Ⅰ型变链素基因片段原核表达的构建

Prokaryotic expression of mutacin Ⅰ gene segments from Streptococcus mutan CH43

目的:获得编码变形链球菌CH43变链素Ⅰ前体肽基因片段,构建Ⅰ型变链素的原核表达载体,优化表达条件。方法:实验于2004-09/2005-06在解放军第四军医大学秦都口腔医院牙体牙髓病实验室地点完成。①实验材料:streptococcusMutanCH43菌株由Dr.P.g.Caufield惠赠。E.coliTOP10E.coliDH5α及表达载体pProEX由秦都口腔医院牙体牙髓病实验室保存。②实验过程:厌氧条件下培养变形链球菌CH43,通过碱裂解法得到其基因组DNA。利用聚合酶链反应技术从变形链球菌CH43基因库中扩增出编码变链素Ⅰ前体肽mutA基因片段相应大小的DNA片段,将片段与pMD18-T载体连接后测序以检测序列正确性。将测序正确的I型变链素mutA按照BamHI和HindIII酶切位点克隆入原核表达载体pProEX-HTb,将连接产物转化入E.coliDH5α,挑出阳性克隆,以A600从0.406～1.666七个不同浓度,异丙基β-D硫代半乳糖苷(终浓度设成1.0,1.4,1.8,2.0,2.5mmol/L5个梯度)诱导表达重组的带有6个组氨酸标签的融合蛋白,诱导时间分别3,6,18h。③实验评估:观察mutA片段的扩增及序列测定结果;表达载体的构建结果;融合蛋白在大肠杆菌中的表达及表达条件的优化。结果:①聚合酶链反应获得的mutA序列与GenBank报道的一致(为147bp)。②Ⅰ型变链素的mutA基因片断被成功克隆入原核表达载体pProEX-HTb中,在A600达到1.000以上,异丙基β-D硫代半乳糖苷终浓度1.2mmol/L,30℃诱导6h,蛋白表达量较佳,稳定可重复。结论:成功克隆变形链球菌CH43变链素ⅠmutA基因片段,构建表达了Ⅰ型变链素的融合蛋白。

AIM： To obtain mutA （coding the prepromutacin Ⅰ ） gene segments of Streptococcus mutan CH43, construct a prokaryotic expression vector of this mut A fragment, and optimize the induced conditions for expressing the prokaryotic vector efficiently. METHODS： The experiment was completed at the Department of Operative Dentistry and Endodontics, Stomatology College of the Fourth Military Medical University of Chinese PLA from September 2004 to June 2005. ①Streptococcus mutan CH43 strain was donated by Dr.P.g.Caufield. E.coliTOP10, E.coli DH5α and pProEX were preserved by the same laboratory above. ②Genome DNA was obtained using alkaline lysis in accordance with the sequence of Streptococcus mutan CH43 cultured under anaerobic condition, and a distinct segment of mut A was amplified by polymerase chain reaction （PCR）. Then the PCR product was inserted into pMD18-T vector, the recombinant plasmid was transformed into E.coli.DH5α, and two-stranded DNA was sequenced after screening. Afterwards the purified PCR products were subcloned into the sequencing vector pProEX-HTb in a certain direction BamHI/HinoⅢ digestion. After identification by PCR and enzyme digestion at A600 as different as 0.406-1.666, the selected positive clone containing the recombinant expression vector was induced by isopropy-β-D-thiogalactoside （IPTG） at the final concentrations of 1.0, 1.4, 1.8, 2.0, 2.5 mmol/L for 3, 6, 18 hours, expressing six histidine-labeled fusion protein.③Amplification and sequencing of mut A segment were detected; the expression vector was determined; the induced expression of fusion protein in E. coil and the induced condition were optimized. RESULTS： ①The length of PCR product （147 bp） was identical with that GenBank reported.②The mut A gene segments of Streptococcus mutan CH43 was cloned into the prokaryotic expression vector pProEX-HTb. The optimal induced condition for the expression included the final concentration of IPTG was 1.2 mmol/L at 30 ℃ for 6 hours with A600 over 1.000. CONCLUSION： The expression vectors containing the mutacin Ⅰ coding gene mutA of Streptococcus mutan CH43 are successfully constructed, and the fusion protein is also constructed.