融合基因CPA—LTB在大肠埃希菌中表达条件的优化

目的在构建含有CPA—LTB融合基因的重组菌株基础上，对其表达条件进行优化，以获得高效表达的CPA—LTB融合蛋白。方法采用限制性内切酶酶切鉴定含CPA—LTB融合基因的重组质粒pCPA—LTB，然后采用均匀设计法对影响目的蛋白表达的因素和水平进行优化：设计实验方案，分别以不同浓度的IPTG和乳糖为诱导剂诱导融合蛋白的表达，SDS-PAGE检测不同条件下融合蛋白的表达情况。结果双酶切鉴定重组质粒含有CPA-LTB融合基因（1700bp），且阅读框架正确。以IPTG为诱导剂在pH7．5、诱导温度31℃、转化菌菌液A-500值为1．2、IPTG终浓度0．4mmol／L诱导6h，目的蛋白表达量最大，占菌体总蛋白相对含量的54％；以乳糖为诱导剂在pH6．5、温度34℃、转化菌菌液A-500值为0．6、乳糖终浓度0．1mmol／L诱导6h，融合蛋白的表达量最大，占菌体总蛋白相对含量的61％。优化前后两种诱导剂诱导表达的蛋白量分别增加63％和42％。结论CPA—LTB融合基因表达质粒转化大肠埃希菌在优化的表达条件下高效表达目的蛋白，为研制α毒素基因工程亚单位疫苗奠定了基础。

Optimization of conditions for expression of the fusion CPA-LTB gene in Escherichia coli

Objective To construct a recombinant strain containing a fusion gene of Clostridium perfringens type A （CPA） and the heat-labile toxin B subunit （LTB） and optimize conditions for its expression in order to obtain a recombi- nant stain expressing high levels of the CPA-LTB fusion protein. Methods The recombinant plasmid pCPA-LTB was i- dentified using restriction endonuclease digestion. Factors for expression of the target protein and levels of its expression were optimized in accordance with the principle of uniform design. CPA-LTB fusion protein was expressed by induction with IPTG and lactose and analyzed using SDS-PAGE. Results Endonuclease digestion indicated that the recombinant plasmid pCPA-LTB contained the CPA-LTB fusion gene （1 700 bp） and had the correct reading frame. The optimal con- ditions for expression of the CPA-LTB fusion gene with IPTG induction were a culture medium pH of 7, 5, a culturing temperature of 31 ℃, and induction with IPTG at a final concentration of 0.4 mmol/L for 6 h when the cell density （OD 6oo ） of the recombinant strain reached 1.2. The level of CPA-LTB protein expression was about 54% of total cellular pro- tein according to SDS-PAGE and gel system analysis. Optimal conditions for expression of the fusion gene with lactose in- duction were a culture medium pH of 6.5, a culturing temperature of 34℃, and induction with lactose at a final concen- tration of 0.1 mmol/L for 6 h when a cell density （OD 600） of 0.6 was reached. The level of protein expression was about 61% of total cellular protein. Optimization increased protein expression with IPTG induction by 63 % and protein expres- sion with lactose induction by 42%. Conclusion The recombinant plasmid pCPA-LTB was highly expressed in Esche- richia coll. This lays the foundation for study of a genetically engineered subunit vaccine against the alpha toxin gene of C. perfringens.