摘要
在大肠杆菌中以可溶性形式高效表达弓形虫膜表面抗原SAG2蛋白,并对其免疫活性进行分析。应用PCR技术从刚地弓形虫RH株的基因组DNA中扩增编码SAG2的基因片段,亚克隆至原核表达载体pET32a(+),在大肠埃希菌(E.coli)BL21内表达,并对其表达条件进行优化,Western blotting和ELISA分析纯化蛋白的免疫原性;纯化的重组蛋白免疫小鼠制备多抗,用间接免疫荧光试验(IFA)分析表达蛋白的免疫反应性。成功构建重组质粒pET32a(+)-tSAG2,所表达的融合蛋白大小约为38kD。在IPTG终浓度为0.1mmol/L、诱导时间4-6h和培养温度32℃条件下,重组SAG2蛋白主要以可溶性形式在大肠杆菌中高效表达,每升培养菌液约获得可溶性重组SAG2蛋白16mg。Western blotting及ELISA结果显示纯化蛋白具有良好的免疫原性。IFA显示重组蛋白的抗血清能够识别刚地弓形虫表面的SAG2天然蛋白,所表达蛋白具有良好的免疫反应性。截断的SAG2基因在大肠杆菌中得到了高效表达,重组蛋白保持了天然蛋白的免疫活性,为进一步利用该重组蛋白进行弓形虫病免疫诊断及基因工程亚单位疫苗的研制奠定基础。
Express the truncated surface antigen SAG2 of Toxoplasma Gondii（T.gondii）in E.coli and analyze the immunological activity of the expressed protein SAG2.The truncated SAG2 gene fragment were amplified by PCR from the total DNA of T.gondii and subcloned into a prokaryotic expression vector pET32a（＋）.After identification by restriction enzymes,the expression of the recombinant pET32a（＋）-tSAG2 was induced by IPTG.After purified by Ni2＋ affinity chromatography,the protein was used to immunize the rat.The immunological activity of the purified SAG2 was analysed by Western blotting,ELISA and IFA analysis.The recombinant pET32a（＋）-tSAG2 was successfully constructed and transformed into E.coli BL21（DE3）,and the recombinant protein was highly expressed in E.coli in conditions of 32℃,0.1 mmol/L IPTG and 4-6 h.About 16 mg soluble recombinant protein can be obtained every litre cultivation.SDS-PAGE and Western blotting analysis showed that the target recombinant protein with the molecular weight of 38 kD.It could be specifically recognized by serum against T.gondii from goat by Western blotting and ELISA.IFA analysis show that the antiserum of SAG2 can recognized the nature protein SAG2 localized in the membrane surface of T.gondii.Trucated SAG2 fragment was highly expressed in E.coli,purified SAG2 maintain the immune activity of natural protein,which can be used for the further research on immunity detection and the genetic engineering subunit vaccine for T.gondii.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第4期173-178,共6页
Biotechnology Bulletin
基金
农业公益性行业科研专项经费项目(200803017)
