摘要
目的为探讨媒介与病原、媒介与宿主的相互作用,建立传播疾病的淡色库蚊性别差异表达cDNA文库,从而筛选阻断疾病传播的疫苗候选分子。方法以淡色库蚊雌蚊成蚊为检测方(Tester)、雄蚊成蚊为驱动方(Driver),进行正向抑制性消减杂交;以雄蚊成蚊为Tester、雌蚊成蚊为Driver,进行反向抑制性消减杂交。将获得的正向抑制性消减杂交产物克隆入pGEM-Teasyvector,并以菌液PCR扩增鉴定插入片段。随机抽取100个阳性克隆进行DNA序列分析,并将所得ESTs序列进行在线BLAST分析。结果从100个阳性克隆中测得98个ESTs序列,在测定的序列中与已知基因具有同源性的有57个,其余41个ESTs与已知基因不具有同源性。结论抑制性消减杂交技术建立雌蚊特异性基因文库,发现了淡色库蚊雌蚊新的ESTs序列,为性别相关基因的功能及性别调控,探讨媒介与病原、媒介与宿主的相互作用及其筛选传播阻断疫苗候选分子的研究奠定了基础。
Objective To construct a sex-differentlally expressed cDNA library for Culex pipiens pallens, and to identify potential vaccine candidate molecules. Method Total RNA samples were separately isolated from pools of adult females and males Culex pipiens paUens, and suppression subtractive hybridization （SSH） was performed by using the PCR-select cDNA Subtraction kit （Clontech） ： the forward subtracted library was constructed by using female as tester and male as driver, and the reverse-subtracted library was constructed by using male as tester and female as driver. The subtracted cDNA was ligated into pGEM（r）-T easy vector. Recombinant plasmids were transformed into competent Escherichia coli cells DH5α. Result 100 random-selected positive clones were sequenced, and the 98 valid EST sequences were analyzed with The Basic Local Alignment Search Tool （BLAST） online （http：//www.ncbi. nlm.nih.gov/BLAST）. 57 ESTs had homologous sequences in the GenBank database, and 41 ESTs had no homologs in the DNA database. Conclusion Sex-differentially expressed cDNA library for Culex pipiens pallens was constructed by SSH, and some female-specific genes were identified which may provide foundation to study the biology of vector-pathogen and vector-host interactions and to identify potential vaccine candidate molecules for blocking transmission of pathogens.
出处
《热带医学杂志》
CAS
2007年第2期105-108,147,共5页
Journal of Tropical Medicine
基金
深圳市科技局重点研究课题(No.JH200505300494A)
关键词
淡色库蚊
抑制消减杂交
性别差异表达基因
Culex pipiens pallens
suppression subtractive hybridization
sex differentially expressed genes
