【作者】 张茜；

【导师】 阚飙；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2009， 硕士

【摘要】 霍乱是由霍乱弧菌(Vibrio cholerae)引起的急性肠道传染病。始于1961年,至今未停息的第七次霍乱大流行由O1群霍乱弧菌El Tor型引起。目前我国利用噬菌体—生物分型相结合的方法对El Tor型菌株进行分型,可以把霍乱弧菌区分为流行株和非流行株,在疾控工作中区别对待两类菌株。El Tor型霍乱弧菌噬菌体VP3是五个分型噬菌体之一。VP3的全基因组测序分析,表明其基因组为线性双链DNA,长39505bp,G+C含量42.62%,预测存在17个保守的启动子序列和52个编码基因。为研究VP3启动子的活性,我们利用海肾荧光素酶基因(rluc)作为报告基因,建立了一种高通量的连续实时监测系统,即在rluc基因上游连接不同的VP3启动子序列得到重组质粒pRLV-clone,将其分别转入霍乱弧菌N16961中,为便于描述,我们将含有上述重组质粒的N16961命名为NPRLV-clone。实验结果显示VP3的17启动子具有明显转录活性,启动子5、6、10和15活性较弱。噬菌体感染宿主菌后,菌体的裂解与噬菌体基因组的大量转录复制有关,该转录过程涉及宿主菌的RNA聚合酶(RNAP)和噬菌体基因组编码的RNAP。为探索VP3 RNAP预测基因的功能和其对自身启动子的识别转录情况,我们把VP3 RNAP的预测编码基因克隆到质粒pBAD33,所得重组质粒pBAD33-VP3-RNAP导入到含有pRLV-clone的大肠杆菌JM109中,通过荧光观测来确定在JM109中VP3的RNAP对于自身启动子的识别和转录情况,结合不感染VP3的NPRLV-clone中观测到的荧光值的结果,来分析VP3和N16961的RNAP对VP3启动子的识别和转录作用。结果提示霍乱弧菌(N16961)的RNAP能够识别和转录绝大多数VP3的启动子区域,与T7家族噬菌体的感染和转录模式相似,霍乱弧菌的RNAP可能在VP3噬菌体感染早期基因组转录过程中发挥重要的作用。JM109的RNAP不能识别和转录霍乱弧菌VP3的绝大多数启动子,这可能与噬菌体严格的宿主特异性有关,但是我们也看到,人为添加或补充VP3的RNAP可以使部分启动子的活性得以表达。VP3噬菌体全基因测序结果和实验证明其染色体为线性双链DNA,多联体形式复制。但是,利用两侧的引物对其DNA的末端进行PCR,能够得到单一条带,PCR产物测序结果显示其序列单一,实验结果提示其基因组可能存在部分环形结构或者是存在特殊结构,需要进一步的研究确认。另外,为研究VP3在霍乱弧菌中的受体和吸附作用,本研究利用自杀质粒pCVD442介导的同源重组技术,缺失了霍乱弧菌N16961中编码O抗原连接酶的VC0237基因。VC0237缺失株(命名为C37),其不被VP3裂解,提示霍乱弧菌O抗原可能与VP3吸附感染宿主菌有关。更多还原

【Abstract】 Cholera is an acute intestinal infectious disease caused by O1 and O139 Vibrio cholerae.There have been seven recorded cholera outbreaks caused by O1 serotype V. cholerae.The seventh and present pandemic of cholera begins in 1961 caused by El Tor strains.A useful phage-biotying approach was used in China.By this approach,V. cholerae El Tor strains is devided into two kinds of strains,i.e,epidemic strains and non-epidemic strains.We deal different group of strains with different processes in our daily work of diseases prevention and control.VP3 is one of the five typing phages used in phage-biotying approach.A shot-gun strategy has been used to determine the complete nucleotide sequence of VP3,combined with the primer walking method to close the sequence gaps.The genome sequence is 39,505bp,with a G+C content of 42.62%,which encodes 52 putative protein encoding open reading frames（CDs） and 17 T7-like promoters.In order to monitor the activity of VP3 promoters,we developed a real-time observation system by useing the rluc as a report gene,along with the VP3 infection of V. cholerae.The result of the test manifests that the 17 predicted VP3 promoters region have promoter activities,but the activity of VP3 promoters No.5,6,10and 15 is weaker than that of the other 13 promoters.After infection,the lysis of host strain is related with generous replication of the VP3 genome which depends on RNAP of V.cholerae and presumed RNAP of VP3. To further determine the role of the two RNAPs,we constructed the plasmid pBAD33-VP3-RNAP by cloning the VP3 RNAP gene to pBAD33.Then the plasmids containing the putative VP3 promoter and RNAP gene respectively were introduced into E.coli JM109 and the fluorescent signal was assayed with the VivRen^TM Live Cell Substrate.We find that the most of the VP3 promoters can be triggered by the RNAP of N16961 in V.cholerae,but not by RNAP of JM109 in JM109.We also found that some VP3 promoters could be triggered in JM109 by introducing VP3 RNAP gene on plasmid.Our results indicate that the infection pattern of VP3 maybe as same as T7 family,in which the host RNAP plays an important role in the transcription of the early genes of T7 phage.The transcription of VP3 genome in V.cholerae may be related to the VP3 DNA terminal sequence.Though the whole genome sequence of VP3 has been sequenced, the real end of it has not been deterimined.We got a single band by using two primers complementary to each end of the VP3 sequence.The results suggests that there maybe some circle or some unique struction at the end of VP3 DNA.In order to study the receptor of VP3 in host cell,we deleted the O antigen encoding gene VC 0237 by homologous recombination mediated by suicide plasmid pCVD442.The generated strain（named C37） can’t be lysed by VP3.Our result indicated that the O antigen of V.cholerae is envolved in the VP3 infection.更多还原