中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2011年
4期
335-340
,共6页
王玲%胡黎平%龙北国%周勇%罗军%袁广明%范宏英
王玲%鬍黎平%龍北國%週勇%囉軍%袁廣明%範宏英
왕령%호려평%룡북국%주용%라군%원엄명%범굉영
大肠埃希菌O157:H7%转位紧密黏附素受体%疫苗
大腸埃希菌O157:H7%轉位緊密黏附素受體%疫苗
대장애희균O157:H7%전위긴밀점부소수체%역묘
EHEC O157:H7%Translocation intimin receptor%Vaccine
目的 克隆、表达和纯化肠出血型大肠埃希菌(enterohaemorrhagic E.coli,EHEC)O157:H7转位紧密黏附素受体蛋白(Tir),观察不同免疫途径对其免疫效价的影响,为EHEC O157:H7亚单位疫苗的研究提供了实验资料.方法 扩增tir基因,克隆到pET-30a(+)载体上,转化至大肠埃希菌BL21/DE3,诱导表达目的 蛋白,通过Ni-IDA亲和层析进行纯化;将重组蛋白Tir免疫小鼠,检测血清和粪便提取物中抗体效价.结果 双酶切和测序鉴定结果均显示重组质粒pET-30a (+)-tir构建成功.SDS-PAGE结果表明,目的 蛋白Tir在大肠埃希菌BL21(DB3)中得到表达.皮下免疫和鼻腔免疫小鼠,血清中均能检测到高效价的IgG类抗体,鼻腔免疫组小鼠血清和粪便lgA类抗体效价明显高于皮下免疫组.结论 Tir蛋白具有一定的免疫原性.
目的 剋隆、錶達和純化腸齣血型大腸埃希菌(enterohaemorrhagic E.coli,EHEC)O157:H7轉位緊密黏附素受體蛋白(Tir),觀察不同免疫途徑對其免疫效價的影響,為EHEC O157:H7亞單位疫苗的研究提供瞭實驗資料.方法 擴增tir基因,剋隆到pET-30a(+)載體上,轉化至大腸埃希菌BL21/DE3,誘導錶達目的 蛋白,通過Ni-IDA親和層析進行純化;將重組蛋白Tir免疫小鼠,檢測血清和糞便提取物中抗體效價.結果 雙酶切和測序鑒定結果均顯示重組質粒pET-30a (+)-tir構建成功.SDS-PAGE結果錶明,目的 蛋白Tir在大腸埃希菌BL21(DB3)中得到錶達.皮下免疫和鼻腔免疫小鼠,血清中均能檢測到高效價的IgG類抗體,鼻腔免疫組小鼠血清和糞便lgA類抗體效價明顯高于皮下免疫組.結論 Tir蛋白具有一定的免疫原性.
목적 극륭、표체화순화장출혈형대장애희균(enterohaemorrhagic E.coli,EHEC)O157:H7전위긴밀점부소수체단백(Tir),관찰불동면역도경대기면역효개적영향,위EHEC O157:H7아단위역묘적연구제공료실험자료.방법 확증tir기인,극륭도pET-30a(+)재체상,전화지대장애희균BL21/DE3,유도표체목적 단백,통과Ni-IDA친화층석진행순화;장중조단백Tir면역소서,검측혈청화분편제취물중항체효개.결과 쌍매절화측서감정결과균현시중조질립pET-30a (+)-tir구건성공.SDS-PAGE결과표명,목적 단백Tir재대장애희균BL21(DB3)중득도표체.피하면역화비강면역소서,혈청중균능검측도고효개적IgG류항체,비강면역조소서혈청화분편lgA류항체효개명현고우피하면역조.결론 Tir단백구유일정적면역원성.
Objective To clone and express translocation intimin receptor(Tir)of enterohemorrhagic Escherichia coli(EHEC)O157:H7,and to analyze the effect of different routes on the induction of immunity to the recombinant protein.Methods The tir eucoding genes were amplified from EHEC O157:H7 strain guangzhou 246 genome,and genes were cloned into the vector pET-30a(+).The pET-30a(+)tir recombinant was transformed into E.coli BL21.and expression was induced bv IPTG.The expressed product was analyzed by SDS-PAGE and purified by Ni-IDA affinity chromatography.The immunized mice sera and fecal against the recombinant protein was detected.Resuits The length of the tir is 1677 bp,with the initiation codon ATG and the termination codon TAA.Double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid pET-30a(+)-tir was constructed.The recombinant protein was expressed in Escherichia coli expression system,and was purified by Ni-IDA affinity chromatography.The mice were able to produce a high serum IgG antibody titer after both subeutaneous and intranasal immunizations.Meanwhile,the intranasal immunization induced serum and fecal IgA antibody titer was significantly higher than that of the subcutaneous immunization group.Conclusion Tir molecule is potential vaccine candidate for preventing EHEC disease.
