结核分枝杆菌ESAT6-CFP10融合蛋白的原核表达及其表达条件的优化

Prokaryotic expression of ESAT6-CFP10 fusion protein of Mycobacterium tuberculosis and optimization of condition for expression

目的原核表达结核分枝杆菌(Mycobacterium tuber-culosis,MTB)ESAT6-CFP10融合蛋白(recombinant ESAT6-CFP10,rEC),并优化表达条件。方法以(GGGGS)3为linker将ESAT6和CFP10两个基因连接,合成rEC全基因,插入至载体pET-28a,构建重组质粒pET-28a-rEC,转化至感受态E. coli BL21(DE3),筛选优势菌株进行诱导表达,并对诱导温度(25、30、37℃)、诱导剂种类及浓度(0. 05、0. 1、0. 2、0. 5、1. 0 mmol/L IPTG及0. 5、1、5、10、15、20 g/L乳糖)、诱导时间(2、3、4、5、6 h)、诱导起始A600(0. 4、0. 6、0. 8、1. 0)进行优化。菌种经5 L发酵罐发酵培养后,采用亲和层析及离子交换层析纯化rEC融合蛋白,于-20℃分别储存0、2、4、6个月,12. 5%SDS-PAGE分析rEC融合蛋白,并对保存0、6月的样品进行HPLC-SEC分析。结果重组质粒pET-28a-rEC经双酶切及测序鉴定,构建正确。rEC融合蛋白最适诱导条件为:于30℃,以0. 1 mmol/L IPTG诱导4 h。纯化后rEC融合蛋白纯度达95%以上,且可与鼠抗ESAT6单克隆抗体发生特异性结合,于-20℃保存6个月未见蛋白降解及聚体产生。结论原核表达了rEC融合蛋白,并优化了其表达条件,获得的rEC融合蛋白具有较高纯度及稳定性。

Objective To express the ESAT6-CFP10 fusion protein of Mycobacterium tuberculosis(MTB)in prokaryotic cells and optimize the condition for expression. Methods ESAT6 and CFP10 genes were linked using(GGGGS)3 as a linker,and the synthetic rEC gene was inserted into vector pET-28 a. The constructed recombinant plasmid pET-28 a-rEC was transformed to E. coli BL21(DE3),and the prominent clones were screened for expresssion. The temperature(25,30 and 37 ℃),inducer and its concentration(0. 05,0. 1,0. 2,0. 5 and 1. 0 mmol/L IPTG and 0. 5,1,5,10,15 and20 g/L lactose),time(2,3,4,5 and 6 h)and original A600 values(0. 4,0. 6,0. 8 and 1. 0)for induction were optimized. After the bacterial strain was cultured in 5 L fermentor, the rEC protein was purified by affinity and ion exchange chromatography,stored at-20 ℃ for 0,2,4 and 6 months and analyzed by SDS-PAGE. The samples 0 and 6 months after storage were analyzed by HPLC-SEC. Results Restriction analysis and sequencing proved that recombinant plasmid p ET-28 a-rEC was constrcuted correctly. The optimal temperature,inducer,concentration and time for induction were 30 ℃,IPTG,0. 1 mmol/L and 4 h respectively. The purified fusion protein reached a purity of more than 95%,and showed specific binding to mouse anti-ESAT6 monoclonal antibody,in which no degenearation or polymer was observed after storage at-20 ℃ for 6 months. Conclusion Fusion protein rEC was expressed in prokaryotic cells,and the condition for expression was optimized. The obtained rEC protein showed high purity and stability.