀中重组蛋白表达并用Western blot鉴定。I-TASSER在线预测重组蛋白HIS-CTLA-4IgV-EgG1Y162的三维结构。结果可溶性重组蛋白HIS-CTLA-4IgV-EgG1Y162在异丙基硫代半乳糖苷(isopropylβ-D-Thiogalactoside,IPTG)(终浓度0.2 mmol/L),28℃4 h诱导条件下表达量较高;包涵体中的重组蛋白在IPTG(终浓度0.8 mmol/L),28℃2 h并37℃2 h诱导条件下表达量最高。Western blot鉴定该蛋白的相对分子量约为32×10^(3),与预期相符。利用I-TASSER在线预测重组蛋白质HIS-CTLA-4IgV-EgG1Y162的3D结构模型,结果显示相邻的蛋白CTLA-4IgV与蛋白EgG1Y162能抵抗空间位阻的影响进行正常折叠。结论探索出重组蛋白HIS-CTLA-4IgV-EgG1Y162的最佳诱导表达条件并对重组蛋白成功鉴定,根据预测的重组蛋白3D结构验证选用linker序列"GTDDDDKAMADIGSEF"能使其前后相邻的两个蛋白独立正常折叠,为进一步深入研究细粒棘球蚴重组疫苗创造了条件。

Objective To explore the best expression conditions of recombinant protein HIS-CTLA-4 IgV-EgG1 Y162 of Echinococcus granulosus,to identify the protein and to predict the three-dimensional(3 D)structure of recombinant protein.Methods The recombinant prokaryotic plasmid pET30 a-CTLA-4 IgV-EgG1 Y162 was transformed into E.coli BL21(DE)strain,The recombinat protein was induced to expressunder different conditions.The expression of recombinat protein in supernatant and precipitation was analyzed by SDS-PAGE and indentified by Western blot.The 3 D structure of recombinant protein HIS-CTLA-4 IgV-EgG1 Y162 was analyzed using online software I-TASSER.Results The quantity of expressed soluble recombinant protein HIS-CTLA-4 IgV-EgG1 Y162 was the highest under 0.2 mmol/L IPTG induction and at 28℃for 4 hours.The expression of recombinant protein in inclusion body was the highest under 0.8 mmol/L final concentration of IPTG induction and at 28℃for 2 hours or at 37℃for 2 hours.The relative molecular weight of the protein identified by Western blot was around 32×10^(3),which was consistent with the expectation.The online I-TASSER analysis for 3 D structure of recombinant protein HIS-CTLA-4 IgV-EgG1 Y162 showed that the adjacent protein CTLA-4 IgV and protein EgG1 Y162 could fold normally against the influence of steric hindrance.Conclusions We have optimized the conditions for induced expression of recombinant protein HIS-CTLA-4 IgV-EgG1 Y162 which has been successfully identified.Based on the predicted 3 Dstructure of the recombinant protein,the linker sequence"GTDDDDKAMADIGSEF"is able to allow the two adjacent proteins folding independently and normally,which created conditions for further study of Echinococcus granulosus recombination vaccines.