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
2014年
9期
702-706
,共5页
金蕾%蒋锦琴%方佳琪%林旭瑷%严杰%孙爱华
金蕾%蔣錦琴%方佳琪%林旭璦%嚴傑%孫愛華
금뢰%장금금%방가기%림욱애%엄걸%손애화
甲型副伤寒沙门菌%h1a-spaO融合基因%重组表达%免疫原性%免疫保护性
甲型副傷寒沙門菌%h1a-spaO融閤基因%重組錶達%免疫原性%免疫保護性
갑형부상한사문균%h1a-spaO융합기인%중조표체%면역원성%면역보호성
Salmonella paratyphi A%h1a-spaO fusion gene%Recombinant expression%Immunoge-nicity%Immunoprotection
目的：构建甲型副伤寒沙门菌h1a-spaO融合基因及其原核表达系统，确定重组表达产物rH1a-SpaO免疫保护作用。方法采用柔性肽序列连接引物PCR构建并扩增h1a与spaO基因的融合基因h1a-spaO，T-A克隆后测序。采用常规基因工程方法构建h1a-spaO融合基因原核表达系统，SDS-PAGE检测其rH1a-SpaO表达情况。采用Western blot鉴定rH1a-SpaO抗原性和免疫反应性，微量肥达试验确定rH1a-SpaO抗血清凝集甲型副伤寒沙门菌的能力。采用小鼠感染模型了解rH1a-SpaO对甲型副伤寒沙门菌致死性感染的保护作用并与等量单一重组表达蛋白H1a（ rH1a）及SpaO （rSpaO）比较。结果所构建的h1a-spaO融合基因与单一h1a或spaO基因核苷酸和氨基酸序列相似性均为100％。 h1a-spaO融合基因原核表达系统能高效表达rH1a-SpaO。 rH1a-SpaO能与rH1a或rSpaO抗血清结合， rH1a-SpaO抗血清也能识别rH1a和rSpaO并经H抗原凝集甲型副伤寒沙门菌。100μg rH1a-SpaO 对甲型副伤寒沙门菌感染小鼠的免疫保护率（93．3％）明显高于等量 rH1a （60．0％）及rSpaO（53．3％）（P＜0．05）。结论重组融合蛋白抗原rH1a-SpaO免疫保护作用较等量单一rH1a或rSpaO更强，可作为甲型副伤寒两价基因工程疫苗或伤寒/副伤寒荚膜多糖-蛋白结合疫苗的有效抗原。
目的：構建甲型副傷寒沙門菌h1a-spaO融閤基因及其原覈錶達繫統，確定重組錶達產物rH1a-SpaO免疫保護作用。方法採用柔性肽序列連接引物PCR構建併擴增h1a與spaO基因的融閤基因h1a-spaO，T-A剋隆後測序。採用常規基因工程方法構建h1a-spaO融閤基因原覈錶達繫統，SDS-PAGE檢測其rH1a-SpaO錶達情況。採用Western blot鑒定rH1a-SpaO抗原性和免疫反應性，微量肥達試驗確定rH1a-SpaO抗血清凝集甲型副傷寒沙門菌的能力。採用小鼠感染模型瞭解rH1a-SpaO對甲型副傷寒沙門菌緻死性感染的保護作用併與等量單一重組錶達蛋白H1a（ rH1a）及SpaO （rSpaO）比較。結果所構建的h1a-spaO融閤基因與單一h1a或spaO基因覈苷痠和氨基痠序列相似性均為100％。 h1a-spaO融閤基因原覈錶達繫統能高效錶達rH1a-SpaO。 rH1a-SpaO能與rH1a或rSpaO抗血清結閤， rH1a-SpaO抗血清也能識彆rH1a和rSpaO併經H抗原凝集甲型副傷寒沙門菌。100μg rH1a-SpaO 對甲型副傷寒沙門菌感染小鼠的免疫保護率（93．3％）明顯高于等量 rH1a （60．0％）及rSpaO（53．3％）（P＜0．05）。結論重組融閤蛋白抗原rH1a-SpaO免疫保護作用較等量單一rH1a或rSpaO更彊，可作為甲型副傷寒兩價基因工程疫苗或傷寒/副傷寒莢膜多糖-蛋白結閤疫苗的有效抗原。
목적：구건갑형부상한사문균h1a-spaO융합기인급기원핵표체계통，학정중조표체산물rH1a-SpaO면역보호작용。방법채용유성태서렬련접인물PCR구건병확증h1a여spaO기인적융합기인h1a-spaO，T-A극륭후측서。채용상규기인공정방법구건h1a-spaO융합기인원핵표체계통，SDS-PAGE검측기rH1a-SpaO표체정황。채용Western blot감정rH1a-SpaO항원성화면역반응성，미량비체시험학정rH1a-SpaO항혈청응집갑형부상한사문균적능력。채용소서감염모형료해rH1a-SpaO대갑형부상한사문균치사성감염적보호작용병여등량단일중조표체단백H1a（ rH1a）급SpaO （rSpaO）비교。결과소구건적h1a-spaO융합기인여단일h1a혹spaO기인핵감산화안기산서렬상사성균위100％。 h1a-spaO융합기인원핵표체계통능고효표체rH1a-SpaO。 rH1a-SpaO능여rH1a혹rSpaO항혈청결합， rH1a-SpaO항혈청야능식별rH1a화rSpaO병경H항원응집갑형부상한사문균。100μg rH1a-SpaO 대갑형부상한사문균감염소서적면역보호솔（93．3％）명현고우등량 rH1a （60．0％）급rSpaO（53．3％）（P＜0．05）。결론중조융합단백항원rH1a-SpaO면역보호작용교등량단일rH1a혹rSpaO경강，가작위갑형부상한량개기인공정역묘혹상한/부상한협막다당-단백결합역묘적유효항원。
