含有分子内佐剂的CIC蛋白表达及免疫活性初步研究

发布时间：2018-03-14 20:29

本文选题：金黄色葡萄球菌　切入点：CTB-IsdBid-Clfais蛋白　出处：《中国畜牧兽医》2017年12期 　论文类型：期刊论文

【摘要】：为了研究含有分子内佐剂的CTB-IsdBid-Clfais(CIC)蛋白表达及其免疫活性,试验采用重叠PCR方法将分子内佐剂CTB与IsdBid-Clfais基因串联,并将CTB-IsdBid-Clfais(CIC)插入到表达载体pET-32a(+)中,构建pET-32a(+)-CTB-IsdBid-Clfais重组质粒,将鉴定正确的重组质粒转化到大肠杆菌BL21感受态细胞中表达目的蛋白CIC,利用Western blotting方法对其进行鉴定,并以ELISA方法检测CIC蛋白的免疫活性。结果发现,试验成功扩增了2 072bp的目的基因CIC,并将CIC正确连接到pET-32a(+)载体上,构建了pET-32a(+)-CTB-IsdBid-Clfais重组质粒;Western blotting结果证实,该重组质粒能够在大肠杆菌BL21感受态细胞中正确表达CIC蛋白,分子质量大小为95.9ku;ELISA结果显示,CIC试验组与IsdBid、Clfais蛋白组间均无显著差异(P0.05),与BSA组间差异极显著(P0.01)。综上所述,本研究成功构建了pET-32a(+)-CTB-IsdBid-Clfais重组质粒,该质粒在大肠杆菌BL21中成功表达了CIC蛋白,且CIC能与IsdBid、Clfais免疫小鼠血清发生反应,具有较强的免疫活性。
[Abstract]:In order to study the expression and immunological activity of CTB-IsdBid-Clfais-CICs containing intramolecular adjuvants, the recombinant plasmid pET-32a (-CTB-IsdBid-Clfais) was constructed by using overlapping PCR method to connect the intramolecular adjuvant CTB with the IsdBid-Clfais gene and insert CTB-IsdBid-Clfais into the expression vector pET-32a (pET-32a), and construct the recombinant plasmid pET-32a (-CTB-IsdBid-Clfais), and construct the recombinant plasmid pET-32a (-CTB-IsdBid-Clfais). The identified recombinant plasmid was transformed into Escherichia coli BL21 competent cells to express the target protein CIC. Western blotting method was used to identify the recombinant plasmid, and the ELISA method was used to detect the immunological activity of CIC protein. The target gene CIC-2072 BP was amplified successfully, and the CIC was correctly ligated to pET-32a () vector. The recombinant plasmid pET-32a (-CTB-IsdBid-Clfais) was constructed by Western blotting. The results showed that the recombinant plasmid could correctly express CIC protein in Escherichia coli BL21 competent cells. The results of Elisa showed that there was no significant difference between CIC test group and Isd Bidn Clfais protein group, but there was very significant difference between CICC test group and BSA group. In conclusion, the recombinant plasmid pET-32a was successfully constructed in this study. The plasmid successfully expressed CIC protein in Escherichia coli BL21, and CIC could react with the serum of Isd Bidsil-Clfais immunized mice, and had strong immune activity.
【作者单位】：黑龙江八一农垦大学生命科学技术学院;
【基金】：黑龙江省大学生创新创业训练计划项目(201510223001) 黑龙江省自然科学基金项目(C201443) 大庆市指导性科技计划项目(S2dfy-2015-44)
【分类号】：S852.611

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