中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2007年
29期
5836-5839
,共4页
李君武%黄泽棋%姚翠婵%周曙光%李晓栋%宋东%黄清华
李君武%黃澤棋%姚翠嬋%週曙光%李曉棟%宋東%黃清華
리군무%황택기%요취선%주서광%리효동%송동%황청화
结核分枝杆菌%Hsp65%Esat6%融合基因%DNA疫苗
結覈分枝桿菌%Hsp65%Esat6%融閤基因%DNA疫苗
결핵분지간균%Hsp65%Esat6%융합기인%DNA역묘
背景:Hsp65和Esat6之间的Linker编码一段疏水性多肽,其具有良好的柔顺性和折叠性,有利于翻译成融合蛋白时融合蛋白之间的空间折叠,使融合蛋白保持与两个天然蛋白空间构象的一致性,从而形成正确的构型而提高它们的免疫原性.目的:从结核分枝杆菌(MTB)H37Rv株中克隆目的融合基因Hsp65-Esat6,与报告基因EGFP一起构建真核表达载体pEGHsp65-Esat6,通过免疫组织化学法分别检测细胞中Hsp65蛋白和ESAT-6蛋白的表达.设计:单一样本实验.单位:暨南大学医学院微生物免疫学教研室.材料:质粒pVAE由华南理工大学曹以诚教授惠赠,MTBH37Rv株、大肠杆菌DH5α、表达质粒pEGFP-C1为本实验室保存,Hela细胞由本教研室胡萍博士惠赠.方法:实验于2005-09/2006-06在暨南大学医学院微生物与免疫教研室和中心实验室完成.以MTB全基因组为模板经聚合酶链反应(PCR)扩增出Hsp65基因(不含终止子),与pEGFP-C1载体进行重组,构建pEGHsp65(不含终止子)重组质粒.再以pVAE质粒为模板,经PCR扩增出Linker-Esat6基因,与pEGHsp65质粒(不含终止子)进行重组,构建真核表达载体pEGHsp65-Esat6.通过限制性内切酶图谱测定pEGHsp65-Esat6融合质粒大小;对质粒pEGFP-C1的多克隆位点内的DNA序列进行序列分析鉴定;将质粒转染Hela细胞,观察荧光的表达情况及转染效率,通过免疫组织化学法分别检测细胞中Hsp65蛋白和ESAT-6蛋白的表达.主要观察指标:①pEGHsp65-Esat6融合质粒大小.②pEGFP-C1DNA序列分析.③转染后Hela细胞的荧光表达情况.④细胞中Hsp65蛋白和ESAT-6蛋白的免疫组织化学结果.结果:①质粒pEGFP-C1的大小约为4.7 kb,而pEGHsp65为6.4 kb,融合质粒pEGHsp65-Esat6约为6.7 kb,在琼脂糖凝胶电泳图上可以分辨出泳动速度的差异.②结果与结核分支杆菌H37Rv株的Hsp65和Esat6的基因序列完全相同.③转染融合质粒24 h后,使用共聚焦显微镜可观察到有部分细胞表达荧光蛋白,说明融合质粒转染成功,转染率约为30%.未转染的Hela细胞则无荧光表达.④通过免疫组织化学法证实融合质粒在Hela细胞内能正确表达出具有生物学活性的Hsp65与ESAT-6蛋白.结论:采用Hsp65和Esat6搭配作为免疫原,成功克隆并构建了结核分枝杆菌融合基因Hsp65-Esat6荧光真核表达质粒.
