热带医学杂志
熱帶醫學雜誌
열대의학잡지
JOURNAL OF TROPICAL MEDICINE
2007年
4期
297-302,306
,共7页
史咏梅%傅玉才%许铭言%徐晓园%许锦阶%邓致刚
史詠梅%傅玉纔%許銘言%徐曉園%許錦階%鄧緻剛
사영매%부옥재%허명언%서효완%허금계%산치강
阴道毛滴虫%Sir2同源基因%基因克隆%免疫定位
陰道毛滴蟲%Sir2同源基因%基因剋隆%免疫定位
음도모적충%Sir2동원기인%기인극륭%면역정위
Trichomonas vaginalis%Sir2 homolognes%gene cloning%immunolocalization
目的 探讨寄生性原虫阴道毛滴虫细胞生长和衰老的相关基因.方法 从阴道毛滴虫的cDNA表达文库中分离出两个与酵母沉寂信息调节因子 (Sir2) 有较高同源性的cDNA克隆,分别命名为TvSir2和TvSir2-like,它们的编码框分别长915 bp和1116 bp.结果 序列分析显示这两个cDNA克隆与酵母Sir2同源性很高,其氨基酸序列中含有Sk2p及其同源蛋白三个特征性保守结构域.分别从这两株cDNA克隆中扩增出表达片段植入表达载体pET-41a,转化宿主菌E.coli BL21并用IPTG(isoprupylthio-β-D-galactoside)诱导表达到大量融合蛋白.用亲和层析法纯化的融合蛋白分别免疫豚鼠,获得的抗血清用Western-blot法识别到滴虫虫体全蛋白中大小为34 000 Mr和42 000 Mr的条带.免疫荧光法检测TvSir2和TvSir2-like蛋白位于细胞核外的内质网和高尔基复合体区域.结论 TvSir2和TvSir2-like克隆是酵酶Sir2的同源基因,为TvSir2和TvSir2-like在模式生物阴道毛滴虫的功能研究奠定了基础.
目的 探討寄生性原蟲陰道毛滴蟲細胞生長和衰老的相關基因.方法 從陰道毛滴蟲的cDNA錶達文庫中分離齣兩箇與酵母沉寂信息調節因子 (Sir2) 有較高同源性的cDNA剋隆,分彆命名為TvSir2和TvSir2-like,它們的編碼框分彆長915 bp和1116 bp.結果 序列分析顯示這兩箇cDNA剋隆與酵母Sir2同源性很高,其氨基痠序列中含有Sk2p及其同源蛋白三箇特徵性保守結構域.分彆從這兩株cDNA剋隆中擴增齣錶達片段植入錶達載體pET-41a,轉化宿主菌E.coli BL21併用IPTG(isoprupylthio-β-D-galactoside)誘導錶達到大量融閤蛋白.用親和層析法純化的融閤蛋白分彆免疫豚鼠,穫得的抗血清用Western-blot法識彆到滴蟲蟲體全蛋白中大小為34 000 Mr和42 000 Mr的條帶.免疫熒光法檢測TvSir2和TvSir2-like蛋白位于細胞覈外的內質網和高爾基複閤體區域.結論 TvSir2和TvSir2-like剋隆是酵酶Sir2的同源基因,為TvSir2和TvSir2-like在模式生物陰道毛滴蟲的功能研究奠定瞭基礎.
목적 탐토기생성원충음도모적충세포생장화쇠로적상관기인.방법 종음도모적충적cDNA표체문고중분리출량개여효모침적신식조절인자 (Sir2) 유교고동원성적cDNA극륭,분별명명위TvSir2화TvSir2-like,타문적편마광분별장915 bp화1116 bp.결과 서렬분석현시저량개cDNA극륭여효모Sir2동원성흔고,기안기산서렬중함유Sk2p급기동원단백삼개특정성보수결구역.분별종저량주cDNA극륭중확증출표체편단식입표체재체pET-41a,전화숙주균E.coli BL21병용IPTG(isoprupylthio-β-D-galactoside)유도표체도대량융합단백.용친화층석법순화적융합단백분별면역돈서,획득적항혈청용Western-blot법식별도적충충체전단백중대소위34 000 Mr화42 000 Mr적조대.면역형광법검측TvSir2화TvSir2-like단백위우세포핵외적내질망화고이기복합체구역.결론 TvSir2화TvSir2-like극륭시효매Sir2적동원기인,위TvSir2화TvSir2-like재모식생물음도모적충적공능연구전정료기출.
Objective To screen cell growth and senescence-related genes of the parasitic pmtist Trichomonas vaginalis,we launched an EST program and isolated two cDNA clones from a T.vaginalis cDNA library,which showed high homology in deduced amino acid sequences to yeast Sir2 and designated as TvSir2 and TvSir2-like.Method The cDNA sequence of TvSIR2 had a length of 1034 base pairs (bp) with an open reading frame of 915 bp,and TvSIR2-like,1214 bp with an open reading frame of 1116 bp.Result The two deduced amino acid sequences shared all the three conserved cole domains with yeast Sir2 and its homologues,suggesting that the two clones were Sir2 homologues. A cDNA fragment from each cDNA clone was subvloned into the expression vector pET-41a.The expression of the fusion proteins in E.coli BL21 stains was induced by isopropylthio-β-D-galactoside (IPTG).Two anti-sera were prepared by immunizing two guinea pigs with the purified fusion proteins, Western-blot analysis demonstrated that each anti-serum reacted with the corresponding recombinant protein and detected a clear band (TvSir2,34 000 Mr;TvSir2-like,42 000 Mr)in protein extracts of the protist.Immunofluolescence techniques showed that TvSir2 and TvSir2-like proteins were both localized in the legions of perinuelear (ER) and Golgi complex.Conclusion Our data suggest that TvSir2 and TvSir2-like were two members of Sir2 family.Their biological functions in the protist would be further studied.