Objective To construct a fusion gene (h1a-spaO) encoding H1a-SpaO protein of Sal-monella paratyphi A ( S.paratyphi A) and to express it in prokaryotic expression system , then to further ana-lyze the immunoprotective effects of the expressed protein rH 1a-SpaO.Methods The h1a-spaO fusion gene formed from separate h1a and spaO genes was amplified by PCR using flexible peptide sequence-containing linking primers and then sequenced after T-A cloning.A prokaryotic expression system for expressing h1a-spaO fusion gene was constructed by using the genetic engineering technique .The expressed protein rH1a-SpaO was examined by SDS-PAGE.The antigenicity and immunoreactivity of rH1a-SpaO protein were deter-mined by Western blot assay .The ability of rH1a-SpaO antiserum agglutinating S.paratyphi A strains was detected by micro-Widal′s test.The immunoprotective effects of rH 1a-SpaO against the lethal dose challenge of S.paratyphi A strains were analyzed in a mouse model and that were compared with those by using equal dose of individual recombinant protein H1a and SpaO (rH1a and rSpaO) as the immunogens, respectively. Results The h1a-spaO fusion gene was 100%identical with the individual h1a or spaO gene in nucleotide and amino acid sequences .The constructed prokaryotic expression system could express the recombinant pro-tein rH1a-SpaO with an advantage of high efficiency .rH1a-SpaO protein was able to react with rH 1a or rSpaO antiserum.Moreover, rH1a-SpaO antiserum also could efficiently recognize rH 1a and rSpaO as well as agglutinate Salmonella paratyphi A strains by binding with H-antigen.The immunoprotective rate (93.3%) in mice pre-immunized with 100 μg of rH1a-SpaO protein was significantly higher than that in those pre-immunized with equal dose of rH1a (60.0%) protein or rSpaO protein(53.3%) (P<0.05).Conclusion The recombinant fusion protein rH 1a-SpaO showed more stronger immunoprotective function than the individ-ual rH1a or rSpaO protein , which could be used as an effective antigen for the development of bi -valent para-typhoid A vaccine or typhoid/paratyphoid capsular polysaccharide-protein combined vaccine .
