生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2013年
3期
351-354
,共4页
韩燃%李涛%王琴%房华丽%罗森%郭峰%李崭%王德慧%李文平%王慧
韓燃%李濤%王琴%房華麗%囉森%郭峰%李嶄%王德慧%李文平%王慧
한연%리도%왕금%방화려%라삼%곽봉%리참%왕덕혜%리문평%왕혜
肠出血性大肠杆菌O157∶H7%EspA%EspB%重组蛋白%免疫效果
腸齣血性大腸桿菌O157∶H7%EspA%EspB%重組蛋白%免疫效果
장출혈성대장간균O157∶H7%EspA%EspB%중조단백%면역효과
enterohemorrhagic Escherichia coli O157∶H7%EspA%EspB%recombinant protein%immune protection
目的：通过DNA重组技术表达肠出血性大肠杆菌（EHEC）O157∶H7的EspA和EspB蛋白，并分析它们的免疫保护性。方法：采用PCR技术从EHEC O157∶H7基因组中扩增espA和espB基因，连接至pET-22b（+）载体上，转化至宿主细胞大肠杆菌BL21（DE3），经IPTG诱导表达，用亲和层析纯化目的蛋白，SDS-PAGE测定其相对分子质量，免疫小鼠分析其免疫保护性。结果：重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致，一致性均为100%；得到了纯度为95%以上的重组EspA和EspB蛋白，免疫小鼠所得到的抗体效价均为106。结论：重组EspA和EspB蛋白获得了可溶性表达，表达的蛋白具有良好的免疫保护性，为进一步制备疫苗奠定了基础。
目的：通過DNA重組技術錶達腸齣血性大腸桿菌（EHEC）O157∶H7的EspA和EspB蛋白，併分析它們的免疫保護性。方法：採用PCR技術從EHEC O157∶H7基因組中擴增espA和espB基因，連接至pET-22b（+）載體上，轉化至宿主細胞大腸桿菌BL21（DE3），經IPTG誘導錶達，用親和層析純化目的蛋白，SDS-PAGE測定其相對分子質量，免疫小鼠分析其免疫保護性。結果：重組espA和espB基因片段的測序結果與GenBank中的相應基因序列完全一緻，一緻性均為100%；得到瞭純度為95%以上的重組EspA和EspB蛋白，免疫小鼠所得到的抗體效價均為106。結論：重組EspA和EspB蛋白穫得瞭可溶性錶達，錶達的蛋白具有良好的免疫保護性，為進一步製備疫苗奠定瞭基礎。
목적：통과DNA중조기술표체장출혈성대장간균（EHEC）O157∶H7적EspA화EspB단백，병분석타문적면역보호성。방법：채용PCR기술종EHEC O157∶H7기인조중확증espA화espB기인，련접지pET-22b（+）재체상，전화지숙주세포대장간균BL21（DE3），경IPTG유도표체，용친화층석순화목적단백，SDS-PAGE측정기상대분자질량，면역소서분석기면역보호성。결과：중조espA화espB기인편단적측서결과여GenBank중적상응기인서렬완전일치，일치성균위100%；득도료순도위95%이상적중조EspA화EspB단백，면역소서소득도적항체효개균위106。결론：중조EspA화EspB단백획득료가용성표체，표체적단백구유량호적면역보호성，위진일보제비역묘전정료기출。
Objective: To express recombinant EspA and EspB from enterohemorrhagic Escherichia coli(EHEC) O157∶H7, and analyze their protective immunity. Methods: The espA and espB gene were amplified by PCR with the genomic DNA from EHEC O157∶H7 strain, the amplified products were cloned into pET-22b(+) vector, then transferred into the host cells E.coli BL21(DE3) strain. The EspA and EspB proteins were expressed induced by IPTG, and purified by affinity chromatography. The relative molecular mass of purified proteins were determined by SDS-PAGE. The immune protection of the two proteins was analyzed by the immunization of mice. Results:The nucleotide sequences of the espA and espB were consistence with the sequence from GenBank by 100%. The purity of recombinant EspA and EspB were above 95%. The highest antibody titer of immunized mice sera was 106. Conclusion: The recombinant EspA and EspB protein of the EHEC O157∶H7 were successfully expressed in soluble form and the recombinant proteins have good protective immunity. These results may provide the founda?tion for the further development on EHEC O157∶H7 vaccine.