揹景:Hsp65和Esat6之間的Linker編碼一段疏水性多肽,其具有良好的柔順性和摺疊性,有利于翻譯成融閤蛋白時融閤蛋白之間的空間摺疊,使融閤蛋白保持與兩箇天然蛋白空間構象的一緻性,從而形成正確的構型而提高它們的免疫原性.目的:從結覈分枝桿菌(MTB)H37Rv株中剋隆目的融閤基因Hsp65-Esat6,與報告基因EGFP一起構建真覈錶達載體pEGHsp65-Esat6,通過免疫組織化學法分彆檢測細胞中Hsp65蛋白和ESAT-6蛋白的錶達.設計:單一樣本實驗.單位:暨南大學醫學院微生物免疫學教研室.材料:質粒pVAE由華南理工大學曹以誠教授惠贈,MTBH37Rv株、大腸桿菌DH5α、錶達質粒pEGFP-C1為本實驗室保存,Hela細胞由本教研室鬍萍博士惠贈.方法:實驗于2005-09/2006-06在暨南大學醫學院微生物與免疫教研室和中心實驗室完成.以MTB全基因組為模闆經聚閤酶鏈反應(PCR)擴增齣Hsp65基因(不含終止子),與pEGFP-C1載體進行重組,構建pEGHsp65(不含終止子)重組質粒.再以pVAE質粒為模闆,經PCR擴增齣Linker-Esat6基因,與pEGHsp65質粒(不含終止子)進行重組,構建真覈錶達載體pEGHsp65-Esat6.通過限製性內切酶圖譜測定pEGHsp65-Esat6融閤質粒大小;對質粒pEGFP-C1的多剋隆位點內的DNA序列進行序列分析鑒定;將質粒轉染Hela細胞,觀察熒光的錶達情況及轉染效率,通過免疫組織化學法分彆檢測細胞中Hsp65蛋白和ESAT-6蛋白的錶達.主要觀察指標:①pEGHsp65-Esat6融閤質粒大小.②pEGFP-C1DNA序列分析.③轉染後Hela細胞的熒光錶達情況.④細胞中Hsp65蛋白和ESAT-6蛋白的免疫組織化學結果.結果:①質粒pEGFP-C1的大小約為4.7 kb,而pEGHsp65為6.4 kb,融閤質粒pEGHsp65-Esat6約為6.7 kb,在瓊脂糖凝膠電泳圖上可以分辨齣泳動速度的差異.②結果與結覈分支桿菌H37Rv株的Hsp65和Esat6的基因序列完全相同.③轉染融閤質粒24 h後,使用共聚焦顯微鏡可觀察到有部分細胞錶達熒光蛋白,說明融閤質粒轉染成功,轉染率約為30%.未轉染的Hela細胞則無熒光錶達.④通過免疫組織化學法證實融閤質粒在Hela細胞內能正確錶達齣具有生物學活性的Hsp65與ESAT-6蛋白.結論:採用Hsp65和Esat6搭配作為免疫原,成功剋隆併構建瞭結覈分枝桿菌融閤基因Hsp65-Esat6熒光真覈錶達質粒.
배경:Hsp65화Esat6지간적Linker편마일단소수성다태,기구유량호적유순성화절첩성,유리우번역성융합단백시융합단백지간적공간절첩,사융합단백보지여량개천연단백공간구상적일치성,종이형성정학적구형이제고타문적면역원성.목적:종결핵분지간균(MTB)H37Rv주중극륭목적융합기인Hsp65-Esat6,여보고기인EGFP일기구건진핵표체재체pEGHsp65-Esat6,통과면역조직화학법분별검측세포중Hsp65단백화ESAT-6단백적표체.설계:단일양본실험.단위:기남대학의학원미생물면역학교연실.재료:질립pVAE유화남리공대학조이성교수혜증,MTBH37Rv주、대장간균DH5α、표체질립pEGFP-C1위본실험실보존,Hela세포유본교연실호평박사혜증.방법:실험우2005-09/2006-06재기남대학의학원미생물여면역교연실화중심실험실완성.이MTB전기인조위모판경취합매련반응(PCR)확증출Hsp65기인(불함종지자),여pEGFP-C1재체진행중조,구건pEGHsp65(불함종지자)중조질립.재이pVAE질립위모판,경PCR확증출Linker-Esat6기인,여pEGHsp65질립(불함종지자)진행중조,구건진핵표체재체pEGHsp65-Esat6.통과한제성내절매도보측정pEGHsp65-Esat6융합질립대소;대질립pEGFP-C1적다극륭위점내적DNA서렬진행서렬분석감정;장질립전염Hela세포,관찰형광적표체정황급전염효솔,통과면역조직화학법분별검측세포중Hsp65단백화ESAT-6단백적표체.주요관찰지표:①pEGHsp65-Esat6융합질립대소.②pEGFP-C1DNA서렬분석.③전염후Hela세포적형광표체정황.④세포중Hsp65단백화ESAT-6단백적면역조직화학결과.결과:①질립pEGFP-C1적대소약위4.7 kb,이pEGHsp65위6.4 kb,융합질립pEGHsp65-Esat6약위6.7 kb,재경지당응효전영도상가이분변출영동속도적차이.②결과여결핵분지간균H37Rv주적Hsp65화Esat6적기인서렬완전상동.③전염융합질립24 h후,사용공취초현미경가관찰도유부분세포표체형광단백,설명융합질립전염성공,전염솔약위30%.미전염적Hela세포칙무형광표체.④통과면역조직화학법증실융합질립재Hela세포내능정학표체출구유생물학활성적Hsp65여ESAT-6단백.결론:채용Hsp65화Esat6탑배작위면역원,성공극륭병구건료결핵분지간균융합기인Hsp65-Esat6형광진핵표체질립.
