华中科技大学学报（医学版）
華中科技大學學報（醫學版）
화중과기대학학보（의학판）
ACTA UNIVERSITATIS MEDICINAE TONGJI
2014年
4期
399-404
,共6页
吴春雪%陈强%李红%余晓君%朱春晖%李岚%何美娟%刘晓艳
吳春雪%陳彊%李紅%餘曉君%硃春暉%李嵐%何美娟%劉曉豔
오춘설%진강%리홍%여효군%주춘휘%리람%하미연%류효염
鼠伤寒沙门菌%spvB%基因缺陷变异%同源重组%生长曲线
鼠傷寒沙門菌%spvB%基因缺陷變異%同源重組%生長麯線
서상한사문균%spvB%기인결함변이%동원중조%생장곡선
Salmonella enterica serovar Typhi%spvB%gene-deleted mutation%homologous recombination%growth curve
目的：为深入研究鼠伤寒沙门菌毒力基因 spvB的功能，制备鼠伤寒沙门菌 spvB基因完全缺陷变异株，观察spvB基因缺陷株在体外酸性环境中的生存能力。方法根据鼠伤寒沙门菌spvB基因序列，设计PCR特异性引物，制备spvB基因缺陷性同源性核苷酸片段，导入自杀质粒 pCVD442后再导入鼠伤寒沙门菌野生株，进行同源重组，用 PCR方法观察重组现象，将完全重组的菌株作为 spvB基因的缺陷变异株，并通过核苷酸序列分析加以确定。绘制生长曲线，对比 spvB基因缺陷变异株与野生株在酸性条件下的生存情况。结果 PCR 及序列分析证实，缺陷变异株的 spvB基因缺失1749 bp。酸性条件下培养1 h及2 h后，野生株的活菌数高于spvB基因缺陷株，差异有统计学意义(P<0.05)；且野生株在酸性环境中的生存率明显高于缺陷株，野生株在1 h 及2 h 后的生存率分别为85.6%、74.9%，缺失株分别为68.0%、42.3%。结论成功构建鼠伤寒沙门菌 spvB基因缺陷变异株，并发现在酸性条件下其生存能力明显降低，为进一步研究其在鼠伤寒沙门菌中的功能奠定了基础。
目的：為深入研究鼠傷寒沙門菌毒力基因 spvB的功能，製備鼠傷寒沙門菌 spvB基因完全缺陷變異株，觀察spvB基因缺陷株在體外痠性環境中的生存能力。方法根據鼠傷寒沙門菌spvB基因序列，設計PCR特異性引物，製備spvB基因缺陷性同源性覈苷痠片段，導入自殺質粒 pCVD442後再導入鼠傷寒沙門菌野生株，進行同源重組，用 PCR方法觀察重組現象，將完全重組的菌株作為 spvB基因的缺陷變異株，併通過覈苷痠序列分析加以確定。繪製生長麯線，對比 spvB基因缺陷變異株與野生株在痠性條件下的生存情況。結果 PCR 及序列分析證實，缺陷變異株的 spvB基因缺失1749 bp。痠性條件下培養1 h及2 h後，野生株的活菌數高于spvB基因缺陷株，差異有統計學意義(P<0.05)；且野生株在痠性環境中的生存率明顯高于缺陷株，野生株在1 h 及2 h 後的生存率分彆為85.6%、74.9%，缺失株分彆為68.0%、42.3%。結論成功構建鼠傷寒沙門菌 spvB基因缺陷變異株，併髮現在痠性條件下其生存能力明顯降低，為進一步研究其在鼠傷寒沙門菌中的功能奠定瞭基礎。
목적：위심입연구서상한사문균독력기인 spvB적공능，제비서상한사문균 spvB기인완전결함변이주，관찰spvB기인결함주재체외산성배경중적생존능력。방법근거서상한사문균spvB기인서렬，설계PCR특이성인물，제비spvB기인결함성동원성핵감산편단，도입자살질립 pCVD442후재도입서상한사문균야생주，진행동원중조，용 PCR방법관찰중조현상，장완전중조적균주작위 spvB기인적결함변이주，병통과핵감산서렬분석가이학정。회제생장곡선，대비 spvB기인결함변이주여야생주재산성조건하적생존정황。결과 PCR 급서렬분석증실，결함변이주적 spvB기인결실1749 bp。산성조건하배양1 h급2 h후，야생주적활균수고우spvB기인결함주，차이유통계학의의(P<0.05)；차야생주재산성배경중적생존솔명현고우결함주，야생주재1 h 급2 h 후적생존솔분별위85.6%、74.9%，결실주분별위68.0%、42.3%。결론성공구건서상한사문균 spvB기인결함변이주，병발현재산성조건하기생존능력명현강저，위진일보연구기재서상한사문균중적공능전정료기출。
Objective To investigate the function of spvB gene,a toxicity gene of Salmonella enterica serovar Typhi,by constructing the spvB gene-deleted mutant and examinimg its survival ability in acid condition.Methods According to the se-quences of spvB gene of Salmonella enterica serovar Typhi,specific primers were designed for PCR.The homologous DNA fragments with spvB gene deleted were constructed,which was cloned into the suicide plasmid pCVD442 and then transferred into the target cells of Salmonella enterica serovar Typhi.The recombination was visualized by PCR,and the complete recombi-nant strain was selected as the spvB gene-deleted mutant strain and confirmed by the corresponding sequencing analysis.Under the acid condition,the survival ability of the spvB mutant and parent was compared by using the growth curve.Results A dele-tion of 1 748 bp of the spvB gene was confirmed by PCR and sequencing analysis in spvB gene-deleted mutants.The number of live wild-type strains were significantly greater than that of spvB gene-deleted mutants under the acid condition for 1 and 2 h, and the difference was statistically significant(P<0.05).The survival rate of the wild-type strains was 85.6% and 74.9% at 1 and 2 h,significantly higher than that of the spvB gene-deleted mutants,which was 68.0% and 42.3%.Conclusion The spvB gene-deleted mutant of Salmonella enterica serovar Typhi was successfully generated and its survival ability was significantly compromised under the acid condition,which lays a foundation for studying the function of the spvB gene in Salmonella enteri-ca serovar Typhi.
