幽门螺杆菌γ谷氨酰转肽酶同源基因的表达与活性检测  

Expression of Helicobacter pylori γ-glutamyltransferase in silkworm cells using a baculovirus expression system

作　　者：孔梅[1] 张尤历[1] 陈鑫[1] 陈慧娟[1] 

机构地区：[1]江苏大学附属医院消化科,江苏省镇江市212001

出　　处：《世界华人消化杂志》2009年第29期2996-3000,共5页World Chinese Journal of Digestology

摘　　要：目的:评价幽门螺杆菌(Hpylori)ggt同源基因的结构特征和功能.方法:通过生物信息学软件分析Hpyloriγ谷氨酰转肽酶(ggt)同源基因产物的结构特征;提取HpyloriDNA,并以之为模板,采用PCR法扩增该基因的全长及去信号肽片段;进行序列分析后,将全长片段与gfp片段连接,分别克隆进杆状病毒的转移质粒pFastBac1中,利用杆状病毒的Bac-to-Bac系统分别获得重组杆状病毒的DNA;通过PCR方法验证这些片段克隆进杆状病毒;将重组病毒的DNA以脂质体为媒介转染家蚕细胞,以获得重组杆状病毒;分别收集这些重组病毒,并再感染家蚕细胞,进行蛋白表达实验和活性检测,以荧光显微镜观察融合蛋白在细胞中的分布.结果:通过PCR方法成功克隆了各种片段;测序的结果表明,未发生突变;基因片段扩增的结果说明,获得了3种重组的杆状病毒,且这3种重组病毒均表达了ggt片段,全长、去信号肽片段及去信号肽片段与gfp融合的片段的表达产物活性分别为3.61,10.50及9.31U/L.Westernblot的结果证实了去信号肽片段与GFP的融合表达.通过荧光显微镜观察去信号肽与gfp的融合片段的表达产物在细胞中的定位,荧光不具有区域的特异性,充满了整个细胞,表明该基因产物并非作用于专一的细胞器.结论:Hpyloriggt同源基因适合于杆状病毒表达,表达产物具有GGT活性,但他对细胞的作用有待进一步研究.AIM： To express the Helicobacter pylori （H pylori） γ-glutamyltransferase （GGT） in silkworm cells using a baculovirus expression system and analyze its activity in vitro. METHODS： The structure characteristics of the H pylori ggt gene was analyzed using biological informatics software. The genomic DNA of H pylori was extracted and used as template to amplify the ggt genes encoding proteins with and without signal peptide by polymerase chain reaction （PCR）. After sequencing, the amplified ggt gene encoding protein without signal peptide was fused with a green fluorescent protein （GFP） sequence. Subsequently, the two amplicons and the fusion sequence were cloned into the baculovirus transfer vector pFastBacl, respectively. Bac-to-Bac baculovirus system was then used to generate recombinant virus DNA. Recombinant virus DNA was transfected into silkworm BmN cells to obtain recombinant viruses. The viruses were harvested and used to infect BmN cells again. After infection, BraN cells were harvested to detect protein expression and enzymatic activity. Fluorescence was observed in infected cells using a fluorescence microscope. RESULTS： The ggt genes encoding proteins with and without signal peptide were success- fully cloned by PCR. Sequencing results indicat- ed that no mutations occurred. All the three re- combinant viruses obtained could express GGT. The activity of GGT expressed by the recombinant viruses harboring the ggt genes encoding proteins with and without signal peptide and the fusion sequence was 3.61, 10.50 and 9.31 U/L, respectively. Western blot assay demonstrated the expression of GGT-GFP fusion protein. Fluo- rescent microscopy showed that the fluorescence was distributed throughout the whole cell, indi- cating that GGT expression is not confined to a certain organelte. CONCLUSION： GGT with enzymatic activity can be expressed in silkworm cells using the baculovirus expression system.

关 键 词：幽门螺杆菌 谷氨酰转肽酶 表达 杆状病毒 

分 类 号：R573[医药卫生—消化系统]