BACKGROUND:There is a linker between Hsp65 and Esat6 coding a segment of water repellent polypeptide.It is very gentle and easy to be folded,which is profitable to translate the keno-folding correctly between the two proteins and to make the space structure of fusional proteins consistent with those two native ones.Then,a correct structure is formed and the immunogenicity of the fusional proteins is improved.OBJECTIVE:This study was to clone the fusional genes Hsp65-Esat6 from mycobacterium tuberculosis(MTB)H37Rv, then to construct into a eukaryotic expressing vector which contains an enhanced green fluorescent protein(EGFP) reporter gene,and finally to identify the expression of Hsp65 protein and Esat6 protein by IHC methods.DESIGN:Single sample experiment.SETTING:Department of Microbiology ＆ Immunology,Medical College of Jinan University.MATERIALS:Plasmid pVAE was donated by Professor Cao from South China University of Technology.MTB H37Rv,E.coil DH5 α,expressing plasmid pEGFP-C1 and Hela cells were donated by Doctor Hu Ping.METHODS:The experiment was conducted at the Department of Microbiology ＆ Immunology and the Medical Experimental Center of Jinan University between September 2005 and June 2006.The whole-genome was extracted from MTB H37Rv by molecular cloning technique,and used it as template to amplify Hsp65 (no terminator) gene by polyrnerase chain reaction (PCR),to recombine it with pEGFP-C1 vector after purification to construct pEGHsp65(no terminator)recombination vector.pVAE vector was used as template to amplify Linker-Esat6 gene(with terminator)by PCR, and then to recombine it with pEGHSP65(no terminator)to construct an eukaryotic expressing vector pEGHsp65-Esat6 with the fusional genes Hsp65-Esat6 inside.Finally,genes Hsp65-Esat was checked by molecule biology methods such as PCR, restriction endonuclease and DNA sequencing.Hela cells were transfected with pEGHsp65-Esat6 and the expression of EGFP and the efficiency of transfection were observed.The expressions of Hsp65 protein and Esat6 protein were detected by IHC methods.MAIN OUTCOME MEASURES:①The size of pEGHsp65-Esat6.②The DNA sequencing result of the pEGFP-C1.③The expression of EGFP in the transfected Hela cells.④The IHC results of Hsp65 protein and Esat6 protein in Hela cells.RESULTS:①The size of pEGFP-C1 was 4.7 kb,that of pEGHsp65 was 6.4 kb,and that of pEGHsp65-Esat6 was 6.7 kb.There were differences between their speeds In agarose electrophoresis.②The results showed that it was the same as reported Hsp65 sequence and Esat6 sequence of MTB H37Rv.③After transfecting pEGHsp65-Esat6 for 24 hours,EGFP was found in 30% of Hela through Laser scanning confocal microscope. But there was no EGFP in non-transfected Hela.④Hsp65 protein and Esat6 protein with biological activities were detected in transfected Hela cells by IHC methods.CONCLUSION: Using Hsp65 and Esat6 as immunogens,we have successfully cloned and constructed a eukaryotic expressing vector which contain fusionaI genes Hsp65-Esat6 of MTB H37Rv and EGFP.
